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Research Article Proteomic Analysis of HDAC3 Selective Inhibitor in the Regulation of Inflammatory Response of Primary Microglia Mingxu Xia, 1 Qiuchen Zhao, 1 He Zhang, 1 Yanting Chen, 1 Zengqiang Yuan, 2 Yun Xu, 1 and Meijuan Zhang 1 1 Department of Neurology, Affiliated Drum Tower Hospital, Nanjing University Medical school, Nanjing, Jiangsu 210008, China 2 e State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China Correspondence should be addressed to Meijuan Zhang; [email protected] Received 23 September 2016; Revised 27 December 2016; Accepted 12 January 2017; Published 15 February 2017 Academic Editor: Michele Fornaro Copyright © 2017 Mingxu Xia et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HDAC3 has been shown to regulate inflammation. However, the role of HDAC3 in primary microglia is largely unknown. RGFP966 is a newly discovered selective HDAC3 inhibitor. In this study, we used protein mass spectrometry to analyze protein alterations in LPS-treated primary microglia with the application of RGFP966. Generally, about 2000 proteins were studied. 168 of 444 (37.8%) LPS-induced proteins were significantly reduced with the treatment of RGFP966, which mainly concentrated on Toll-like receptor signaling pathway. In this regard, we selected Toll-like receptor 2 (TLR2), TLR3, TLR6, MAPK p38, CD36, and spleen tyrosine kinase (SYK) for further validation and found that they were all significantly upregulated aſter LPS stimulation and downregulated in the presence of RGFP966. Additionally, RGFP966 inhibited supernatant tumor necrosis factor (TNF)- and Interleukin 6 (IL-6) concentrations. Activation of STAT3 and STAT5 was partially blocked by RGFP966 at 2 h aſter LPS-stimulation. e fluorescence intensity of CD16/32 was significantly decreased in LPS + RGFP966-treated group. In conclusion, our data provided a hint that RGFP966 may be a potential therapeutic medication combating microglia activation and inflammatory response in central nervous system, which was probably related to its repressive impacts on TLR signaling pathways and STAT3/STAT5 pathways. 1. Introduction Microglia are resident immune cells in the brain and play a pivotal role in immune surveillance. ey are activated in diverse neurological diseases including encephalitis, stroke, Parkinson’s disease, and Alzheimer’s disease, resulting in the subsequent inflammatory cascade [1]. It is undisputable that inflammation is beneficial for homeostasis restoration and tissue repair by means of clearing pathogens and harmful cell components. However, excessive inflammation causes damage to brain tissues and exacerbates the initial insult. erefore, the magnitude of microglia activation must be tightly controlled to avoid the collateral tissue damage and to regulate the progression of neurological diseases [2, 3]. Histone deacetylases (HDACs) are conserved metallo- proteases which aim to remove acetyl groups from lysine residues of targeted proteins. In accordance with their struc- tural diversity, HDACs are divided into four subtypes: Class I (HDAC1, HDAC2, HDAC3, and HDAC8), Class II consisting of IIa (HDAC4, HDAC5, HDAC7, and HDAC9) and IIb (HDAC6 and HDAC10), Class III (a family of sirtuins), and Class IV (HDAC11) [4]. Together with histone acetylases (HATs), HDACs regulate acetylation level of histones (H3, H2AK5, H4K5, H4K12, H2B, H4K8, and H4K16) as well as some other proteins (p65 and myocyte enhancer factor 2) [5]. Recently, several studies proposed that HDAC inhibitors are involved in modulating innate immune activity [6, 7] and could be potentially applied in various human diseases [8]. Hindawi Neural Plasticity Volume 2017, Article ID 6237351, 13 pages https://doi.org/10.1155/2017/6237351

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Page 1: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

Research ArticleProteomic Analysis of HDAC3 SelectiveInhibitor in the Regulation of Inflammatory Response ofPrimary Microglia

Mingxu Xia1 Qiuchen Zhao1 He Zhang1 Yanting Chen1 Zengqiang Yuan2

Yun Xu1 andMeijuan Zhang1

1Department of Neurology Affiliated Drum Tower Hospital Nanjing University Medical school NanjingJiangsu 210008 China2The State Key Laboratory of Brain and Cognitive Sciences Institute of Biophysics Chinese Academy of SciencesBeijing 100101 China

Correspondence should be addressed to Meijuan Zhang juanzi1986126163com

Received 23 September 2016 Revised 27 December 2016 Accepted 12 January 2017 Published 15 February 2017

Academic Editor Michele Fornaro

Copyright copy 2017 Mingxu Xia et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

HDAC3 has been shown to regulate inflammationHowever the role ofHDAC3 in primarymicroglia is largely unknown RGFP966is a newly discovered selective HDAC3 inhibitor In this study we used protein mass spectrometry to analyze protein alterations inLPS-treated primary microglia with the application of RGFP966 Generally about 2000 proteins were studied 168 of 444 (378)LPS-induced proteins were significantly reduced with the treatment of RGFP966 which mainly concentrated on Toll-like receptorsignaling pathway In this regard we selected Toll-like receptor 2 (TLR2) TLR3 TLR6 MAPK p38 CD36 and spleen tyrosinekinase (SYK) for further validation and found that they were all significantly upregulated after LPS stimulation and downregulatedin the presence of RGFP966 Additionally RGFP966 inhibited supernatant tumor necrosis factor (TNF)-120572 and Interleukin 6 (IL-6)concentrations Activation of STAT3 and STAT5 was partially blocked by RGFP966 at 2 h after LPS-stimulation The fluorescenceintensity of CD1632 was significantly decreased in LPS + RGFP966-treated group In conclusion our data provided a hint thatRGFP966may be a potential therapeuticmedication combatingmicroglia activation and inflammatory response in central nervoussystem which was probably related to its repressive impacts on TLR signaling pathways and STAT3STAT5 pathways

1 Introduction

Microglia are resident immune cells in the brain and playa pivotal role in immune surveillance They are activated indiverse neurological diseases including encephalitis strokeParkinsonrsquos disease and Alzheimerrsquos disease resulting in thesubsequent inflammatory cascade [1] It is undisputable thatinflammation is beneficial for homeostasis restoration andtissue repair by means of clearing pathogens and harmfulcell components However excessive inflammation causesdamage to brain tissues and exacerbates the initial insultTherefore the magnitude of microglia activation must betightly controlled to avoid the collateral tissue damage andto regulate the progression of neurological diseases [2 3]

Histone deacetylases (HDACs) are conserved metallo-proteases which aim to remove acetyl groups from lysineresidues of targeted proteins In accordance with their struc-tural diversity HDACs are divided into four subtypes Class I(HDAC1 HDAC2 HDAC3 andHDAC8) Class II consistingof IIa (HDAC4 HDAC5 HDAC7 and HDAC9) and IIb(HDAC6 and HDAC10) Class III (a family of sirtuins) andClass IV (HDAC11) [4] Together with histone acetylases(HATs) HDACs regulate acetylation level of histones (H3H2AK5 H4K5 H4K12 H2B H4K8 and H4K16) as well assome other proteins (p65 and myocyte enhancer factor 2)[5] Recently several studies proposed that HDAC inhibitorsare involved in modulating innate immune activity [6 7] andcould be potentially applied in various human diseases [8]

HindawiNeural PlasticityVolume 2017 Article ID 6237351 13 pageshttpsdoiorg10115520176237351

2 Neural Plasticity

However previous used broad-spectrum HDAC inhibi-tors target several HDACs and it is difficult to define exactrole of each subtype Additionally clinical trials with pan-HDACi in cancer patients suffered undesired effects includ-ing increased susceptibility to pneumonia thrombocytope-nia anorexia [9] Thus it is necessary to focus on the specificHDAC inhibitor

HDAC3 is themost widely expressedHDACs in the brain[10] and is thought to play a role in Huntington [4] SCA [11]and dementia diseases [12] HDAC3-deficient macrophagespossessed decreased ability to activate inflammatory geneexpression in response to LPS stimulation [13] Concomi-tantly HDAC3 was found to be an epigenomic brake inmacrophage alternative activation [14] while inflammationrepressive repertoire of HDAC3 in primary microglia islargely unknown RGFP966 is a selective HDAC3 inhibitorwith an IC50 of 008120583M and no effective inhibition of otherHDACs at concentrations up to 15 120583M and could cross brainblood barrier when administrated peripherally [15] Pharma-cological inhibition of HDAC3 may bring more evidencesand prosperities for clinical applications This insight nowgives us the opportunity to study possible inflammatoryconsequences of HDAC3 in central nervous system

In this issue we used protein mass spectrometry toprofile global molecular alterations in primary microgliaexposed toRGFP966 exploring a potential signaling pathwaythroughwhichHDAC3 specific inhibitor RGFP966 regulatedinflammation

2 Materials and Methods

21 Primary Microglia Culture and Treatment Primarymicroglia cells were prepared from C57BL6 mice bornwithin 24 hours as previously described Briefly cerebralcortex tissue was digested in TrypLE for 10 minutes at 37∘CThen Minimum Essential Medium (MEM) (Hyclone USA)supplemented with 10 fetal bovine serum (FBS) (BiologicalIndustries Israel) 100UmL penicillin and 100 ugmL strep-tomycinwas used to terminate digestionAfterwards the cellswere centrifuged at 1500 rpm for 5minutes resuspended andseeded in the 75 cm2 flasks After 36ndash48 hours the culturemedia were replaced by Dulbeccorsquos Modified Eagle Media(DMEM) (HycloneUSA)with 10FBS 100UmLpenicillinand 100 ugmL streptomycin At day 11ndash13 microglia cellswere suspended and obtained by shaking the flasks at 180 rpmfor 10min at 37∘C The mature microglia cells were seededinto plates at a density of 2 times 105cm2 and placed for 36ndash48 hours before further treatment RGFP966 (Selleckchem)was dissolved in dimethyl sulfoxide (DMSO) to make 55mMstock solution In purified enzyme assays inhibition IC50values of RGFP966 for HDAC1 HDAC2 and HDAC3 weregt15 120583Mgt15 120583M and 008 120583M[15 16] Primarymicroglia cellswere pretreated with DMSO or RGFP966 at a concentrationof 15 120583M for an hour Then LPS (Sigma-Aldrich USA) wasadded to the culture media at a dose of 500 ngmL Proteins

mRNA and supernatant were collected for proteomic anal-ysis western blotting CBA and Q-PCR at indicated timepoints

22 Proteomic Analysis Proteomic analysis was performedby AB SCIEX TripleTOF 5600 mass spectrometer (ABSCIEX USA) equipped with a liquid chromatography-tandem mass spectrometry (LC-MSMS) system Proteins(200120583g) of four samples were resolved on 10 SDS polyacry-lamide gels The gels were stained with Coomassie Blue G-250 for 1 hour and then cut into blocks after distaining inultrapure H

2O The gel blocks were digested using trypsin

and peptides were extracted from them After being driedand redissolved the peptides were analyzedThemass spectrawere annotated against the Uniprot proteome databaseBy means of the Software DAVID coupled with STRING(version 100) the Gene Ontology analysis KEGG pathwayanalysis and protein-protein interactions were completed

23 Real-Time PCR As described previously Trizol reagent(InvitrogenUSA)was used for the total RNA extraction frommicroglia cells and then RNA was reverse-transcribed intocDNA with a PrimeScript RT reagent Kit (Takara DalianChina) The quantitative measurements were performed onan ABI 7500 PCR instrument (Applied Biosystems USA)with a SYBR green Kit (Takara Dalian China) Relative geneexpressions were normalized to glyceraldehyde-3-phosphatedehydrogenase (GAPDH) and mRNA expression levels werepresented as fold changes versus DMSO group The primers(Invitrogen) used are as follows

TLR-2 primers

Forward 51015840-TCACATGGCAGAAGATGTGTC-31015840

Reverse 51015840-GGTGATGCAATTCGGATGCT-31015840

TLR-3 primers

Forward 51015840-TGAGAAGAGCCACAGTGATAGA-31015840

Reverse 51015840-CTCTCCAGCAGAAGAGACACAA-31015840

TLR6 primers

Forward 51015840-AATGGTACCGTCAGTGCTGGA-31015840

Reverse 51015840-CTTGGCTCATGTTGCAGAGG-31015840

CD36 primers

Forward 51015840-TGAATGGTTGAGACCCCGTG-31015840

Reverse 51015840-TAGAACAGCTTGCTTGCCCA-31015840

GAPDH primers

Forward 51015840-GCCAAGGCTGTGGGCAAGGT-31015840

Reverse 51015840-TCTCCAGGCGGCACGTCAGA-31015840

24 Western Blot Western blot was performed as previ-ously described Equal amounts of proteins were separated

Neural Plasticity 3

by sodium dodecyl sulfate-PAGE electrophoresis and thenblotted onto polyvinylidene fluoride membranes After beingblocked in 5 fat-free milk for 2 hours membranes wereincubated with primary antibodies against TLR2 (1 1000Abcam UK) TLR3 (1 1000 Abcam UK) TLR6 (1 1000Cell Signaling Technology USA) MAPK p38 (1 500 CellSignaling Technology USA) phospho-p38 (1 500 Cell Sig-naling Technology USA) CD36 (1 1000 Abcam UK) SYK(1 1000 AbcamUK) STAT3 (1 500 Cell Signaling Technol-ogy USA) phospho-STAT3 (1 500 Cell Signaling Technol-ogy USA) STAT5 (1 500 Cell Signaling Technology USA)phospho-STAT5 (1 500 Cell Signaling Technology USA)and GAPDH (1 5000 Bioworld USA) in 4∘C overnight toprobe targeted proteins Horseradish peroxidase-conjugatedsecondary antibodies (1 5000 Bioworld USA) were used tocombine primary antibodies and the reaction was detectedwith an ECL Kit (Bioworld USA) The intensities of blotswere quantified by densitometry

25 Immunofluorescence Primary microglia cells seeded oncover slips were fixed with 4 polyformaldehyde for 15 min-utes and permeabilizedwith 02Triton-X100 for 20minutesat room temperature After being blocked in 2 BSA in PBSfor 2 hours microglia cells were incubated with donkey anti-CD1632 antibody (1 500 BD Pharmingen USA) overnightin 4∘C and subsequently incubated with FITC-conjugatedanti-rat IgG (Invitrogen USA) at room temperature for 2hours DAPI staining was used to localize the nuclei Imageswere taken by a fluorescence microscope (Olympus Japan)and fluorescence intensities were analyzed by ImageJ software(version 139 National Institutes of Health USA)

26 Quantification of Secreted Cytokines Cytokines in thesupernatants were measured using the Cytometric BeadArray (CBA) Mouse Inflammation Kit (BD BiosciencesUSA) Briefly after incubation with capture beads on whichanti-cytokine antibodies are coated and PE-conjugated anti-cytokine antibodies cytokine levels can be quantitativelyanalyzed The assays were performed by BD Accuri C6 flowcytometer (BD Biosciences USA) following the manufac-turerrsquos instructions and the data were generated with FCAPArray version 301 Software

27 Statistical Analysis Data were expressed as mean plusmn SDof three independent experiments Comparisons betweengroups were conducted with SPSS 220 software Differenceswere analyzed by two-way analysis of variance (ANOVA)followed by Bonferronirsquos post hoc test and considered to bestatistically significant if 119901 lt 005

3 Results

31 Overview of Proteomic Analysis In this study 1883 pro-teins were detected in DMSO group and 1967 proteins werein RGFP966 group In LPS-stimulated groups there were1806 proteins in DMSO + LPS group and 2024 proteins in

Table 1 Summary of LC-MSMS data

Treatment Total proteinsDMSO 1883RGFP966 1967DMSO + LPS 1806RGFP966 + LPS 2024

DMSO

DMSORGFP9

66

RGFP966

+ LPS

+ LPS

169

70

138

35

48

28

82

87

107

1361

58

53

120

64

174

Figure 1 Overview of expressed proteins in four groups The bluecircle representedDMSOgroup the red circle representedRGFP966group the green circle represented DMSO + LPS group and theyellow circle represented RGFP966 + LPS group

RGFP966 + LPS group (Table 1) A Venn diagram showed therelationship of expressed proteins in four groups (Figure 1)

32 Alterations in Proteins Expression We identified gt 15fold peptide gt 1 as upregulated proteins and lt 066 foldpeptide gt 1 as downregulated proteins The numbers of dif-ferently expressed proteins between two groups were listed inTable 2 Specifically 168 of 444 (378) LPS-induced proteinswere significantly reduced with the treatment of RGFP966(Figure 2(a)) Likewise when analyzing proteins downreg-ulated by LPS the impact of RGFP966 was comparablewith 134 of 404 (332) proteins being rescued by RGFP966(Figure 2(b)) The heat map presented detailed informationof the upregulated as well as downregulated proteins whichwere caused by LPS and simultaneously reversed byRGFP966(Figure 3)

33 Gene Ontology Analysis GO analysis was used to revealthe function of proteins in three aspects cellular compo-nent molecular function and biological process The 168differentially expressed proteins overlapped in Figure 2(a)were mainly involved in biological process cellular processimmune system process and establishment of localizationcellular component organellemacromolecular complex andorganelle part molecular function binding catalytic activity

4 Neural Plasticity

Table 2 Summary of upregulateddownregulated proteins candidates

Upregulated protein candidates Downregulated protein candidatesgt 15 fold peptide gt 1 lt 066 fold peptide gt 1

RGFP966 versus DMSO 493 338DMSO + LPS versus DMSO 444 404RGFP966 + LPS versus DMSO + LPS 401 275

DMSO + LPSDMSO upregulated proteins

107168276

RGFP966 + LPSDMSO + LPS downregulated proteins

(a)

DMSO + LPSDMSO downregulated proteins

267134270

RGFP966 + LPSDMSO + LPS upregulated proteins

(b)

Figure 2 Venn diagrams of the differentially expressed proteins between LPS-stimulated (DMSO + LPSDMSO) and RGFP966-treated(RGFP966 + LPSDMSO + LPS) microglia cells (a) 168 proteins were upregulated by LPS and reversed with the treatment of RGFP966 (b)134 proteins were downregulated after the stimulation of LPS and rescued by RGFP966

and enzyme regulator activity (Figure 4(a)) However the 134differentially expressed proteins overlapped in Figure 2(b)were mainly involved in biological process cellular processmetabolic process and establishment of localization cellularcomponent organelle macromolecular complex and cellpart molecular function catalytic activity binding andelectron carrier activity (Figure 4(b))

34 KEGG Pathway Analysis To explore the potential sig-naling pathway through which HDAC3 specific inhibitorRGFP966 regulates inflammatory response KEGG pathwayanalysis was performed As shown in Figure 5(a) the pathwaymap depicted that the 168 differentially expressed proteinsoverlapped in Figure 2(a) were related to Toll-like recep-tor signaling pathway Alzheimerrsquos disease cytosolic DNA-sensing pathway spliceosome RIG-I-like receptor signalingapoptosis cell cycle insulin signaling pathway Huntingtonrsquosdisease calcium signaling pathway and pathways in cancerHowever the 134 differentially expressed proteins over-lapped in Figure 2(b) belonged to the following pathwaysaminoacyl-tRNAbiosynthesis Fc gammaR-mediated phago-cytosis mTOR signaling pathway spliceosome Parkinsonrsquosdisease and so forth (Figure 5(b))

35 mRNA Verifications of Discrepant Proteins between LPSGroup and LPS + RGFP966 Group RT-PCR was performed

to detect the mRNA levels of TLR2 TLR3 TLR6 CD36and SYK at 6 hours after LPS stimulation As expectedLPS stimulation potentiated profound mRNA increase ofTLR2 (924 folds versus DMSO group) TLR3 (2881 foldsversus DMSO group) TLR6 (173 folds versus DMSO group)and CD36 (878 folds versus DMSO group) However theseaugments were significantly decreased by RGFP966 with thedecline amplitudes at 7358 in TLR2 (119901 = 0002) 3551in TLR3 (119901 lt 0001) 3757 in TLR6 (119901 = 0007) 8532 inCD36 (119901 lt 0001) respectively (Figures 6(a)ndash6(d))Howeverthe mRNA expression level of SYK was not significantlyaltered (data not shown)

36 Protein Verifications of Discrepant Proteins between LPSGroup and LPS + RGFP966 Group Then we verified thechanges of TLR2 TLR3 TLR6 CD36 and SYK at the proteinlevel by western blotting (Figures 7(a)ndash7(g)) Consistent withthe results of mRNA detection the protein expression levelsof TLR2 (831 plusmn 054 in DMSO + LPS versus 100 plusmn 107 inDMSO 119901 lt 0001 187 plusmn 020 in RGFP966 + LPS versus831 plusmn 054 in DMSO + LPS 119901 lt 0001) TLR6 (1009 plusmn 066in DMSO + LPS versus 100 plusmn 028 in DMSO 119901 lt 0001388 plusmn 107 in RGFP966 + LPS versus 1009 plusmn 066 in DMSO+ LPS 119901 lt 0001) CD36 (375 plusmn 013 in DMSO + LPS versus100 plusmn 009 in DMSO 119901 lt 0001 141 plusmn 018 in RGFP966 +LPS versus 375 plusmn 013 in DMSO + LPS 119901 lt 0001) and SYK

Neural Plasticity 5

RSAD2_MOUSEIFIT2_MOUSEIL1B_MOUSE

SAP18_MOUSEIL1A_MOUSE

TMED5_MOUSERL30_MOUSE

MMP13_MOUSECKLF3_MOUSE

TRABD_MOUSETMCO1_MOUSE

NOS2_MOUSES15A3_MOUSERIPL2_MOUSE

COMD9_MOUSEHSC20_MOUSETIM50_MOUSEGMDS_MOUSEMECR_MOUSE

ADAP2_MOUSERM10_MOUSE

APOOL_MOUSECUTC_MOUSESTXB3_MOUSEGORS2_MOUSENNRD_MOUSECOG2_MOUSE

COASY_MOUSEH1BP3_MOUSEILVBL_MOUSE

PKHA2_MOUSEKHDR1_MOUSEMMP12_MOUSE

TOIP2_MOUSETGAP1_MOUSEPCAT1_MOUSEABCF2_MOUSE

LOX5_MOUSESCFD2_MOUSE

TAP2_MOUSEACSL4_MOUSE

CNNM3_MOUSEMD1L1_MOUSEBMP2K_MOUSEBICD2_MOUSE

EPS8_MOUSEVPS53_MOUSETLR3_MOUSENRP2_MOUSE

NAT10_MOUSEDIAP2_MOUSEAT8A1_MOUSEPI3R4_MOUSEU520_MOUSE

MCM7_MOUSECO3_MOUSE

SPCS2_MOUSE3BP1_MOUSE

AIMP1_MOUSETLR2_MOUSE

NUBP2_MOUSERM12_MOUSE

UN45A_MOUSERAB31_MOUSEC2D1B_MOUSETOR4A_MOUSERASL2_MOUSEVPS41_MOUSEPRAF3_MOUSE

STRUM_MOUSESWP70_MOUSE

ZPR1_MOUSECD47_MOUSE

SAR1A_MOUSEGTPB1_MOUSERBM25_MOUSENSUN2_MOUSE

LA_MOUSETHMS2_MOUSE

SH3L3_MOUSE2ABA_MOUSEPSB8_MOUSE

TWF2_MOUSECBL_MOUSE

FRIH_MOUSEUSO1_MOUSE

CASP8_MOUSECOMDA_MOUSE

KCMF1_MOUSEHOOK2_MOUSE

FKB15_MOUSEBAG1_MOUSE

ABRAL_MOUSEPDE12_MOUSESTING_MOUSE

SPB8_MOUSELYPA2_MOUSEPEX14_MOUSE

STAP1_MOUSECLC6A_MOUSE

ROA0_MOUSEPPP6_MOUSE

RBM47_MOUSEMA2C1_MOUSENUPL1_MOUSE

BPHL_MOUSEBRE_MOUSE

UFL1_MOUSETHOP1_MOUSEKANK2_MOUSE

SPS1_MOUSECCD93_MOUSESPAG7_MOUSEPON2_MOUSETR150_MOUSE

NCBP1_MOUSEMCCB_MOUSETTL12_MOUSE

IF2P_MOUSEASM_MOUSEIF4E_MOUSE

COPZ1_MOUSEPFD3_MOUSE

TBCA_MOUSERAGP1_MOUSECX6A1_MOUSEFMR1_MOUSEUMPS_MOUSE

GOSR1_MOUSEPPT2_MOUSEIMA7_MOUSEIMA3_MOUSEIMA4_MOUSE

VMA5A_MOUSEIF4H_MOUSE

ACADS_MOUSEKSYK_MOUSE

CELF2_MOUSETBCD_MOUSE

PIGS_MOUSEADAS_MOUSETITIN_MOUSECALM_MOUSEDC1I2_MOUSE

RL23_MOUSECOPG2_MOUSE

ILK_MOUSESF3B1_MOUSEISOC1_MOUSESGTA_MOUSE

HCFC1_MOUSEHMHA1_MOUSE

EMC8_MOUSEGSH0_MOUSE

MCTS1_MOUSEIF4B_MOUSEVIP2_MOUSEFXR1_MOUSEASNS_MOUSE

LC7L3_MOUSENIBAN_MOUSEMCM6_MOUSE

IRF3_MOUSEIF5A1_MOUSE

GNAQ_MOUSENU4M_MOUSE

GAK_MOUSESTK10_MOUSESTXB2_MOUSEVIGLN_MOUSE

EIF3B_MOUSEAGAL_MOUSE

SYIC_MOUSESCFD1_MOUSE

SCMC1_MOUSEVASP_MOUSE

EIF3D_MOUSEACSF2_MOUSEDNJA1_MOUSEAHSA1_MOUSE

APMAP_MOUSENDUAA_MOUSE

PGES2_MOUSESEPT9_MOUSE

CRK_MOUSEBAG6_MOUSEPIN1_MOUSE

DAZP1_MOUSEGTF2I_MOUSESDF2L_MOUSE

UB2V1_MOUSETPD54_MOUSE

SSRA_MOUSEGMFB_MOUSEPRP19_MOUSERCC1_MOUSE

NUP85_MOUSEDIAC_MOUSE

MARE2_MOUSEPDCL3_MOUSE

TM209_MOUSERCC2_MOUSE

NRDC_MOUSECLC7A_MOUSELPPRC_MOUSE

TTYH3_MOUSESORCN_MOUSE

PA1B2_MOUSESC31A_MOUSE

PCY1A_MOUSELDHB_MOUSE

TCEA1_MOUSEFYB_MOUSE

RL21_MOUSEAT1A1_MOUSE

ASC_MOUSERGS10_MOUSEVINC_MOUSESBP1_MOUSEGGH_MOUSE

CP062_MOUSERD23B_MOUSE

ACADV_MOUSESYHC_MOUSE

VDAC1_MOUSESMCA5_MOUSE

SIR2_MOUSEENASE_MOUSEFA98B_MOUSESRSF1_MOUSE

ASM3B_MOUSEPRDX3_MOUSEPLOD3_MOUSEMYG1_MOUSEARSK_MOUSEEI2BA_MOUSESYLC_MOUSE

FCGR3_MOUSEDOPD_MOUSE

RENT1_MOUSENPS3B_MOUSE

TOM22_MOUSEEIF2A_MOUSE

CAB39_MOUSEHINT1_MOUSEABCE1_MOUSEUCHL4_MOUSE

ACBP_MOUSEARHG7_MOUSE

PON3_MOUSEWASH7_MOUSE

STAB1_MOUSEPSME3_MOUSE

MANBA_MOUSEDYHC1_MOUSEETUD1_MOUSEPK3CG_MOUSETCRG1_MOUSEPARP1_MOUSEITIH2_MOUSE

NOMO1_MOUSECLCN5_MOUSEVPS52_MOUSE

EXOC5_MOUSEDP13B_MOUSESART3_MOUSE

PEX5_MOUSEPGM2L_MOUSEHP1B3_MOUSE

T2FA_MOUSEADPGK_MOUSE

DDB1_MOUSETMM43_MOUSE

TS101_MOUSESIAE_MOUSE

CSK22_MOUSELMAN1_MOUSEUCHL5_MOUSE

PGP_MOUSEDEOC_MOUSEPEX19_MOUSECTBP1_MOUSEAOAH_MOUSEKAP2_MOUSE

ETHE1_MOUSEADHX_MOUSEIF4E2_MOUSEDRG1_MOUSE

HNRPC_MOUSEDOCK2_MOUSENDUB8_MOUSEC1QBP_MOUSECNPY2_MOUSE

HDHD2_MOUSENFU1_MOUSE

NQO2_MOUSELKHA4_MOUSE

EDF1_MOUSENDUS8_MOUSERAB18_MOUSERS15A_MOUSE

RS21_MOUSE

DMSO + LPSversus DMSO

10

0

minus10

log10(ratio)

RGFP966 + LPS

DMSO + LPSversus

Figure 3 Heat map generated by MeV regarding proteins subjected to RGFP966 and LPS regulation Experimental groups were presentedon the horizontal axis and proteins were on the vertical axis Colors were consistent with protein expression levels red indicated upregulatedratio and green indicated downregulated ratio

6 Neural Plasticity

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4

5

Biological process Cellular component Molecular function

Cel

lula

r pro

cess

Met

abol

ic p

roce

ssEs

tabl

ishm

ent o

f loc

aliz

atio

nLo

caliz

atio

n

Mul

tiorg

anism

pro

cess

Resp

onse

to st

imul

us

Dea

th

Loco

mot

ion

Cel

lula

r com

pone

nt o

rgan

izat

ion

Dev

elopm

enta

l pro

cess

Imm

une s

yste

m p

roce

ss

Cel

lula

r com

pone

nt b

ioge

nesis

Biol

ogic

al ad

hesio

n

Repr

oduc

tive p

roce

ssRe

prod

uctio

n

Mul

ticel

lula

r org

anism

al p

roce

ssBi

olog

ical

regu

latio

nO

rgan

elle

Mac

rom

olec

ular

com

plex

Org

anel

le p

art

Enve

lope

Cel

l par

tC

ell

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

part

Extr

acel

lula

r reg

ion

Bind

ing

Cata

lytic

activ

ity

Enzy

me r

egul

ator

activ

itySt

ruct

ural

mol

ecul

e act

ivity

Elec

tron

carr

ier a

ctiv

ity

Tran

spor

ter a

ctiv

ityTr

ansc

riptio

n re

gula

tor a

ctiv

ity

Mol

ecul

ar tr

ansd

ucer

activ

ity

(b)

Figure 4 The GO analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The GO analysis involvedbiological process cellular component and molecular function The vertical axis values equaled minus log(119901)

(272 plusmn 013 in DMSO + LPS versus 100 plusmn 007 in DMSO119901 lt 0001 145 plusmn 015 in RGFP966 + LPS versus 272 plusmn 013in DMSO + LPS 119901 lt 0001) were all significantly increasedat the stimulation time of 12 hours and decreased with thetreatment of RGFP966 In our study though there was nosignificant change seen in total MAPK p38 the protein levelof phospho-p38 (331 plusmn 040 in DMSO + LPS versus 100 plusmn023 in DMSO 119901 lt 0001 256 plusmn 058 in RGFP966 + LPSversus 331 plusmn 040 in DMSO + LPS 119901 = 0021) showed a

similar trend as the proteins mentioned above TLR3 (634 plusmn033 in DMSO + LPS versus 100 plusmn 115 in DMSO 119901 = 0003196 plusmn 093 in RGFP966 + LPS versus 634 plusmn 033 in DMSO+ LPS 119901 = 0011) an exceptional protein was significantlyaggregated after LPS stimulation and reduced by RGFP966 at24 hours

37 RGFP966 Inhibited LPS-Induced Microglia ActivationWe further verified the effect of HDAC3 inhibition on

Neural Plasticity 7

1

Pathways in cancerCalcium signaling pathway

Huntingtonrsquos diseaseInsulin signaling pathway

Cell cycleApoptosis

RIG-I-like receptor signaling pathwaySpliceosome

Cytosolic DNA-sensing pathwayAlzheimerrsquos disease

Toll-like receptor signaling pathway

15 20 05(a)

1 150 05Pathways in cancerAlzheimerrsquos disease

Chemokine signaling pathwayOxidative phosphorylation

Regulation of actin cytoskeletonLeukocyte transendothelial migration

Focal adhesionHuntingtonrsquos disease

RibosomeChronic myeloid leukemiaInsulin signaling pathway

Parkinsonrsquos diseaseSpliceosome

mTOR signaling pathwayFc gamma R-mediated phagocytosis

Aminoacyl-tRNA biosynthesis

(b)

Figure 5 The KEGG pathway analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The horizontalaxis values equaled minus log(119901)

functional microglia activation We first stained primarycultured microglia with CD1632 as shown in Figure 8(a)microglia demonstrated bipolar shape in intact status whilein LPS stimulation group microglia enlarged and grew lotsof branches The fluorescence intensity of CD1632 was sig-nificantly decreased in LPS + RGFP966-treated group (Fig-ure 8(c)) We then detected classical inflammatory cytokinesin the supernatants of cultured cells exposed to LPS for 1 h2 h 3 h 8 h 12 h and 24 h with or without RGFP966 Weobserved profound inhibition of IL-6 (Figure 8(d)) (24 h15230 plusmn 5213 pgmL in RGFP966 + LPS versus 25538 plusmn8741 pgmL in DMSO + LPS 119901 = 0012) and TNF-120572(Figure 8(e)) (12 h 5189 plusmn 1613 pgmL in RGFP966 + LPSversus 17241 plusmn 4716 pgmL in DMSO + LPS 119901 lt 000124 h 7332 plusmn 2436 pgmL in RGFP966 + LPS versus 20977 plusmn1091 pgmL in DMSO + LPS 119901 lt 0001) secretion byRGFP966 Additionally STAT3 and STAT5 were two funda-mental signaling pathways governing inflammatory responsein various neurological diseases [17] In order to found out themechanism responsible for its anti-inflammatory responses

we detected phosphorylation levels of STAT3 and STAT5As shown in Figure 8(b) phosphorylation of STAT3 andSTAT5 increased 2 h after LPS treatment and declined quickly4 h after LPS RGFP966 inhibited not only phosphorylationof STAT3 and STAT5 but also decreased STAT5 expressionlevel whichmay partially contribute to its anti-inflammatoryresponse

4 Discussion

In summary we analyzed the differentially expressed proteinsof LPS-stimulated primary microglia with the treatmentof RGFP966 by proteomics A total of 168 LPS-inducedproteins were significantly reduced by RGFP966 in thisstudy The altered proteins mainly concentrated on thefollowing categories Toll-like receptor signaling pathwayAlzheimerrsquos disease cytosolic DNA-sensing pathway andfunctioned in cellular process immune system process andorganelle For Alzheimerrsquos disease preliminary evidencesdemonstrated inhibition of HDAC3 enzyme in neurons

8 Neural Plasticity

DMSORGFP966

TLR2 mRNA

Vehicle LPS0

5

10

15lowastlowast

lowastlowast(fo

ld o

f con

trol)

Relat

ive m

RNA

leve

l

(a)

DMSORGFP966

TLR3 mRNA

Vehicle LPS

lowastlowast

lowastlowast

0

10

20

30

40

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(b)

DMSORGFP966

TLR6 mRNA

Vehicle LPS

lowastlowast

lowastlowast

00

05

10

15

20

25

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(c)

DMSORGFP966

CD36 mRNA

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(d)

Figure 6 RT-PCR analysis of TLR2 TLR3 TLR6 and CD36 in four experimental groups The mRNA levels of TLR2 (a) TLR3 (b) TLR6(c) and CD36 (d) were significantly reduced by RGFP966 at the stimulation time of 6 hours lowastlowast119901 lt 001119873 = 3 repeats

prevented amyloid-beta oligomer-induced synaptic plasticityimpairments [18] and enhanced memory process [15] ForDNA-sensing pathways HDAC3 was thought to participatein IRF3IFN-120573 signaling pathways [13] In this study wemainly analyzed the differentially expressed TLR relative pro-teins of LPS-stimulated primarymicroglia with the treatmentof RGFP966 Toll-like receptor signaling pathway played animportant role in the regulation of inflammation In thisregard we selected TLR2 TLR3 TLR6 CD36 and SYK forfurther validation and found that they were all significantlyupregulated after LPS stimulation and downregulated inthe presence of RGFP966 Additionally morphological andfunctional alterations of microglia exposure to LPS werealso relieved by RGFP966 Our data provided a hint thatHDAC3 inhibitor may be a potential therapeutic target

combating microglia activation and inflammatory responsein neurological diseases

TLRs are a family of type I transmembrane recep-tors which share the multiple leucine-rich repeats (LRRs)domains in the extracellular space and the similar cytosolicdomains of the interleukin-1 (IL-1) receptors [19] TLRsare considered to recognize the distinct pathogen-associatedmolecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) which then drivea cascade of inflammatory signaling and converge at tran-scription factors nuclear factor-120581B (NF-120581B) [20] MAPK[21 22] and JAKSTAT pathways [23] In addition to TLR236 TLR coreceptorCD36 and regulatory protein SYKwereall decreased by RGFP966 Spleen tyrosine kinase (SYK) anonreceptor tyrosine kinase is typically regarded as a vital

Neural Plasticity 9

GAPDH

TLR2

DMSO RGFP966DMSO+ LPS

RGFP966+ LPS DMSO RGFP966

DMSO+ LPS

RGFP966+ LPS

TLR3

GAPDH

TLR6

GAPDH

CD36

GAPDH

phospho-p38

GAPDH

p38

SYK

GAPDH

(a)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR2 Protein level

0

2

4

6

8

10

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

(b)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)TLR3 Protein level

0

2

4

6

8

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(c)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR6 Protein level

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(d)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

CD36 Protein level

0

1

2

3

4

5

DMSORGFP966

Vehicle LPS

lowastlowast

lowastlowast

(e)

Figure 7 Continued

10 Neural Plasticity

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)phospho-p38 Protein level

0

1

2

3

4

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(f)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

SYK Protein level

0

1

2

3

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

(g)

Figure 7Western blot analysis of TLR2 TLR3 TLR6MAPKp38 phospho-p38 SYK andCD36 in four experimental groups (a)The proteinlevels of TLR2 (b) TLR6 (d) SYK (g) phospho-p38 (f) and CD36 (e) were significantly altered by RGFP966 after 12 hoursrsquo stimulation of LPSwhile TLR3 was at 24 hours There was no significant difference of MAPK p38 protein expression among four groups lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

regulator in adaptive immunity [24] It consists of two tandemSH2 domains and a C-terminal tyrosine kinase domain SH2domains selectively bind to the phosphorylated immunore-ceptor tyrosine-based activation motifs (ITAMs) of immunereceptors such as MyD88 TRAF6 and TRIF transmittinginitiative signals to downstream pathways SYK is found to befundamental for Toll-like receptor signaling pathway and theinhibition of SYK suppresses the release of proinflammatorycytokines [25 26] In this study we detected alterations ofSYK in protein level but not mRNA level We surmisedthat posttranscriptional modification may also contributeto HDAC3rsquos functions Preliminary evidences demonstratethat HDAC family played a role in protein degradation[27] while inadequate time points of mRNA detection couldalso be an explanation for this discrepancy CD36 is a TLRcoreceptor playing a pivotal role in inflammatory responsesinitiated by TLRs [28] Previous researches reported thatCD36 contributed to the recognition of diacylglycerol ligandsby forming CD36-CD14-TLR2-TLR6 complex thus it con-trolled gram-positive bacterial infectionWhat ismore CD36was found to be involved in the formation of TLR4-TLR6heterodimers which induced the production of proinflam-matory cytokines nitric oxide and reactive oxygen speciesin response to endogenous ligands [29] Overall RGFP966demonstrated profound inhibitory effects on TLR signalingpathway and proinflammatory cytokines including IL-6 andTNF-120572

HDAC inhibitors have been reported to interfere withthe activation of the mitogen-activated protein kinasesSTAT1 AP-1 or NF-120581B signal transduction pathways [30

31] Here we observed transient STAT3 and STAT5 acti-vation at 2 hours after LPS stimulation which was aheadof the alteration of IL-6 and TNF-120572 Exposed to inflam-matory stimulation STAT35 was phosphorylated by JAKsleading to transcriptional activation Activation of STATselicited the expression of acute-phase proteins as well asa number of cytokines and chemokines including IL-6and TNF-120572 [17] Our study was in keeping with previousstudy inHDAC3 knockoutmacrophage which demonstratedthat HDAC3 knockout impaired STAT1STAT3STAT5 path-ways [13] Figure 9 presented a diagram showing theprotein and protein interactions among proteins Brieflyinitial change of STAT3STAT5 or NF-120581B [5] caused byHDAC3i may further elicit later alterations of TLR sig-naling pathway and proinflammatory cytokines includingIL-6 and TNF-120572 The underlying mechanism for HDAC3in the regulation of STAT3STAT5 is unknown Some evi-dences from Pan-HDAC inhibitors indicated that promoter-associated histone acetylation of SOCS1 and SOCS3 caused byHDACi may further downregulated JAK2STAT3 signaling[32]

In conclusions we identified TLR signaling pathwaymicroglia activation and STAT35 pathway which wereinhibited by RGFP966 Identification of these changes mayprovide valuable clues for the future applications of selectiveHDAC3 inhibitor in the clinic

Competing Interests

The authors declare no conflict of interests

Neural Plasticity 11

DMSO RGFP966

DMSO + LPS RGFP966 + LPS

(a)

p-STAT3

STAT3

p-STAT5

GAPDH

GAPDH

DMSO + LPS RGFP966 + LPS

STAT5

DMSO RGFP966 1h 2h 4h 1h 2h 4h

(b)

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

0

20000

40000

60000

80000

100000

Fluo

resc

ence

inte

nsity

of C

D16

32

(c)

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

0200400600800

100001500020000250003000035000 IL-6

LPSLPS + RGFP966

lowast

1 2 3 8 12 240(h)

(d)

TNF-120572

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

050

100

8000

3000

13000

18000

23000

LPSLPS + RGFP966

lowastlowastlowastlowast

1 2 3 8 12 240(h)

(e)

Figure 8 RGFP966 inhibited LPS-inducedmicroglia activation (a) Representativemicrophotographs of primarymicroglia cultures 24 hoursafter LPS and RGFP966 treatmentThe cultures were stained with CD1632 a marker of activatedmicroglia Scale bar = 10120583m (b) Expressionand phosphorylation of STAT3 and STAT5 in microglia pretreated with RGFP966 (c) Quantitative analysis of fluorescence intensity ofCD1632 All the photographs used for quantitative analysis were taken under the same exposure time and data were expressed as fluorescenceintensity per cell of randomly selected regions lowastlowast119901 lt 001119873 = 3 repeats Supernatant concentration of IL-6 (d) and TNF-120572 (e) detected atvarious stimulation times lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 2: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

2 Neural Plasticity

However previous used broad-spectrum HDAC inhibi-tors target several HDACs and it is difficult to define exactrole of each subtype Additionally clinical trials with pan-HDACi in cancer patients suffered undesired effects includ-ing increased susceptibility to pneumonia thrombocytope-nia anorexia [9] Thus it is necessary to focus on the specificHDAC inhibitor

HDAC3 is themost widely expressedHDACs in the brain[10] and is thought to play a role in Huntington [4] SCA [11]and dementia diseases [12] HDAC3-deficient macrophagespossessed decreased ability to activate inflammatory geneexpression in response to LPS stimulation [13] Concomi-tantly HDAC3 was found to be an epigenomic brake inmacrophage alternative activation [14] while inflammationrepressive repertoire of HDAC3 in primary microglia islargely unknown RGFP966 is a selective HDAC3 inhibitorwith an IC50 of 008120583M and no effective inhibition of otherHDACs at concentrations up to 15 120583M and could cross brainblood barrier when administrated peripherally [15] Pharma-cological inhibition of HDAC3 may bring more evidencesand prosperities for clinical applications This insight nowgives us the opportunity to study possible inflammatoryconsequences of HDAC3 in central nervous system

In this issue we used protein mass spectrometry toprofile global molecular alterations in primary microgliaexposed toRGFP966 exploring a potential signaling pathwaythroughwhichHDAC3 specific inhibitor RGFP966 regulatedinflammation

2 Materials and Methods

21 Primary Microglia Culture and Treatment Primarymicroglia cells were prepared from C57BL6 mice bornwithin 24 hours as previously described Briefly cerebralcortex tissue was digested in TrypLE for 10 minutes at 37∘CThen Minimum Essential Medium (MEM) (Hyclone USA)supplemented with 10 fetal bovine serum (FBS) (BiologicalIndustries Israel) 100UmL penicillin and 100 ugmL strep-tomycinwas used to terminate digestionAfterwards the cellswere centrifuged at 1500 rpm for 5minutes resuspended andseeded in the 75 cm2 flasks After 36ndash48 hours the culturemedia were replaced by Dulbeccorsquos Modified Eagle Media(DMEM) (HycloneUSA)with 10FBS 100UmLpenicillinand 100 ugmL streptomycin At day 11ndash13 microglia cellswere suspended and obtained by shaking the flasks at 180 rpmfor 10min at 37∘C The mature microglia cells were seededinto plates at a density of 2 times 105cm2 and placed for 36ndash48 hours before further treatment RGFP966 (Selleckchem)was dissolved in dimethyl sulfoxide (DMSO) to make 55mMstock solution In purified enzyme assays inhibition IC50values of RGFP966 for HDAC1 HDAC2 and HDAC3 weregt15 120583Mgt15 120583M and 008 120583M[15 16] Primarymicroglia cellswere pretreated with DMSO or RGFP966 at a concentrationof 15 120583M for an hour Then LPS (Sigma-Aldrich USA) wasadded to the culture media at a dose of 500 ngmL Proteins

mRNA and supernatant were collected for proteomic anal-ysis western blotting CBA and Q-PCR at indicated timepoints

22 Proteomic Analysis Proteomic analysis was performedby AB SCIEX TripleTOF 5600 mass spectrometer (ABSCIEX USA) equipped with a liquid chromatography-tandem mass spectrometry (LC-MSMS) system Proteins(200120583g) of four samples were resolved on 10 SDS polyacry-lamide gels The gels were stained with Coomassie Blue G-250 for 1 hour and then cut into blocks after distaining inultrapure H

2O The gel blocks were digested using trypsin

and peptides were extracted from them After being driedand redissolved the peptides were analyzedThemass spectrawere annotated against the Uniprot proteome databaseBy means of the Software DAVID coupled with STRING(version 100) the Gene Ontology analysis KEGG pathwayanalysis and protein-protein interactions were completed

23 Real-Time PCR As described previously Trizol reagent(InvitrogenUSA)was used for the total RNA extraction frommicroglia cells and then RNA was reverse-transcribed intocDNA with a PrimeScript RT reagent Kit (Takara DalianChina) The quantitative measurements were performed onan ABI 7500 PCR instrument (Applied Biosystems USA)with a SYBR green Kit (Takara Dalian China) Relative geneexpressions were normalized to glyceraldehyde-3-phosphatedehydrogenase (GAPDH) and mRNA expression levels werepresented as fold changes versus DMSO group The primers(Invitrogen) used are as follows

TLR-2 primers

Forward 51015840-TCACATGGCAGAAGATGTGTC-31015840

Reverse 51015840-GGTGATGCAATTCGGATGCT-31015840

TLR-3 primers

Forward 51015840-TGAGAAGAGCCACAGTGATAGA-31015840

Reverse 51015840-CTCTCCAGCAGAAGAGACACAA-31015840

TLR6 primers

Forward 51015840-AATGGTACCGTCAGTGCTGGA-31015840

Reverse 51015840-CTTGGCTCATGTTGCAGAGG-31015840

CD36 primers

Forward 51015840-TGAATGGTTGAGACCCCGTG-31015840

Reverse 51015840-TAGAACAGCTTGCTTGCCCA-31015840

GAPDH primers

Forward 51015840-GCCAAGGCTGTGGGCAAGGT-31015840

Reverse 51015840-TCTCCAGGCGGCACGTCAGA-31015840

24 Western Blot Western blot was performed as previ-ously described Equal amounts of proteins were separated

Neural Plasticity 3

by sodium dodecyl sulfate-PAGE electrophoresis and thenblotted onto polyvinylidene fluoride membranes After beingblocked in 5 fat-free milk for 2 hours membranes wereincubated with primary antibodies against TLR2 (1 1000Abcam UK) TLR3 (1 1000 Abcam UK) TLR6 (1 1000Cell Signaling Technology USA) MAPK p38 (1 500 CellSignaling Technology USA) phospho-p38 (1 500 Cell Sig-naling Technology USA) CD36 (1 1000 Abcam UK) SYK(1 1000 AbcamUK) STAT3 (1 500 Cell Signaling Technol-ogy USA) phospho-STAT3 (1 500 Cell Signaling Technol-ogy USA) STAT5 (1 500 Cell Signaling Technology USA)phospho-STAT5 (1 500 Cell Signaling Technology USA)and GAPDH (1 5000 Bioworld USA) in 4∘C overnight toprobe targeted proteins Horseradish peroxidase-conjugatedsecondary antibodies (1 5000 Bioworld USA) were used tocombine primary antibodies and the reaction was detectedwith an ECL Kit (Bioworld USA) The intensities of blotswere quantified by densitometry

25 Immunofluorescence Primary microglia cells seeded oncover slips were fixed with 4 polyformaldehyde for 15 min-utes and permeabilizedwith 02Triton-X100 for 20minutesat room temperature After being blocked in 2 BSA in PBSfor 2 hours microglia cells were incubated with donkey anti-CD1632 antibody (1 500 BD Pharmingen USA) overnightin 4∘C and subsequently incubated with FITC-conjugatedanti-rat IgG (Invitrogen USA) at room temperature for 2hours DAPI staining was used to localize the nuclei Imageswere taken by a fluorescence microscope (Olympus Japan)and fluorescence intensities were analyzed by ImageJ software(version 139 National Institutes of Health USA)

26 Quantification of Secreted Cytokines Cytokines in thesupernatants were measured using the Cytometric BeadArray (CBA) Mouse Inflammation Kit (BD BiosciencesUSA) Briefly after incubation with capture beads on whichanti-cytokine antibodies are coated and PE-conjugated anti-cytokine antibodies cytokine levels can be quantitativelyanalyzed The assays were performed by BD Accuri C6 flowcytometer (BD Biosciences USA) following the manufac-turerrsquos instructions and the data were generated with FCAPArray version 301 Software

27 Statistical Analysis Data were expressed as mean plusmn SDof three independent experiments Comparisons betweengroups were conducted with SPSS 220 software Differenceswere analyzed by two-way analysis of variance (ANOVA)followed by Bonferronirsquos post hoc test and considered to bestatistically significant if 119901 lt 005

3 Results

31 Overview of Proteomic Analysis In this study 1883 pro-teins were detected in DMSO group and 1967 proteins werein RGFP966 group In LPS-stimulated groups there were1806 proteins in DMSO + LPS group and 2024 proteins in

Table 1 Summary of LC-MSMS data

Treatment Total proteinsDMSO 1883RGFP966 1967DMSO + LPS 1806RGFP966 + LPS 2024

DMSO

DMSORGFP9

66

RGFP966

+ LPS

+ LPS

169

70

138

35

48

28

82

87

107

1361

58

53

120

64

174

Figure 1 Overview of expressed proteins in four groups The bluecircle representedDMSOgroup the red circle representedRGFP966group the green circle represented DMSO + LPS group and theyellow circle represented RGFP966 + LPS group

RGFP966 + LPS group (Table 1) A Venn diagram showed therelationship of expressed proteins in four groups (Figure 1)

32 Alterations in Proteins Expression We identified gt 15fold peptide gt 1 as upregulated proteins and lt 066 foldpeptide gt 1 as downregulated proteins The numbers of dif-ferently expressed proteins between two groups were listed inTable 2 Specifically 168 of 444 (378) LPS-induced proteinswere significantly reduced with the treatment of RGFP966(Figure 2(a)) Likewise when analyzing proteins downreg-ulated by LPS the impact of RGFP966 was comparablewith 134 of 404 (332) proteins being rescued by RGFP966(Figure 2(b)) The heat map presented detailed informationof the upregulated as well as downregulated proteins whichwere caused by LPS and simultaneously reversed byRGFP966(Figure 3)

33 Gene Ontology Analysis GO analysis was used to revealthe function of proteins in three aspects cellular compo-nent molecular function and biological process The 168differentially expressed proteins overlapped in Figure 2(a)were mainly involved in biological process cellular processimmune system process and establishment of localizationcellular component organellemacromolecular complex andorganelle part molecular function binding catalytic activity

4 Neural Plasticity

Table 2 Summary of upregulateddownregulated proteins candidates

Upregulated protein candidates Downregulated protein candidatesgt 15 fold peptide gt 1 lt 066 fold peptide gt 1

RGFP966 versus DMSO 493 338DMSO + LPS versus DMSO 444 404RGFP966 + LPS versus DMSO + LPS 401 275

DMSO + LPSDMSO upregulated proteins

107168276

RGFP966 + LPSDMSO + LPS downregulated proteins

(a)

DMSO + LPSDMSO downregulated proteins

267134270

RGFP966 + LPSDMSO + LPS upregulated proteins

(b)

Figure 2 Venn diagrams of the differentially expressed proteins between LPS-stimulated (DMSO + LPSDMSO) and RGFP966-treated(RGFP966 + LPSDMSO + LPS) microglia cells (a) 168 proteins were upregulated by LPS and reversed with the treatment of RGFP966 (b)134 proteins were downregulated after the stimulation of LPS and rescued by RGFP966

and enzyme regulator activity (Figure 4(a)) However the 134differentially expressed proteins overlapped in Figure 2(b)were mainly involved in biological process cellular processmetabolic process and establishment of localization cellularcomponent organelle macromolecular complex and cellpart molecular function catalytic activity binding andelectron carrier activity (Figure 4(b))

34 KEGG Pathway Analysis To explore the potential sig-naling pathway through which HDAC3 specific inhibitorRGFP966 regulates inflammatory response KEGG pathwayanalysis was performed As shown in Figure 5(a) the pathwaymap depicted that the 168 differentially expressed proteinsoverlapped in Figure 2(a) were related to Toll-like recep-tor signaling pathway Alzheimerrsquos disease cytosolic DNA-sensing pathway spliceosome RIG-I-like receptor signalingapoptosis cell cycle insulin signaling pathway Huntingtonrsquosdisease calcium signaling pathway and pathways in cancerHowever the 134 differentially expressed proteins over-lapped in Figure 2(b) belonged to the following pathwaysaminoacyl-tRNAbiosynthesis Fc gammaR-mediated phago-cytosis mTOR signaling pathway spliceosome Parkinsonrsquosdisease and so forth (Figure 5(b))

35 mRNA Verifications of Discrepant Proteins between LPSGroup and LPS + RGFP966 Group RT-PCR was performed

to detect the mRNA levels of TLR2 TLR3 TLR6 CD36and SYK at 6 hours after LPS stimulation As expectedLPS stimulation potentiated profound mRNA increase ofTLR2 (924 folds versus DMSO group) TLR3 (2881 foldsversus DMSO group) TLR6 (173 folds versus DMSO group)and CD36 (878 folds versus DMSO group) However theseaugments were significantly decreased by RGFP966 with thedecline amplitudes at 7358 in TLR2 (119901 = 0002) 3551in TLR3 (119901 lt 0001) 3757 in TLR6 (119901 = 0007) 8532 inCD36 (119901 lt 0001) respectively (Figures 6(a)ndash6(d))Howeverthe mRNA expression level of SYK was not significantlyaltered (data not shown)

36 Protein Verifications of Discrepant Proteins between LPSGroup and LPS + RGFP966 Group Then we verified thechanges of TLR2 TLR3 TLR6 CD36 and SYK at the proteinlevel by western blotting (Figures 7(a)ndash7(g)) Consistent withthe results of mRNA detection the protein expression levelsof TLR2 (831 plusmn 054 in DMSO + LPS versus 100 plusmn 107 inDMSO 119901 lt 0001 187 plusmn 020 in RGFP966 + LPS versus831 plusmn 054 in DMSO + LPS 119901 lt 0001) TLR6 (1009 plusmn 066in DMSO + LPS versus 100 plusmn 028 in DMSO 119901 lt 0001388 plusmn 107 in RGFP966 + LPS versus 1009 plusmn 066 in DMSO+ LPS 119901 lt 0001) CD36 (375 plusmn 013 in DMSO + LPS versus100 plusmn 009 in DMSO 119901 lt 0001 141 plusmn 018 in RGFP966 +LPS versus 375 plusmn 013 in DMSO + LPS 119901 lt 0001) and SYK

Neural Plasticity 5

RSAD2_MOUSEIFIT2_MOUSEIL1B_MOUSE

SAP18_MOUSEIL1A_MOUSE

TMED5_MOUSERL30_MOUSE

MMP13_MOUSECKLF3_MOUSE

TRABD_MOUSETMCO1_MOUSE

NOS2_MOUSES15A3_MOUSERIPL2_MOUSE

COMD9_MOUSEHSC20_MOUSETIM50_MOUSEGMDS_MOUSEMECR_MOUSE

ADAP2_MOUSERM10_MOUSE

APOOL_MOUSECUTC_MOUSESTXB3_MOUSEGORS2_MOUSENNRD_MOUSECOG2_MOUSE

COASY_MOUSEH1BP3_MOUSEILVBL_MOUSE

PKHA2_MOUSEKHDR1_MOUSEMMP12_MOUSE

TOIP2_MOUSETGAP1_MOUSEPCAT1_MOUSEABCF2_MOUSE

LOX5_MOUSESCFD2_MOUSE

TAP2_MOUSEACSL4_MOUSE

CNNM3_MOUSEMD1L1_MOUSEBMP2K_MOUSEBICD2_MOUSE

EPS8_MOUSEVPS53_MOUSETLR3_MOUSENRP2_MOUSE

NAT10_MOUSEDIAP2_MOUSEAT8A1_MOUSEPI3R4_MOUSEU520_MOUSE

MCM7_MOUSECO3_MOUSE

SPCS2_MOUSE3BP1_MOUSE

AIMP1_MOUSETLR2_MOUSE

NUBP2_MOUSERM12_MOUSE

UN45A_MOUSERAB31_MOUSEC2D1B_MOUSETOR4A_MOUSERASL2_MOUSEVPS41_MOUSEPRAF3_MOUSE

STRUM_MOUSESWP70_MOUSE

ZPR1_MOUSECD47_MOUSE

SAR1A_MOUSEGTPB1_MOUSERBM25_MOUSENSUN2_MOUSE

LA_MOUSETHMS2_MOUSE

SH3L3_MOUSE2ABA_MOUSEPSB8_MOUSE

TWF2_MOUSECBL_MOUSE

FRIH_MOUSEUSO1_MOUSE

CASP8_MOUSECOMDA_MOUSE

KCMF1_MOUSEHOOK2_MOUSE

FKB15_MOUSEBAG1_MOUSE

ABRAL_MOUSEPDE12_MOUSESTING_MOUSE

SPB8_MOUSELYPA2_MOUSEPEX14_MOUSE

STAP1_MOUSECLC6A_MOUSE

ROA0_MOUSEPPP6_MOUSE

RBM47_MOUSEMA2C1_MOUSENUPL1_MOUSE

BPHL_MOUSEBRE_MOUSE

UFL1_MOUSETHOP1_MOUSEKANK2_MOUSE

SPS1_MOUSECCD93_MOUSESPAG7_MOUSEPON2_MOUSETR150_MOUSE

NCBP1_MOUSEMCCB_MOUSETTL12_MOUSE

IF2P_MOUSEASM_MOUSEIF4E_MOUSE

COPZ1_MOUSEPFD3_MOUSE

TBCA_MOUSERAGP1_MOUSECX6A1_MOUSEFMR1_MOUSEUMPS_MOUSE

GOSR1_MOUSEPPT2_MOUSEIMA7_MOUSEIMA3_MOUSEIMA4_MOUSE

VMA5A_MOUSEIF4H_MOUSE

ACADS_MOUSEKSYK_MOUSE

CELF2_MOUSETBCD_MOUSE

PIGS_MOUSEADAS_MOUSETITIN_MOUSECALM_MOUSEDC1I2_MOUSE

RL23_MOUSECOPG2_MOUSE

ILK_MOUSESF3B1_MOUSEISOC1_MOUSESGTA_MOUSE

HCFC1_MOUSEHMHA1_MOUSE

EMC8_MOUSEGSH0_MOUSE

MCTS1_MOUSEIF4B_MOUSEVIP2_MOUSEFXR1_MOUSEASNS_MOUSE

LC7L3_MOUSENIBAN_MOUSEMCM6_MOUSE

IRF3_MOUSEIF5A1_MOUSE

GNAQ_MOUSENU4M_MOUSE

GAK_MOUSESTK10_MOUSESTXB2_MOUSEVIGLN_MOUSE

EIF3B_MOUSEAGAL_MOUSE

SYIC_MOUSESCFD1_MOUSE

SCMC1_MOUSEVASP_MOUSE

EIF3D_MOUSEACSF2_MOUSEDNJA1_MOUSEAHSA1_MOUSE

APMAP_MOUSENDUAA_MOUSE

PGES2_MOUSESEPT9_MOUSE

CRK_MOUSEBAG6_MOUSEPIN1_MOUSE

DAZP1_MOUSEGTF2I_MOUSESDF2L_MOUSE

UB2V1_MOUSETPD54_MOUSE

SSRA_MOUSEGMFB_MOUSEPRP19_MOUSERCC1_MOUSE

NUP85_MOUSEDIAC_MOUSE

MARE2_MOUSEPDCL3_MOUSE

TM209_MOUSERCC2_MOUSE

NRDC_MOUSECLC7A_MOUSELPPRC_MOUSE

TTYH3_MOUSESORCN_MOUSE

PA1B2_MOUSESC31A_MOUSE

PCY1A_MOUSELDHB_MOUSE

TCEA1_MOUSEFYB_MOUSE

RL21_MOUSEAT1A1_MOUSE

ASC_MOUSERGS10_MOUSEVINC_MOUSESBP1_MOUSEGGH_MOUSE

CP062_MOUSERD23B_MOUSE

ACADV_MOUSESYHC_MOUSE

VDAC1_MOUSESMCA5_MOUSE

SIR2_MOUSEENASE_MOUSEFA98B_MOUSESRSF1_MOUSE

ASM3B_MOUSEPRDX3_MOUSEPLOD3_MOUSEMYG1_MOUSEARSK_MOUSEEI2BA_MOUSESYLC_MOUSE

FCGR3_MOUSEDOPD_MOUSE

RENT1_MOUSENPS3B_MOUSE

TOM22_MOUSEEIF2A_MOUSE

CAB39_MOUSEHINT1_MOUSEABCE1_MOUSEUCHL4_MOUSE

ACBP_MOUSEARHG7_MOUSE

PON3_MOUSEWASH7_MOUSE

STAB1_MOUSEPSME3_MOUSE

MANBA_MOUSEDYHC1_MOUSEETUD1_MOUSEPK3CG_MOUSETCRG1_MOUSEPARP1_MOUSEITIH2_MOUSE

NOMO1_MOUSECLCN5_MOUSEVPS52_MOUSE

EXOC5_MOUSEDP13B_MOUSESART3_MOUSE

PEX5_MOUSEPGM2L_MOUSEHP1B3_MOUSE

T2FA_MOUSEADPGK_MOUSE

DDB1_MOUSETMM43_MOUSE

TS101_MOUSESIAE_MOUSE

CSK22_MOUSELMAN1_MOUSEUCHL5_MOUSE

PGP_MOUSEDEOC_MOUSEPEX19_MOUSECTBP1_MOUSEAOAH_MOUSEKAP2_MOUSE

ETHE1_MOUSEADHX_MOUSEIF4E2_MOUSEDRG1_MOUSE

HNRPC_MOUSEDOCK2_MOUSENDUB8_MOUSEC1QBP_MOUSECNPY2_MOUSE

HDHD2_MOUSENFU1_MOUSE

NQO2_MOUSELKHA4_MOUSE

EDF1_MOUSENDUS8_MOUSERAB18_MOUSERS15A_MOUSE

RS21_MOUSE

DMSO + LPSversus DMSO

10

0

minus10

log10(ratio)

RGFP966 + LPS

DMSO + LPSversus

Figure 3 Heat map generated by MeV regarding proteins subjected to RGFP966 and LPS regulation Experimental groups were presentedon the horizontal axis and proteins were on the vertical axis Colors were consistent with protein expression levels red indicated upregulatedratio and green indicated downregulated ratio

6 Neural Plasticity

Biological process Cellular component Molecular function

0

1

2

3

4

5

6

7

Cel

lula

r pro

cess

Imm

une s

yste

m p

roce

ssEs

tabl

ishm

ent o

f loc

aliz

atio

nLo

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atio

nM

ultio

rgan

ism p

roce

ssRe

spon

se to

stim

ulus

Met

abol

ic p

roce

ssD

eath

Loco

mot

ion

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r com

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rgan

izat

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Dev

elopm

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Cel

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nt b

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Biol

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Repr

oduc

tion

Mul

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r org

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ssBi

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ical

regu

latio

nO

rgan

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Mac

rom

olec

ular

com

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Enve

lope

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e-en

close

d lu

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part

Extr

acel

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Bind

ing

Cata

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activ

ityEn

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e reg

ulat

or ac

tivity

Stru

ctur

al m

olec

ule a

ctiv

ityEl

ectro

n ca

rrie

r act

ivity

Tran

spor

ter a

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ityTr

ansc

riptio

n re

gula

tor a

ctiv

ityM

olec

ular

tran

sduc

er ac

tivity

(a)

0

1

2

3

4

5

Biological process Cellular component Molecular function

Cel

lula

r pro

cess

Met

abol

ic p

roce

ssEs

tabl

ishm

ent o

f loc

aliz

atio

nLo

caliz

atio

n

Mul

tiorg

anism

pro

cess

Resp

onse

to st

imul

us

Dea

th

Loco

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ion

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r com

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nt o

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izat

ion

Dev

elopm

enta

l pro

cess

Imm

une s

yste

m p

roce

ss

Cel

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r com

pone

nt b

ioge

nesis

Biol

ogic

al ad

hesio

n

Repr

oduc

tive p

roce

ssRe

prod

uctio

n

Mul

ticel

lula

r org

anism

al p

roce

ssBi

olog

ical

regu

latio

nO

rgan

elle

Mac

rom

olec

ular

com

plex

Org

anel

le p

art

Enve

lope

Cel

l par

tC

ell

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

part

Extr

acel

lula

r reg

ion

Bind

ing

Cata

lytic

activ

ity

Enzy

me r

egul

ator

activ

itySt

ruct

ural

mol

ecul

e act

ivity

Elec

tron

carr

ier a

ctiv

ity

Tran

spor

ter a

ctiv

ityTr

ansc

riptio

n re

gula

tor a

ctiv

ity

Mol

ecul

ar tr

ansd

ucer

activ

ity

(b)

Figure 4 The GO analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The GO analysis involvedbiological process cellular component and molecular function The vertical axis values equaled minus log(119901)

(272 plusmn 013 in DMSO + LPS versus 100 plusmn 007 in DMSO119901 lt 0001 145 plusmn 015 in RGFP966 + LPS versus 272 plusmn 013in DMSO + LPS 119901 lt 0001) were all significantly increasedat the stimulation time of 12 hours and decreased with thetreatment of RGFP966 In our study though there was nosignificant change seen in total MAPK p38 the protein levelof phospho-p38 (331 plusmn 040 in DMSO + LPS versus 100 plusmn023 in DMSO 119901 lt 0001 256 plusmn 058 in RGFP966 + LPSversus 331 plusmn 040 in DMSO + LPS 119901 = 0021) showed a

similar trend as the proteins mentioned above TLR3 (634 plusmn033 in DMSO + LPS versus 100 plusmn 115 in DMSO 119901 = 0003196 plusmn 093 in RGFP966 + LPS versus 634 plusmn 033 in DMSO+ LPS 119901 = 0011) an exceptional protein was significantlyaggregated after LPS stimulation and reduced by RGFP966 at24 hours

37 RGFP966 Inhibited LPS-Induced Microglia ActivationWe further verified the effect of HDAC3 inhibition on

Neural Plasticity 7

1

Pathways in cancerCalcium signaling pathway

Huntingtonrsquos diseaseInsulin signaling pathway

Cell cycleApoptosis

RIG-I-like receptor signaling pathwaySpliceosome

Cytosolic DNA-sensing pathwayAlzheimerrsquos disease

Toll-like receptor signaling pathway

15 20 05(a)

1 150 05Pathways in cancerAlzheimerrsquos disease

Chemokine signaling pathwayOxidative phosphorylation

Regulation of actin cytoskeletonLeukocyte transendothelial migration

Focal adhesionHuntingtonrsquos disease

RibosomeChronic myeloid leukemiaInsulin signaling pathway

Parkinsonrsquos diseaseSpliceosome

mTOR signaling pathwayFc gamma R-mediated phagocytosis

Aminoacyl-tRNA biosynthesis

(b)

Figure 5 The KEGG pathway analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The horizontalaxis values equaled minus log(119901)

functional microglia activation We first stained primarycultured microglia with CD1632 as shown in Figure 8(a)microglia demonstrated bipolar shape in intact status whilein LPS stimulation group microglia enlarged and grew lotsof branches The fluorescence intensity of CD1632 was sig-nificantly decreased in LPS + RGFP966-treated group (Fig-ure 8(c)) We then detected classical inflammatory cytokinesin the supernatants of cultured cells exposed to LPS for 1 h2 h 3 h 8 h 12 h and 24 h with or without RGFP966 Weobserved profound inhibition of IL-6 (Figure 8(d)) (24 h15230 plusmn 5213 pgmL in RGFP966 + LPS versus 25538 plusmn8741 pgmL in DMSO + LPS 119901 = 0012) and TNF-120572(Figure 8(e)) (12 h 5189 plusmn 1613 pgmL in RGFP966 + LPSversus 17241 plusmn 4716 pgmL in DMSO + LPS 119901 lt 000124 h 7332 plusmn 2436 pgmL in RGFP966 + LPS versus 20977 plusmn1091 pgmL in DMSO + LPS 119901 lt 0001) secretion byRGFP966 Additionally STAT3 and STAT5 were two funda-mental signaling pathways governing inflammatory responsein various neurological diseases [17] In order to found out themechanism responsible for its anti-inflammatory responses

we detected phosphorylation levels of STAT3 and STAT5As shown in Figure 8(b) phosphorylation of STAT3 andSTAT5 increased 2 h after LPS treatment and declined quickly4 h after LPS RGFP966 inhibited not only phosphorylationof STAT3 and STAT5 but also decreased STAT5 expressionlevel whichmay partially contribute to its anti-inflammatoryresponse

4 Discussion

In summary we analyzed the differentially expressed proteinsof LPS-stimulated primary microglia with the treatmentof RGFP966 by proteomics A total of 168 LPS-inducedproteins were significantly reduced by RGFP966 in thisstudy The altered proteins mainly concentrated on thefollowing categories Toll-like receptor signaling pathwayAlzheimerrsquos disease cytosolic DNA-sensing pathway andfunctioned in cellular process immune system process andorganelle For Alzheimerrsquos disease preliminary evidencesdemonstrated inhibition of HDAC3 enzyme in neurons

8 Neural Plasticity

DMSORGFP966

TLR2 mRNA

Vehicle LPS0

5

10

15lowastlowast

lowastlowast(fo

ld o

f con

trol)

Relat

ive m

RNA

leve

l

(a)

DMSORGFP966

TLR3 mRNA

Vehicle LPS

lowastlowast

lowastlowast

0

10

20

30

40

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(b)

DMSORGFP966

TLR6 mRNA

Vehicle LPS

lowastlowast

lowastlowast

00

05

10

15

20

25

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(c)

DMSORGFP966

CD36 mRNA

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(d)

Figure 6 RT-PCR analysis of TLR2 TLR3 TLR6 and CD36 in four experimental groups The mRNA levels of TLR2 (a) TLR3 (b) TLR6(c) and CD36 (d) were significantly reduced by RGFP966 at the stimulation time of 6 hours lowastlowast119901 lt 001119873 = 3 repeats

prevented amyloid-beta oligomer-induced synaptic plasticityimpairments [18] and enhanced memory process [15] ForDNA-sensing pathways HDAC3 was thought to participatein IRF3IFN-120573 signaling pathways [13] In this study wemainly analyzed the differentially expressed TLR relative pro-teins of LPS-stimulated primarymicroglia with the treatmentof RGFP966 Toll-like receptor signaling pathway played animportant role in the regulation of inflammation In thisregard we selected TLR2 TLR3 TLR6 CD36 and SYK forfurther validation and found that they were all significantlyupregulated after LPS stimulation and downregulated inthe presence of RGFP966 Additionally morphological andfunctional alterations of microglia exposure to LPS werealso relieved by RGFP966 Our data provided a hint thatHDAC3 inhibitor may be a potential therapeutic target

combating microglia activation and inflammatory responsein neurological diseases

TLRs are a family of type I transmembrane recep-tors which share the multiple leucine-rich repeats (LRRs)domains in the extracellular space and the similar cytosolicdomains of the interleukin-1 (IL-1) receptors [19] TLRsare considered to recognize the distinct pathogen-associatedmolecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) which then drivea cascade of inflammatory signaling and converge at tran-scription factors nuclear factor-120581B (NF-120581B) [20] MAPK[21 22] and JAKSTAT pathways [23] In addition to TLR236 TLR coreceptorCD36 and regulatory protein SYKwereall decreased by RGFP966 Spleen tyrosine kinase (SYK) anonreceptor tyrosine kinase is typically regarded as a vital

Neural Plasticity 9

GAPDH

TLR2

DMSO RGFP966DMSO+ LPS

RGFP966+ LPS DMSO RGFP966

DMSO+ LPS

RGFP966+ LPS

TLR3

GAPDH

TLR6

GAPDH

CD36

GAPDH

phospho-p38

GAPDH

p38

SYK

GAPDH

(a)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR2 Protein level

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DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

(b)

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ive p

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in b

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(fold

of c

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l)TLR3 Protein level

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Vehicle LPS

lowastlowastlowast

(c)

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of c

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l)

TLR6 Protein level

DMSORGFP966

Vehicle LPS

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(d)

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CD36 Protein level

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DMSORGFP966

Vehicle LPS

lowastlowast

lowastlowast

(e)

Figure 7 Continued

10 Neural Plasticity

Relat

ive p

rote

in b

and

inte

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(fold

of c

ontro

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(f)

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SYK Protein level

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2

3

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

(g)

Figure 7Western blot analysis of TLR2 TLR3 TLR6MAPKp38 phospho-p38 SYK andCD36 in four experimental groups (a)The proteinlevels of TLR2 (b) TLR6 (d) SYK (g) phospho-p38 (f) and CD36 (e) were significantly altered by RGFP966 after 12 hoursrsquo stimulation of LPSwhile TLR3 was at 24 hours There was no significant difference of MAPK p38 protein expression among four groups lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

regulator in adaptive immunity [24] It consists of two tandemSH2 domains and a C-terminal tyrosine kinase domain SH2domains selectively bind to the phosphorylated immunore-ceptor tyrosine-based activation motifs (ITAMs) of immunereceptors such as MyD88 TRAF6 and TRIF transmittinginitiative signals to downstream pathways SYK is found to befundamental for Toll-like receptor signaling pathway and theinhibition of SYK suppresses the release of proinflammatorycytokines [25 26] In this study we detected alterations ofSYK in protein level but not mRNA level We surmisedthat posttranscriptional modification may also contributeto HDAC3rsquos functions Preliminary evidences demonstratethat HDAC family played a role in protein degradation[27] while inadequate time points of mRNA detection couldalso be an explanation for this discrepancy CD36 is a TLRcoreceptor playing a pivotal role in inflammatory responsesinitiated by TLRs [28] Previous researches reported thatCD36 contributed to the recognition of diacylglycerol ligandsby forming CD36-CD14-TLR2-TLR6 complex thus it con-trolled gram-positive bacterial infectionWhat ismore CD36was found to be involved in the formation of TLR4-TLR6heterodimers which induced the production of proinflam-matory cytokines nitric oxide and reactive oxygen speciesin response to endogenous ligands [29] Overall RGFP966demonstrated profound inhibitory effects on TLR signalingpathway and proinflammatory cytokines including IL-6 andTNF-120572

HDAC inhibitors have been reported to interfere withthe activation of the mitogen-activated protein kinasesSTAT1 AP-1 or NF-120581B signal transduction pathways [30

31] Here we observed transient STAT3 and STAT5 acti-vation at 2 hours after LPS stimulation which was aheadof the alteration of IL-6 and TNF-120572 Exposed to inflam-matory stimulation STAT35 was phosphorylated by JAKsleading to transcriptional activation Activation of STATselicited the expression of acute-phase proteins as well asa number of cytokines and chemokines including IL-6and TNF-120572 [17] Our study was in keeping with previousstudy inHDAC3 knockoutmacrophage which demonstratedthat HDAC3 knockout impaired STAT1STAT3STAT5 path-ways [13] Figure 9 presented a diagram showing theprotein and protein interactions among proteins Brieflyinitial change of STAT3STAT5 or NF-120581B [5] caused byHDAC3i may further elicit later alterations of TLR sig-naling pathway and proinflammatory cytokines includingIL-6 and TNF-120572 The underlying mechanism for HDAC3in the regulation of STAT3STAT5 is unknown Some evi-dences from Pan-HDAC inhibitors indicated that promoter-associated histone acetylation of SOCS1 and SOCS3 caused byHDACi may further downregulated JAK2STAT3 signaling[32]

In conclusions we identified TLR signaling pathwaymicroglia activation and STAT35 pathway which wereinhibited by RGFP966 Identification of these changes mayprovide valuable clues for the future applications of selectiveHDAC3 inhibitor in the clinic

Competing Interests

The authors declare no conflict of interests

Neural Plasticity 11

DMSO RGFP966

DMSO + LPS RGFP966 + LPS

(a)

p-STAT3

STAT3

p-STAT5

GAPDH

GAPDH

DMSO + LPS RGFP966 + LPS

STAT5

DMSO RGFP966 1h 2h 4h 1h 2h 4h

(b)

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

0

20000

40000

60000

80000

100000

Fluo

resc

ence

inte

nsity

of C

D16

32

(c)

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

0200400600800

100001500020000250003000035000 IL-6

LPSLPS + RGFP966

lowast

1 2 3 8 12 240(h)

(d)

TNF-120572

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

050

100

8000

3000

13000

18000

23000

LPSLPS + RGFP966

lowastlowastlowastlowast

1 2 3 8 12 240(h)

(e)

Figure 8 RGFP966 inhibited LPS-inducedmicroglia activation (a) Representativemicrophotographs of primarymicroglia cultures 24 hoursafter LPS and RGFP966 treatmentThe cultures were stained with CD1632 a marker of activatedmicroglia Scale bar = 10120583m (b) Expressionand phosphorylation of STAT3 and STAT5 in microglia pretreated with RGFP966 (c) Quantitative analysis of fluorescence intensity ofCD1632 All the photographs used for quantitative analysis were taken under the same exposure time and data were expressed as fluorescenceintensity per cell of randomly selected regions lowastlowast119901 lt 001119873 = 3 repeats Supernatant concentration of IL-6 (d) and TNF-120572 (e) detected atvarious stimulation times lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 3: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

Neural Plasticity 3

by sodium dodecyl sulfate-PAGE electrophoresis and thenblotted onto polyvinylidene fluoride membranes After beingblocked in 5 fat-free milk for 2 hours membranes wereincubated with primary antibodies against TLR2 (1 1000Abcam UK) TLR3 (1 1000 Abcam UK) TLR6 (1 1000Cell Signaling Technology USA) MAPK p38 (1 500 CellSignaling Technology USA) phospho-p38 (1 500 Cell Sig-naling Technology USA) CD36 (1 1000 Abcam UK) SYK(1 1000 AbcamUK) STAT3 (1 500 Cell Signaling Technol-ogy USA) phospho-STAT3 (1 500 Cell Signaling Technol-ogy USA) STAT5 (1 500 Cell Signaling Technology USA)phospho-STAT5 (1 500 Cell Signaling Technology USA)and GAPDH (1 5000 Bioworld USA) in 4∘C overnight toprobe targeted proteins Horseradish peroxidase-conjugatedsecondary antibodies (1 5000 Bioworld USA) were used tocombine primary antibodies and the reaction was detectedwith an ECL Kit (Bioworld USA) The intensities of blotswere quantified by densitometry

25 Immunofluorescence Primary microglia cells seeded oncover slips were fixed with 4 polyformaldehyde for 15 min-utes and permeabilizedwith 02Triton-X100 for 20minutesat room temperature After being blocked in 2 BSA in PBSfor 2 hours microglia cells were incubated with donkey anti-CD1632 antibody (1 500 BD Pharmingen USA) overnightin 4∘C and subsequently incubated with FITC-conjugatedanti-rat IgG (Invitrogen USA) at room temperature for 2hours DAPI staining was used to localize the nuclei Imageswere taken by a fluorescence microscope (Olympus Japan)and fluorescence intensities were analyzed by ImageJ software(version 139 National Institutes of Health USA)

26 Quantification of Secreted Cytokines Cytokines in thesupernatants were measured using the Cytometric BeadArray (CBA) Mouse Inflammation Kit (BD BiosciencesUSA) Briefly after incubation with capture beads on whichanti-cytokine antibodies are coated and PE-conjugated anti-cytokine antibodies cytokine levels can be quantitativelyanalyzed The assays were performed by BD Accuri C6 flowcytometer (BD Biosciences USA) following the manufac-turerrsquos instructions and the data were generated with FCAPArray version 301 Software

27 Statistical Analysis Data were expressed as mean plusmn SDof three independent experiments Comparisons betweengroups were conducted with SPSS 220 software Differenceswere analyzed by two-way analysis of variance (ANOVA)followed by Bonferronirsquos post hoc test and considered to bestatistically significant if 119901 lt 005

3 Results

31 Overview of Proteomic Analysis In this study 1883 pro-teins were detected in DMSO group and 1967 proteins werein RGFP966 group In LPS-stimulated groups there were1806 proteins in DMSO + LPS group and 2024 proteins in

Table 1 Summary of LC-MSMS data

Treatment Total proteinsDMSO 1883RGFP966 1967DMSO + LPS 1806RGFP966 + LPS 2024

DMSO

DMSORGFP9

66

RGFP966

+ LPS

+ LPS

169

70

138

35

48

28

82

87

107

1361

58

53

120

64

174

Figure 1 Overview of expressed proteins in four groups The bluecircle representedDMSOgroup the red circle representedRGFP966group the green circle represented DMSO + LPS group and theyellow circle represented RGFP966 + LPS group

RGFP966 + LPS group (Table 1) A Venn diagram showed therelationship of expressed proteins in four groups (Figure 1)

32 Alterations in Proteins Expression We identified gt 15fold peptide gt 1 as upregulated proteins and lt 066 foldpeptide gt 1 as downregulated proteins The numbers of dif-ferently expressed proteins between two groups were listed inTable 2 Specifically 168 of 444 (378) LPS-induced proteinswere significantly reduced with the treatment of RGFP966(Figure 2(a)) Likewise when analyzing proteins downreg-ulated by LPS the impact of RGFP966 was comparablewith 134 of 404 (332) proteins being rescued by RGFP966(Figure 2(b)) The heat map presented detailed informationof the upregulated as well as downregulated proteins whichwere caused by LPS and simultaneously reversed byRGFP966(Figure 3)

33 Gene Ontology Analysis GO analysis was used to revealthe function of proteins in three aspects cellular compo-nent molecular function and biological process The 168differentially expressed proteins overlapped in Figure 2(a)were mainly involved in biological process cellular processimmune system process and establishment of localizationcellular component organellemacromolecular complex andorganelle part molecular function binding catalytic activity

4 Neural Plasticity

Table 2 Summary of upregulateddownregulated proteins candidates

Upregulated protein candidates Downregulated protein candidatesgt 15 fold peptide gt 1 lt 066 fold peptide gt 1

RGFP966 versus DMSO 493 338DMSO + LPS versus DMSO 444 404RGFP966 + LPS versus DMSO + LPS 401 275

DMSO + LPSDMSO upregulated proteins

107168276

RGFP966 + LPSDMSO + LPS downregulated proteins

(a)

DMSO + LPSDMSO downregulated proteins

267134270

RGFP966 + LPSDMSO + LPS upregulated proteins

(b)

Figure 2 Venn diagrams of the differentially expressed proteins between LPS-stimulated (DMSO + LPSDMSO) and RGFP966-treated(RGFP966 + LPSDMSO + LPS) microglia cells (a) 168 proteins were upregulated by LPS and reversed with the treatment of RGFP966 (b)134 proteins were downregulated after the stimulation of LPS and rescued by RGFP966

and enzyme regulator activity (Figure 4(a)) However the 134differentially expressed proteins overlapped in Figure 2(b)were mainly involved in biological process cellular processmetabolic process and establishment of localization cellularcomponent organelle macromolecular complex and cellpart molecular function catalytic activity binding andelectron carrier activity (Figure 4(b))

34 KEGG Pathway Analysis To explore the potential sig-naling pathway through which HDAC3 specific inhibitorRGFP966 regulates inflammatory response KEGG pathwayanalysis was performed As shown in Figure 5(a) the pathwaymap depicted that the 168 differentially expressed proteinsoverlapped in Figure 2(a) were related to Toll-like recep-tor signaling pathway Alzheimerrsquos disease cytosolic DNA-sensing pathway spliceosome RIG-I-like receptor signalingapoptosis cell cycle insulin signaling pathway Huntingtonrsquosdisease calcium signaling pathway and pathways in cancerHowever the 134 differentially expressed proteins over-lapped in Figure 2(b) belonged to the following pathwaysaminoacyl-tRNAbiosynthesis Fc gammaR-mediated phago-cytosis mTOR signaling pathway spliceosome Parkinsonrsquosdisease and so forth (Figure 5(b))

35 mRNA Verifications of Discrepant Proteins between LPSGroup and LPS + RGFP966 Group RT-PCR was performed

to detect the mRNA levels of TLR2 TLR3 TLR6 CD36and SYK at 6 hours after LPS stimulation As expectedLPS stimulation potentiated profound mRNA increase ofTLR2 (924 folds versus DMSO group) TLR3 (2881 foldsversus DMSO group) TLR6 (173 folds versus DMSO group)and CD36 (878 folds versus DMSO group) However theseaugments were significantly decreased by RGFP966 with thedecline amplitudes at 7358 in TLR2 (119901 = 0002) 3551in TLR3 (119901 lt 0001) 3757 in TLR6 (119901 = 0007) 8532 inCD36 (119901 lt 0001) respectively (Figures 6(a)ndash6(d))Howeverthe mRNA expression level of SYK was not significantlyaltered (data not shown)

36 Protein Verifications of Discrepant Proteins between LPSGroup and LPS + RGFP966 Group Then we verified thechanges of TLR2 TLR3 TLR6 CD36 and SYK at the proteinlevel by western blotting (Figures 7(a)ndash7(g)) Consistent withthe results of mRNA detection the protein expression levelsof TLR2 (831 plusmn 054 in DMSO + LPS versus 100 plusmn 107 inDMSO 119901 lt 0001 187 plusmn 020 in RGFP966 + LPS versus831 plusmn 054 in DMSO + LPS 119901 lt 0001) TLR6 (1009 plusmn 066in DMSO + LPS versus 100 plusmn 028 in DMSO 119901 lt 0001388 plusmn 107 in RGFP966 + LPS versus 1009 plusmn 066 in DMSO+ LPS 119901 lt 0001) CD36 (375 plusmn 013 in DMSO + LPS versus100 plusmn 009 in DMSO 119901 lt 0001 141 plusmn 018 in RGFP966 +LPS versus 375 plusmn 013 in DMSO + LPS 119901 lt 0001) and SYK

Neural Plasticity 5

RSAD2_MOUSEIFIT2_MOUSEIL1B_MOUSE

SAP18_MOUSEIL1A_MOUSE

TMED5_MOUSERL30_MOUSE

MMP13_MOUSECKLF3_MOUSE

TRABD_MOUSETMCO1_MOUSE

NOS2_MOUSES15A3_MOUSERIPL2_MOUSE

COMD9_MOUSEHSC20_MOUSETIM50_MOUSEGMDS_MOUSEMECR_MOUSE

ADAP2_MOUSERM10_MOUSE

APOOL_MOUSECUTC_MOUSESTXB3_MOUSEGORS2_MOUSENNRD_MOUSECOG2_MOUSE

COASY_MOUSEH1BP3_MOUSEILVBL_MOUSE

PKHA2_MOUSEKHDR1_MOUSEMMP12_MOUSE

TOIP2_MOUSETGAP1_MOUSEPCAT1_MOUSEABCF2_MOUSE

LOX5_MOUSESCFD2_MOUSE

TAP2_MOUSEACSL4_MOUSE

CNNM3_MOUSEMD1L1_MOUSEBMP2K_MOUSEBICD2_MOUSE

EPS8_MOUSEVPS53_MOUSETLR3_MOUSENRP2_MOUSE

NAT10_MOUSEDIAP2_MOUSEAT8A1_MOUSEPI3R4_MOUSEU520_MOUSE

MCM7_MOUSECO3_MOUSE

SPCS2_MOUSE3BP1_MOUSE

AIMP1_MOUSETLR2_MOUSE

NUBP2_MOUSERM12_MOUSE

UN45A_MOUSERAB31_MOUSEC2D1B_MOUSETOR4A_MOUSERASL2_MOUSEVPS41_MOUSEPRAF3_MOUSE

STRUM_MOUSESWP70_MOUSE

ZPR1_MOUSECD47_MOUSE

SAR1A_MOUSEGTPB1_MOUSERBM25_MOUSENSUN2_MOUSE

LA_MOUSETHMS2_MOUSE

SH3L3_MOUSE2ABA_MOUSEPSB8_MOUSE

TWF2_MOUSECBL_MOUSE

FRIH_MOUSEUSO1_MOUSE

CASP8_MOUSECOMDA_MOUSE

KCMF1_MOUSEHOOK2_MOUSE

FKB15_MOUSEBAG1_MOUSE

ABRAL_MOUSEPDE12_MOUSESTING_MOUSE

SPB8_MOUSELYPA2_MOUSEPEX14_MOUSE

STAP1_MOUSECLC6A_MOUSE

ROA0_MOUSEPPP6_MOUSE

RBM47_MOUSEMA2C1_MOUSENUPL1_MOUSE

BPHL_MOUSEBRE_MOUSE

UFL1_MOUSETHOP1_MOUSEKANK2_MOUSE

SPS1_MOUSECCD93_MOUSESPAG7_MOUSEPON2_MOUSETR150_MOUSE

NCBP1_MOUSEMCCB_MOUSETTL12_MOUSE

IF2P_MOUSEASM_MOUSEIF4E_MOUSE

COPZ1_MOUSEPFD3_MOUSE

TBCA_MOUSERAGP1_MOUSECX6A1_MOUSEFMR1_MOUSEUMPS_MOUSE

GOSR1_MOUSEPPT2_MOUSEIMA7_MOUSEIMA3_MOUSEIMA4_MOUSE

VMA5A_MOUSEIF4H_MOUSE

ACADS_MOUSEKSYK_MOUSE

CELF2_MOUSETBCD_MOUSE

PIGS_MOUSEADAS_MOUSETITIN_MOUSECALM_MOUSEDC1I2_MOUSE

RL23_MOUSECOPG2_MOUSE

ILK_MOUSESF3B1_MOUSEISOC1_MOUSESGTA_MOUSE

HCFC1_MOUSEHMHA1_MOUSE

EMC8_MOUSEGSH0_MOUSE

MCTS1_MOUSEIF4B_MOUSEVIP2_MOUSEFXR1_MOUSEASNS_MOUSE

LC7L3_MOUSENIBAN_MOUSEMCM6_MOUSE

IRF3_MOUSEIF5A1_MOUSE

GNAQ_MOUSENU4M_MOUSE

GAK_MOUSESTK10_MOUSESTXB2_MOUSEVIGLN_MOUSE

EIF3B_MOUSEAGAL_MOUSE

SYIC_MOUSESCFD1_MOUSE

SCMC1_MOUSEVASP_MOUSE

EIF3D_MOUSEACSF2_MOUSEDNJA1_MOUSEAHSA1_MOUSE

APMAP_MOUSENDUAA_MOUSE

PGES2_MOUSESEPT9_MOUSE

CRK_MOUSEBAG6_MOUSEPIN1_MOUSE

DAZP1_MOUSEGTF2I_MOUSESDF2L_MOUSE

UB2V1_MOUSETPD54_MOUSE

SSRA_MOUSEGMFB_MOUSEPRP19_MOUSERCC1_MOUSE

NUP85_MOUSEDIAC_MOUSE

MARE2_MOUSEPDCL3_MOUSE

TM209_MOUSERCC2_MOUSE

NRDC_MOUSECLC7A_MOUSELPPRC_MOUSE

TTYH3_MOUSESORCN_MOUSE

PA1B2_MOUSESC31A_MOUSE

PCY1A_MOUSELDHB_MOUSE

TCEA1_MOUSEFYB_MOUSE

RL21_MOUSEAT1A1_MOUSE

ASC_MOUSERGS10_MOUSEVINC_MOUSESBP1_MOUSEGGH_MOUSE

CP062_MOUSERD23B_MOUSE

ACADV_MOUSESYHC_MOUSE

VDAC1_MOUSESMCA5_MOUSE

SIR2_MOUSEENASE_MOUSEFA98B_MOUSESRSF1_MOUSE

ASM3B_MOUSEPRDX3_MOUSEPLOD3_MOUSEMYG1_MOUSEARSK_MOUSEEI2BA_MOUSESYLC_MOUSE

FCGR3_MOUSEDOPD_MOUSE

RENT1_MOUSENPS3B_MOUSE

TOM22_MOUSEEIF2A_MOUSE

CAB39_MOUSEHINT1_MOUSEABCE1_MOUSEUCHL4_MOUSE

ACBP_MOUSEARHG7_MOUSE

PON3_MOUSEWASH7_MOUSE

STAB1_MOUSEPSME3_MOUSE

MANBA_MOUSEDYHC1_MOUSEETUD1_MOUSEPK3CG_MOUSETCRG1_MOUSEPARP1_MOUSEITIH2_MOUSE

NOMO1_MOUSECLCN5_MOUSEVPS52_MOUSE

EXOC5_MOUSEDP13B_MOUSESART3_MOUSE

PEX5_MOUSEPGM2L_MOUSEHP1B3_MOUSE

T2FA_MOUSEADPGK_MOUSE

DDB1_MOUSETMM43_MOUSE

TS101_MOUSESIAE_MOUSE

CSK22_MOUSELMAN1_MOUSEUCHL5_MOUSE

PGP_MOUSEDEOC_MOUSEPEX19_MOUSECTBP1_MOUSEAOAH_MOUSEKAP2_MOUSE

ETHE1_MOUSEADHX_MOUSEIF4E2_MOUSEDRG1_MOUSE

HNRPC_MOUSEDOCK2_MOUSENDUB8_MOUSEC1QBP_MOUSECNPY2_MOUSE

HDHD2_MOUSENFU1_MOUSE

NQO2_MOUSELKHA4_MOUSE

EDF1_MOUSENDUS8_MOUSERAB18_MOUSERS15A_MOUSE

RS21_MOUSE

DMSO + LPSversus DMSO

10

0

minus10

log10(ratio)

RGFP966 + LPS

DMSO + LPSversus

Figure 3 Heat map generated by MeV regarding proteins subjected to RGFP966 and LPS regulation Experimental groups were presentedon the horizontal axis and proteins were on the vertical axis Colors were consistent with protein expression levels red indicated upregulatedratio and green indicated downregulated ratio

6 Neural Plasticity

Biological process Cellular component Molecular function

0

1

2

3

4

5

6

7

Cel

lula

r pro

cess

Imm

une s

yste

m p

roce

ssEs

tabl

ishm

ent o

f loc

aliz

atio

nLo

caliz

atio

nM

ultio

rgan

ism p

roce

ssRe

spon

se to

stim

ulus

Met

abol

ic p

roce

ssD

eath

Loco

mot

ion

Cel

lula

r com

pone

nt o

rgan

izat

ion

Dev

elopm

enta

l pro

cess

Cel

lula

r com

pone

nt b

ioge

nesis

Biol

ogic

al ad

hesio

nRe

prod

uctiv

e pro

cess

Repr

oduc

tion

Mul

ticel

lula

r org

anism

al p

roce

ssBi

olog

ical

regu

latio

nO

rgan

elle

Mac

rom

olec

ular

com

plex

Org

anel

le p

art

Enve

lope

Cel

l par

tC

ell

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

part

Extr

acel

lula

r reg

ion

Bind

ing

Cata

lytic

activ

ityEn

zym

e reg

ulat

or ac

tivity

Stru

ctur

al m

olec

ule a

ctiv

ityEl

ectro

n ca

rrie

r act

ivity

Tran

spor

ter a

ctiv

ityTr

ansc

riptio

n re

gula

tor a

ctiv

ityM

olec

ular

tran

sduc

er ac

tivity

(a)

0

1

2

3

4

5

Biological process Cellular component Molecular function

Cel

lula

r pro

cess

Met

abol

ic p

roce

ssEs

tabl

ishm

ent o

f loc

aliz

atio

nLo

caliz

atio

n

Mul

tiorg

anism

pro

cess

Resp

onse

to st

imul

us

Dea

th

Loco

mot

ion

Cel

lula

r com

pone

nt o

rgan

izat

ion

Dev

elopm

enta

l pro

cess

Imm

une s

yste

m p

roce

ss

Cel

lula

r com

pone

nt b

ioge

nesis

Biol

ogic

al ad

hesio

n

Repr

oduc

tive p

roce

ssRe

prod

uctio

n

Mul

ticel

lula

r org

anism

al p

roce

ssBi

olog

ical

regu

latio

nO

rgan

elle

Mac

rom

olec

ular

com

plex

Org

anel

le p

art

Enve

lope

Cel

l par

tC

ell

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

part

Extr

acel

lula

r reg

ion

Bind

ing

Cata

lytic

activ

ity

Enzy

me r

egul

ator

activ

itySt

ruct

ural

mol

ecul

e act

ivity

Elec

tron

carr

ier a

ctiv

ity

Tran

spor

ter a

ctiv

ityTr

ansc

riptio

n re

gula

tor a

ctiv

ity

Mol

ecul

ar tr

ansd

ucer

activ

ity

(b)

Figure 4 The GO analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The GO analysis involvedbiological process cellular component and molecular function The vertical axis values equaled minus log(119901)

(272 plusmn 013 in DMSO + LPS versus 100 plusmn 007 in DMSO119901 lt 0001 145 plusmn 015 in RGFP966 + LPS versus 272 plusmn 013in DMSO + LPS 119901 lt 0001) were all significantly increasedat the stimulation time of 12 hours and decreased with thetreatment of RGFP966 In our study though there was nosignificant change seen in total MAPK p38 the protein levelof phospho-p38 (331 plusmn 040 in DMSO + LPS versus 100 plusmn023 in DMSO 119901 lt 0001 256 plusmn 058 in RGFP966 + LPSversus 331 plusmn 040 in DMSO + LPS 119901 = 0021) showed a

similar trend as the proteins mentioned above TLR3 (634 plusmn033 in DMSO + LPS versus 100 plusmn 115 in DMSO 119901 = 0003196 plusmn 093 in RGFP966 + LPS versus 634 plusmn 033 in DMSO+ LPS 119901 = 0011) an exceptional protein was significantlyaggregated after LPS stimulation and reduced by RGFP966 at24 hours

37 RGFP966 Inhibited LPS-Induced Microglia ActivationWe further verified the effect of HDAC3 inhibition on

Neural Plasticity 7

1

Pathways in cancerCalcium signaling pathway

Huntingtonrsquos diseaseInsulin signaling pathway

Cell cycleApoptosis

RIG-I-like receptor signaling pathwaySpliceosome

Cytosolic DNA-sensing pathwayAlzheimerrsquos disease

Toll-like receptor signaling pathway

15 20 05(a)

1 150 05Pathways in cancerAlzheimerrsquos disease

Chemokine signaling pathwayOxidative phosphorylation

Regulation of actin cytoskeletonLeukocyte transendothelial migration

Focal adhesionHuntingtonrsquos disease

RibosomeChronic myeloid leukemiaInsulin signaling pathway

Parkinsonrsquos diseaseSpliceosome

mTOR signaling pathwayFc gamma R-mediated phagocytosis

Aminoacyl-tRNA biosynthesis

(b)

Figure 5 The KEGG pathway analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The horizontalaxis values equaled minus log(119901)

functional microglia activation We first stained primarycultured microglia with CD1632 as shown in Figure 8(a)microglia demonstrated bipolar shape in intact status whilein LPS stimulation group microglia enlarged and grew lotsof branches The fluorescence intensity of CD1632 was sig-nificantly decreased in LPS + RGFP966-treated group (Fig-ure 8(c)) We then detected classical inflammatory cytokinesin the supernatants of cultured cells exposed to LPS for 1 h2 h 3 h 8 h 12 h and 24 h with or without RGFP966 Weobserved profound inhibition of IL-6 (Figure 8(d)) (24 h15230 plusmn 5213 pgmL in RGFP966 + LPS versus 25538 plusmn8741 pgmL in DMSO + LPS 119901 = 0012) and TNF-120572(Figure 8(e)) (12 h 5189 plusmn 1613 pgmL in RGFP966 + LPSversus 17241 plusmn 4716 pgmL in DMSO + LPS 119901 lt 000124 h 7332 plusmn 2436 pgmL in RGFP966 + LPS versus 20977 plusmn1091 pgmL in DMSO + LPS 119901 lt 0001) secretion byRGFP966 Additionally STAT3 and STAT5 were two funda-mental signaling pathways governing inflammatory responsein various neurological diseases [17] In order to found out themechanism responsible for its anti-inflammatory responses

we detected phosphorylation levels of STAT3 and STAT5As shown in Figure 8(b) phosphorylation of STAT3 andSTAT5 increased 2 h after LPS treatment and declined quickly4 h after LPS RGFP966 inhibited not only phosphorylationof STAT3 and STAT5 but also decreased STAT5 expressionlevel whichmay partially contribute to its anti-inflammatoryresponse

4 Discussion

In summary we analyzed the differentially expressed proteinsof LPS-stimulated primary microglia with the treatmentof RGFP966 by proteomics A total of 168 LPS-inducedproteins were significantly reduced by RGFP966 in thisstudy The altered proteins mainly concentrated on thefollowing categories Toll-like receptor signaling pathwayAlzheimerrsquos disease cytosolic DNA-sensing pathway andfunctioned in cellular process immune system process andorganelle For Alzheimerrsquos disease preliminary evidencesdemonstrated inhibition of HDAC3 enzyme in neurons

8 Neural Plasticity

DMSORGFP966

TLR2 mRNA

Vehicle LPS0

5

10

15lowastlowast

lowastlowast(fo

ld o

f con

trol)

Relat

ive m

RNA

leve

l

(a)

DMSORGFP966

TLR3 mRNA

Vehicle LPS

lowastlowast

lowastlowast

0

10

20

30

40

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(b)

DMSORGFP966

TLR6 mRNA

Vehicle LPS

lowastlowast

lowastlowast

00

05

10

15

20

25

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(c)

DMSORGFP966

CD36 mRNA

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(d)

Figure 6 RT-PCR analysis of TLR2 TLR3 TLR6 and CD36 in four experimental groups The mRNA levels of TLR2 (a) TLR3 (b) TLR6(c) and CD36 (d) were significantly reduced by RGFP966 at the stimulation time of 6 hours lowastlowast119901 lt 001119873 = 3 repeats

prevented amyloid-beta oligomer-induced synaptic plasticityimpairments [18] and enhanced memory process [15] ForDNA-sensing pathways HDAC3 was thought to participatein IRF3IFN-120573 signaling pathways [13] In this study wemainly analyzed the differentially expressed TLR relative pro-teins of LPS-stimulated primarymicroglia with the treatmentof RGFP966 Toll-like receptor signaling pathway played animportant role in the regulation of inflammation In thisregard we selected TLR2 TLR3 TLR6 CD36 and SYK forfurther validation and found that they were all significantlyupregulated after LPS stimulation and downregulated inthe presence of RGFP966 Additionally morphological andfunctional alterations of microglia exposure to LPS werealso relieved by RGFP966 Our data provided a hint thatHDAC3 inhibitor may be a potential therapeutic target

combating microglia activation and inflammatory responsein neurological diseases

TLRs are a family of type I transmembrane recep-tors which share the multiple leucine-rich repeats (LRRs)domains in the extracellular space and the similar cytosolicdomains of the interleukin-1 (IL-1) receptors [19] TLRsare considered to recognize the distinct pathogen-associatedmolecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) which then drivea cascade of inflammatory signaling and converge at tran-scription factors nuclear factor-120581B (NF-120581B) [20] MAPK[21 22] and JAKSTAT pathways [23] In addition to TLR236 TLR coreceptorCD36 and regulatory protein SYKwereall decreased by RGFP966 Spleen tyrosine kinase (SYK) anonreceptor tyrosine kinase is typically regarded as a vital

Neural Plasticity 9

GAPDH

TLR2

DMSO RGFP966DMSO+ LPS

RGFP966+ LPS DMSO RGFP966

DMSO+ LPS

RGFP966+ LPS

TLR3

GAPDH

TLR6

GAPDH

CD36

GAPDH

phospho-p38

GAPDH

p38

SYK

GAPDH

(a)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR2 Protein level

0

2

4

6

8

10

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

(b)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)TLR3 Protein level

0

2

4

6

8

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(c)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR6 Protein level

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(d)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

CD36 Protein level

0

1

2

3

4

5

DMSORGFP966

Vehicle LPS

lowastlowast

lowastlowast

(e)

Figure 7 Continued

10 Neural Plasticity

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)phospho-p38 Protein level

0

1

2

3

4

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(f)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

SYK Protein level

0

1

2

3

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

(g)

Figure 7Western blot analysis of TLR2 TLR3 TLR6MAPKp38 phospho-p38 SYK andCD36 in four experimental groups (a)The proteinlevels of TLR2 (b) TLR6 (d) SYK (g) phospho-p38 (f) and CD36 (e) were significantly altered by RGFP966 after 12 hoursrsquo stimulation of LPSwhile TLR3 was at 24 hours There was no significant difference of MAPK p38 protein expression among four groups lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

regulator in adaptive immunity [24] It consists of two tandemSH2 domains and a C-terminal tyrosine kinase domain SH2domains selectively bind to the phosphorylated immunore-ceptor tyrosine-based activation motifs (ITAMs) of immunereceptors such as MyD88 TRAF6 and TRIF transmittinginitiative signals to downstream pathways SYK is found to befundamental for Toll-like receptor signaling pathway and theinhibition of SYK suppresses the release of proinflammatorycytokines [25 26] In this study we detected alterations ofSYK in protein level but not mRNA level We surmisedthat posttranscriptional modification may also contributeto HDAC3rsquos functions Preliminary evidences demonstratethat HDAC family played a role in protein degradation[27] while inadequate time points of mRNA detection couldalso be an explanation for this discrepancy CD36 is a TLRcoreceptor playing a pivotal role in inflammatory responsesinitiated by TLRs [28] Previous researches reported thatCD36 contributed to the recognition of diacylglycerol ligandsby forming CD36-CD14-TLR2-TLR6 complex thus it con-trolled gram-positive bacterial infectionWhat ismore CD36was found to be involved in the formation of TLR4-TLR6heterodimers which induced the production of proinflam-matory cytokines nitric oxide and reactive oxygen speciesin response to endogenous ligands [29] Overall RGFP966demonstrated profound inhibitory effects on TLR signalingpathway and proinflammatory cytokines including IL-6 andTNF-120572

HDAC inhibitors have been reported to interfere withthe activation of the mitogen-activated protein kinasesSTAT1 AP-1 or NF-120581B signal transduction pathways [30

31] Here we observed transient STAT3 and STAT5 acti-vation at 2 hours after LPS stimulation which was aheadof the alteration of IL-6 and TNF-120572 Exposed to inflam-matory stimulation STAT35 was phosphorylated by JAKsleading to transcriptional activation Activation of STATselicited the expression of acute-phase proteins as well asa number of cytokines and chemokines including IL-6and TNF-120572 [17] Our study was in keeping with previousstudy inHDAC3 knockoutmacrophage which demonstratedthat HDAC3 knockout impaired STAT1STAT3STAT5 path-ways [13] Figure 9 presented a diagram showing theprotein and protein interactions among proteins Brieflyinitial change of STAT3STAT5 or NF-120581B [5] caused byHDAC3i may further elicit later alterations of TLR sig-naling pathway and proinflammatory cytokines includingIL-6 and TNF-120572 The underlying mechanism for HDAC3in the regulation of STAT3STAT5 is unknown Some evi-dences from Pan-HDAC inhibitors indicated that promoter-associated histone acetylation of SOCS1 and SOCS3 caused byHDACi may further downregulated JAK2STAT3 signaling[32]

In conclusions we identified TLR signaling pathwaymicroglia activation and STAT35 pathway which wereinhibited by RGFP966 Identification of these changes mayprovide valuable clues for the future applications of selectiveHDAC3 inhibitor in the clinic

Competing Interests

The authors declare no conflict of interests

Neural Plasticity 11

DMSO RGFP966

DMSO + LPS RGFP966 + LPS

(a)

p-STAT3

STAT3

p-STAT5

GAPDH

GAPDH

DMSO + LPS RGFP966 + LPS

STAT5

DMSO RGFP966 1h 2h 4h 1h 2h 4h

(b)

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

0

20000

40000

60000

80000

100000

Fluo

resc

ence

inte

nsity

of C

D16

32

(c)

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

0200400600800

100001500020000250003000035000 IL-6

LPSLPS + RGFP966

lowast

1 2 3 8 12 240(h)

(d)

TNF-120572

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

050

100

8000

3000

13000

18000

23000

LPSLPS + RGFP966

lowastlowastlowastlowast

1 2 3 8 12 240(h)

(e)

Figure 8 RGFP966 inhibited LPS-inducedmicroglia activation (a) Representativemicrophotographs of primarymicroglia cultures 24 hoursafter LPS and RGFP966 treatmentThe cultures were stained with CD1632 a marker of activatedmicroglia Scale bar = 10120583m (b) Expressionand phosphorylation of STAT3 and STAT5 in microglia pretreated with RGFP966 (c) Quantitative analysis of fluorescence intensity ofCD1632 All the photographs used for quantitative analysis were taken under the same exposure time and data were expressed as fluorescenceintensity per cell of randomly selected regions lowastlowast119901 lt 001119873 = 3 repeats Supernatant concentration of IL-6 (d) and TNF-120572 (e) detected atvarious stimulation times lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 4: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

4 Neural Plasticity

Table 2 Summary of upregulateddownregulated proteins candidates

Upregulated protein candidates Downregulated protein candidatesgt 15 fold peptide gt 1 lt 066 fold peptide gt 1

RGFP966 versus DMSO 493 338DMSO + LPS versus DMSO 444 404RGFP966 + LPS versus DMSO + LPS 401 275

DMSO + LPSDMSO upregulated proteins

107168276

RGFP966 + LPSDMSO + LPS downregulated proteins

(a)

DMSO + LPSDMSO downregulated proteins

267134270

RGFP966 + LPSDMSO + LPS upregulated proteins

(b)

Figure 2 Venn diagrams of the differentially expressed proteins between LPS-stimulated (DMSO + LPSDMSO) and RGFP966-treated(RGFP966 + LPSDMSO + LPS) microglia cells (a) 168 proteins were upregulated by LPS and reversed with the treatment of RGFP966 (b)134 proteins were downregulated after the stimulation of LPS and rescued by RGFP966

and enzyme regulator activity (Figure 4(a)) However the 134differentially expressed proteins overlapped in Figure 2(b)were mainly involved in biological process cellular processmetabolic process and establishment of localization cellularcomponent organelle macromolecular complex and cellpart molecular function catalytic activity binding andelectron carrier activity (Figure 4(b))

34 KEGG Pathway Analysis To explore the potential sig-naling pathway through which HDAC3 specific inhibitorRGFP966 regulates inflammatory response KEGG pathwayanalysis was performed As shown in Figure 5(a) the pathwaymap depicted that the 168 differentially expressed proteinsoverlapped in Figure 2(a) were related to Toll-like recep-tor signaling pathway Alzheimerrsquos disease cytosolic DNA-sensing pathway spliceosome RIG-I-like receptor signalingapoptosis cell cycle insulin signaling pathway Huntingtonrsquosdisease calcium signaling pathway and pathways in cancerHowever the 134 differentially expressed proteins over-lapped in Figure 2(b) belonged to the following pathwaysaminoacyl-tRNAbiosynthesis Fc gammaR-mediated phago-cytosis mTOR signaling pathway spliceosome Parkinsonrsquosdisease and so forth (Figure 5(b))

35 mRNA Verifications of Discrepant Proteins between LPSGroup and LPS + RGFP966 Group RT-PCR was performed

to detect the mRNA levels of TLR2 TLR3 TLR6 CD36and SYK at 6 hours after LPS stimulation As expectedLPS stimulation potentiated profound mRNA increase ofTLR2 (924 folds versus DMSO group) TLR3 (2881 foldsversus DMSO group) TLR6 (173 folds versus DMSO group)and CD36 (878 folds versus DMSO group) However theseaugments were significantly decreased by RGFP966 with thedecline amplitudes at 7358 in TLR2 (119901 = 0002) 3551in TLR3 (119901 lt 0001) 3757 in TLR6 (119901 = 0007) 8532 inCD36 (119901 lt 0001) respectively (Figures 6(a)ndash6(d))Howeverthe mRNA expression level of SYK was not significantlyaltered (data not shown)

36 Protein Verifications of Discrepant Proteins between LPSGroup and LPS + RGFP966 Group Then we verified thechanges of TLR2 TLR3 TLR6 CD36 and SYK at the proteinlevel by western blotting (Figures 7(a)ndash7(g)) Consistent withthe results of mRNA detection the protein expression levelsof TLR2 (831 plusmn 054 in DMSO + LPS versus 100 plusmn 107 inDMSO 119901 lt 0001 187 plusmn 020 in RGFP966 + LPS versus831 plusmn 054 in DMSO + LPS 119901 lt 0001) TLR6 (1009 plusmn 066in DMSO + LPS versus 100 plusmn 028 in DMSO 119901 lt 0001388 plusmn 107 in RGFP966 + LPS versus 1009 plusmn 066 in DMSO+ LPS 119901 lt 0001) CD36 (375 plusmn 013 in DMSO + LPS versus100 plusmn 009 in DMSO 119901 lt 0001 141 plusmn 018 in RGFP966 +LPS versus 375 plusmn 013 in DMSO + LPS 119901 lt 0001) and SYK

Neural Plasticity 5

RSAD2_MOUSEIFIT2_MOUSEIL1B_MOUSE

SAP18_MOUSEIL1A_MOUSE

TMED5_MOUSERL30_MOUSE

MMP13_MOUSECKLF3_MOUSE

TRABD_MOUSETMCO1_MOUSE

NOS2_MOUSES15A3_MOUSERIPL2_MOUSE

COMD9_MOUSEHSC20_MOUSETIM50_MOUSEGMDS_MOUSEMECR_MOUSE

ADAP2_MOUSERM10_MOUSE

APOOL_MOUSECUTC_MOUSESTXB3_MOUSEGORS2_MOUSENNRD_MOUSECOG2_MOUSE

COASY_MOUSEH1BP3_MOUSEILVBL_MOUSE

PKHA2_MOUSEKHDR1_MOUSEMMP12_MOUSE

TOIP2_MOUSETGAP1_MOUSEPCAT1_MOUSEABCF2_MOUSE

LOX5_MOUSESCFD2_MOUSE

TAP2_MOUSEACSL4_MOUSE

CNNM3_MOUSEMD1L1_MOUSEBMP2K_MOUSEBICD2_MOUSE

EPS8_MOUSEVPS53_MOUSETLR3_MOUSENRP2_MOUSE

NAT10_MOUSEDIAP2_MOUSEAT8A1_MOUSEPI3R4_MOUSEU520_MOUSE

MCM7_MOUSECO3_MOUSE

SPCS2_MOUSE3BP1_MOUSE

AIMP1_MOUSETLR2_MOUSE

NUBP2_MOUSERM12_MOUSE

UN45A_MOUSERAB31_MOUSEC2D1B_MOUSETOR4A_MOUSERASL2_MOUSEVPS41_MOUSEPRAF3_MOUSE

STRUM_MOUSESWP70_MOUSE

ZPR1_MOUSECD47_MOUSE

SAR1A_MOUSEGTPB1_MOUSERBM25_MOUSENSUN2_MOUSE

LA_MOUSETHMS2_MOUSE

SH3L3_MOUSE2ABA_MOUSEPSB8_MOUSE

TWF2_MOUSECBL_MOUSE

FRIH_MOUSEUSO1_MOUSE

CASP8_MOUSECOMDA_MOUSE

KCMF1_MOUSEHOOK2_MOUSE

FKB15_MOUSEBAG1_MOUSE

ABRAL_MOUSEPDE12_MOUSESTING_MOUSE

SPB8_MOUSELYPA2_MOUSEPEX14_MOUSE

STAP1_MOUSECLC6A_MOUSE

ROA0_MOUSEPPP6_MOUSE

RBM47_MOUSEMA2C1_MOUSENUPL1_MOUSE

BPHL_MOUSEBRE_MOUSE

UFL1_MOUSETHOP1_MOUSEKANK2_MOUSE

SPS1_MOUSECCD93_MOUSESPAG7_MOUSEPON2_MOUSETR150_MOUSE

NCBP1_MOUSEMCCB_MOUSETTL12_MOUSE

IF2P_MOUSEASM_MOUSEIF4E_MOUSE

COPZ1_MOUSEPFD3_MOUSE

TBCA_MOUSERAGP1_MOUSECX6A1_MOUSEFMR1_MOUSEUMPS_MOUSE

GOSR1_MOUSEPPT2_MOUSEIMA7_MOUSEIMA3_MOUSEIMA4_MOUSE

VMA5A_MOUSEIF4H_MOUSE

ACADS_MOUSEKSYK_MOUSE

CELF2_MOUSETBCD_MOUSE

PIGS_MOUSEADAS_MOUSETITIN_MOUSECALM_MOUSEDC1I2_MOUSE

RL23_MOUSECOPG2_MOUSE

ILK_MOUSESF3B1_MOUSEISOC1_MOUSESGTA_MOUSE

HCFC1_MOUSEHMHA1_MOUSE

EMC8_MOUSEGSH0_MOUSE

MCTS1_MOUSEIF4B_MOUSEVIP2_MOUSEFXR1_MOUSEASNS_MOUSE

LC7L3_MOUSENIBAN_MOUSEMCM6_MOUSE

IRF3_MOUSEIF5A1_MOUSE

GNAQ_MOUSENU4M_MOUSE

GAK_MOUSESTK10_MOUSESTXB2_MOUSEVIGLN_MOUSE

EIF3B_MOUSEAGAL_MOUSE

SYIC_MOUSESCFD1_MOUSE

SCMC1_MOUSEVASP_MOUSE

EIF3D_MOUSEACSF2_MOUSEDNJA1_MOUSEAHSA1_MOUSE

APMAP_MOUSENDUAA_MOUSE

PGES2_MOUSESEPT9_MOUSE

CRK_MOUSEBAG6_MOUSEPIN1_MOUSE

DAZP1_MOUSEGTF2I_MOUSESDF2L_MOUSE

UB2V1_MOUSETPD54_MOUSE

SSRA_MOUSEGMFB_MOUSEPRP19_MOUSERCC1_MOUSE

NUP85_MOUSEDIAC_MOUSE

MARE2_MOUSEPDCL3_MOUSE

TM209_MOUSERCC2_MOUSE

NRDC_MOUSECLC7A_MOUSELPPRC_MOUSE

TTYH3_MOUSESORCN_MOUSE

PA1B2_MOUSESC31A_MOUSE

PCY1A_MOUSELDHB_MOUSE

TCEA1_MOUSEFYB_MOUSE

RL21_MOUSEAT1A1_MOUSE

ASC_MOUSERGS10_MOUSEVINC_MOUSESBP1_MOUSEGGH_MOUSE

CP062_MOUSERD23B_MOUSE

ACADV_MOUSESYHC_MOUSE

VDAC1_MOUSESMCA5_MOUSE

SIR2_MOUSEENASE_MOUSEFA98B_MOUSESRSF1_MOUSE

ASM3B_MOUSEPRDX3_MOUSEPLOD3_MOUSEMYG1_MOUSEARSK_MOUSEEI2BA_MOUSESYLC_MOUSE

FCGR3_MOUSEDOPD_MOUSE

RENT1_MOUSENPS3B_MOUSE

TOM22_MOUSEEIF2A_MOUSE

CAB39_MOUSEHINT1_MOUSEABCE1_MOUSEUCHL4_MOUSE

ACBP_MOUSEARHG7_MOUSE

PON3_MOUSEWASH7_MOUSE

STAB1_MOUSEPSME3_MOUSE

MANBA_MOUSEDYHC1_MOUSEETUD1_MOUSEPK3CG_MOUSETCRG1_MOUSEPARP1_MOUSEITIH2_MOUSE

NOMO1_MOUSECLCN5_MOUSEVPS52_MOUSE

EXOC5_MOUSEDP13B_MOUSESART3_MOUSE

PEX5_MOUSEPGM2L_MOUSEHP1B3_MOUSE

T2FA_MOUSEADPGK_MOUSE

DDB1_MOUSETMM43_MOUSE

TS101_MOUSESIAE_MOUSE

CSK22_MOUSELMAN1_MOUSEUCHL5_MOUSE

PGP_MOUSEDEOC_MOUSEPEX19_MOUSECTBP1_MOUSEAOAH_MOUSEKAP2_MOUSE

ETHE1_MOUSEADHX_MOUSEIF4E2_MOUSEDRG1_MOUSE

HNRPC_MOUSEDOCK2_MOUSENDUB8_MOUSEC1QBP_MOUSECNPY2_MOUSE

HDHD2_MOUSENFU1_MOUSE

NQO2_MOUSELKHA4_MOUSE

EDF1_MOUSENDUS8_MOUSERAB18_MOUSERS15A_MOUSE

RS21_MOUSE

DMSO + LPSversus DMSO

10

0

minus10

log10(ratio)

RGFP966 + LPS

DMSO + LPSversus

Figure 3 Heat map generated by MeV regarding proteins subjected to RGFP966 and LPS regulation Experimental groups were presentedon the horizontal axis and proteins were on the vertical axis Colors were consistent with protein expression levels red indicated upregulatedratio and green indicated downregulated ratio

6 Neural Plasticity

Biological process Cellular component Molecular function

0

1

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Cel

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r pro

cess

Imm

une s

yste

m p

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ishm

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f loc

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nLo

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atio

nM

ultio

rgan

ism p

roce

ssRe

spon

se to

stim

ulus

Met

abol

ic p

roce

ssD

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Loco

mot

ion

Cel

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r com

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rgan

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Dev

elopm

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l pro

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Cel

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pone

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Biol

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al ad

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Repr

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tion

Mul

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r org

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ical

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Mac

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ular

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Org

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le p

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Enve

lope

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Extr

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Bind

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Cata

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ityEn

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e reg

ulat

or ac

tivity

Stru

ctur

al m

olec

ule a

ctiv

ityEl

ectro

n ca

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r act

ivity

Tran

spor

ter a

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ityTr

ansc

riptio

n re

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tor a

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ityM

olec

ular

tran

sduc

er ac

tivity

(a)

0

1

2

3

4

5

Biological process Cellular component Molecular function

Cel

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r pro

cess

Met

abol

ic p

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tabl

ishm

ent o

f loc

aliz

atio

nLo

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Mul

tiorg

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cess

Resp

onse

to st

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th

Loco

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ion

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r com

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Dev

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une s

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Biol

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Repr

oduc

tive p

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ssRe

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r org

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al p

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ical

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Mac

rom

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ular

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Org

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le p

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lope

Cel

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e-en

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d lu

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Extr

acel

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r reg

ion

part

Extr

acel

lula

r reg

ion

Bind

ing

Cata

lytic

activ

ity

Enzy

me r

egul

ator

activ

itySt

ruct

ural

mol

ecul

e act

ivity

Elec

tron

carr

ier a

ctiv

ity

Tran

spor

ter a

ctiv

ityTr

ansc

riptio

n re

gula

tor a

ctiv

ity

Mol

ecul

ar tr

ansd

ucer

activ

ity

(b)

Figure 4 The GO analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The GO analysis involvedbiological process cellular component and molecular function The vertical axis values equaled minus log(119901)

(272 plusmn 013 in DMSO + LPS versus 100 plusmn 007 in DMSO119901 lt 0001 145 plusmn 015 in RGFP966 + LPS versus 272 plusmn 013in DMSO + LPS 119901 lt 0001) were all significantly increasedat the stimulation time of 12 hours and decreased with thetreatment of RGFP966 In our study though there was nosignificant change seen in total MAPK p38 the protein levelof phospho-p38 (331 plusmn 040 in DMSO + LPS versus 100 plusmn023 in DMSO 119901 lt 0001 256 plusmn 058 in RGFP966 + LPSversus 331 plusmn 040 in DMSO + LPS 119901 = 0021) showed a

similar trend as the proteins mentioned above TLR3 (634 plusmn033 in DMSO + LPS versus 100 plusmn 115 in DMSO 119901 = 0003196 plusmn 093 in RGFP966 + LPS versus 634 plusmn 033 in DMSO+ LPS 119901 = 0011) an exceptional protein was significantlyaggregated after LPS stimulation and reduced by RGFP966 at24 hours

37 RGFP966 Inhibited LPS-Induced Microglia ActivationWe further verified the effect of HDAC3 inhibition on

Neural Plasticity 7

1

Pathways in cancerCalcium signaling pathway

Huntingtonrsquos diseaseInsulin signaling pathway

Cell cycleApoptosis

RIG-I-like receptor signaling pathwaySpliceosome

Cytosolic DNA-sensing pathwayAlzheimerrsquos disease

Toll-like receptor signaling pathway

15 20 05(a)

1 150 05Pathways in cancerAlzheimerrsquos disease

Chemokine signaling pathwayOxidative phosphorylation

Regulation of actin cytoskeletonLeukocyte transendothelial migration

Focal adhesionHuntingtonrsquos disease

RibosomeChronic myeloid leukemiaInsulin signaling pathway

Parkinsonrsquos diseaseSpliceosome

mTOR signaling pathwayFc gamma R-mediated phagocytosis

Aminoacyl-tRNA biosynthesis

(b)

Figure 5 The KEGG pathway analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The horizontalaxis values equaled minus log(119901)

functional microglia activation We first stained primarycultured microglia with CD1632 as shown in Figure 8(a)microglia demonstrated bipolar shape in intact status whilein LPS stimulation group microglia enlarged and grew lotsof branches The fluorescence intensity of CD1632 was sig-nificantly decreased in LPS + RGFP966-treated group (Fig-ure 8(c)) We then detected classical inflammatory cytokinesin the supernatants of cultured cells exposed to LPS for 1 h2 h 3 h 8 h 12 h and 24 h with or without RGFP966 Weobserved profound inhibition of IL-6 (Figure 8(d)) (24 h15230 plusmn 5213 pgmL in RGFP966 + LPS versus 25538 plusmn8741 pgmL in DMSO + LPS 119901 = 0012) and TNF-120572(Figure 8(e)) (12 h 5189 plusmn 1613 pgmL in RGFP966 + LPSversus 17241 plusmn 4716 pgmL in DMSO + LPS 119901 lt 000124 h 7332 plusmn 2436 pgmL in RGFP966 + LPS versus 20977 plusmn1091 pgmL in DMSO + LPS 119901 lt 0001) secretion byRGFP966 Additionally STAT3 and STAT5 were two funda-mental signaling pathways governing inflammatory responsein various neurological diseases [17] In order to found out themechanism responsible for its anti-inflammatory responses

we detected phosphorylation levels of STAT3 and STAT5As shown in Figure 8(b) phosphorylation of STAT3 andSTAT5 increased 2 h after LPS treatment and declined quickly4 h after LPS RGFP966 inhibited not only phosphorylationof STAT3 and STAT5 but also decreased STAT5 expressionlevel whichmay partially contribute to its anti-inflammatoryresponse

4 Discussion

In summary we analyzed the differentially expressed proteinsof LPS-stimulated primary microglia with the treatmentof RGFP966 by proteomics A total of 168 LPS-inducedproteins were significantly reduced by RGFP966 in thisstudy The altered proteins mainly concentrated on thefollowing categories Toll-like receptor signaling pathwayAlzheimerrsquos disease cytosolic DNA-sensing pathway andfunctioned in cellular process immune system process andorganelle For Alzheimerrsquos disease preliminary evidencesdemonstrated inhibition of HDAC3 enzyme in neurons

8 Neural Plasticity

DMSORGFP966

TLR2 mRNA

Vehicle LPS0

5

10

15lowastlowast

lowastlowast(fo

ld o

f con

trol)

Relat

ive m

RNA

leve

l

(a)

DMSORGFP966

TLR3 mRNA

Vehicle LPS

lowastlowast

lowastlowast

0

10

20

30

40

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(b)

DMSORGFP966

TLR6 mRNA

Vehicle LPS

lowastlowast

lowastlowast

00

05

10

15

20

25

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(c)

DMSORGFP966

CD36 mRNA

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(d)

Figure 6 RT-PCR analysis of TLR2 TLR3 TLR6 and CD36 in four experimental groups The mRNA levels of TLR2 (a) TLR3 (b) TLR6(c) and CD36 (d) were significantly reduced by RGFP966 at the stimulation time of 6 hours lowastlowast119901 lt 001119873 = 3 repeats

prevented amyloid-beta oligomer-induced synaptic plasticityimpairments [18] and enhanced memory process [15] ForDNA-sensing pathways HDAC3 was thought to participatein IRF3IFN-120573 signaling pathways [13] In this study wemainly analyzed the differentially expressed TLR relative pro-teins of LPS-stimulated primarymicroglia with the treatmentof RGFP966 Toll-like receptor signaling pathway played animportant role in the regulation of inflammation In thisregard we selected TLR2 TLR3 TLR6 CD36 and SYK forfurther validation and found that they were all significantlyupregulated after LPS stimulation and downregulated inthe presence of RGFP966 Additionally morphological andfunctional alterations of microglia exposure to LPS werealso relieved by RGFP966 Our data provided a hint thatHDAC3 inhibitor may be a potential therapeutic target

combating microglia activation and inflammatory responsein neurological diseases

TLRs are a family of type I transmembrane recep-tors which share the multiple leucine-rich repeats (LRRs)domains in the extracellular space and the similar cytosolicdomains of the interleukin-1 (IL-1) receptors [19] TLRsare considered to recognize the distinct pathogen-associatedmolecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) which then drivea cascade of inflammatory signaling and converge at tran-scription factors nuclear factor-120581B (NF-120581B) [20] MAPK[21 22] and JAKSTAT pathways [23] In addition to TLR236 TLR coreceptorCD36 and regulatory protein SYKwereall decreased by RGFP966 Spleen tyrosine kinase (SYK) anonreceptor tyrosine kinase is typically regarded as a vital

Neural Plasticity 9

GAPDH

TLR2

DMSO RGFP966DMSO+ LPS

RGFP966+ LPS DMSO RGFP966

DMSO+ LPS

RGFP966+ LPS

TLR3

GAPDH

TLR6

GAPDH

CD36

GAPDH

phospho-p38

GAPDH

p38

SYK

GAPDH

(a)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR2 Protein level

0

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DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

(b)

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ive p

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in b

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(fold

of c

ontro

l)TLR3 Protein level

0

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DMSORGFP966

Vehicle LPS

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(c)

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ive p

rote

in b

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(fold

of c

ontro

l)

TLR6 Protein level

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

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(d)

Relat

ive p

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in b

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(fold

of c

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l)

CD36 Protein level

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DMSORGFP966

Vehicle LPS

lowastlowast

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(e)

Figure 7 Continued

10 Neural Plasticity

Relat

ive p

rote

in b

and

inte

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(fold

of c

ontro

l)phospho-p38 Protein level

0

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DMSORGFP966

Vehicle LPS

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(f)

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(fold

of c

ontro

l)

SYK Protein level

0

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DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

(g)

Figure 7Western blot analysis of TLR2 TLR3 TLR6MAPKp38 phospho-p38 SYK andCD36 in four experimental groups (a)The proteinlevels of TLR2 (b) TLR6 (d) SYK (g) phospho-p38 (f) and CD36 (e) were significantly altered by RGFP966 after 12 hoursrsquo stimulation of LPSwhile TLR3 was at 24 hours There was no significant difference of MAPK p38 protein expression among four groups lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

regulator in adaptive immunity [24] It consists of two tandemSH2 domains and a C-terminal tyrosine kinase domain SH2domains selectively bind to the phosphorylated immunore-ceptor tyrosine-based activation motifs (ITAMs) of immunereceptors such as MyD88 TRAF6 and TRIF transmittinginitiative signals to downstream pathways SYK is found to befundamental for Toll-like receptor signaling pathway and theinhibition of SYK suppresses the release of proinflammatorycytokines [25 26] In this study we detected alterations ofSYK in protein level but not mRNA level We surmisedthat posttranscriptional modification may also contributeto HDAC3rsquos functions Preliminary evidences demonstratethat HDAC family played a role in protein degradation[27] while inadequate time points of mRNA detection couldalso be an explanation for this discrepancy CD36 is a TLRcoreceptor playing a pivotal role in inflammatory responsesinitiated by TLRs [28] Previous researches reported thatCD36 contributed to the recognition of diacylglycerol ligandsby forming CD36-CD14-TLR2-TLR6 complex thus it con-trolled gram-positive bacterial infectionWhat ismore CD36was found to be involved in the formation of TLR4-TLR6heterodimers which induced the production of proinflam-matory cytokines nitric oxide and reactive oxygen speciesin response to endogenous ligands [29] Overall RGFP966demonstrated profound inhibitory effects on TLR signalingpathway and proinflammatory cytokines including IL-6 andTNF-120572

HDAC inhibitors have been reported to interfere withthe activation of the mitogen-activated protein kinasesSTAT1 AP-1 or NF-120581B signal transduction pathways [30

31] Here we observed transient STAT3 and STAT5 acti-vation at 2 hours after LPS stimulation which was aheadof the alteration of IL-6 and TNF-120572 Exposed to inflam-matory stimulation STAT35 was phosphorylated by JAKsleading to transcriptional activation Activation of STATselicited the expression of acute-phase proteins as well asa number of cytokines and chemokines including IL-6and TNF-120572 [17] Our study was in keeping with previousstudy inHDAC3 knockoutmacrophage which demonstratedthat HDAC3 knockout impaired STAT1STAT3STAT5 path-ways [13] Figure 9 presented a diagram showing theprotein and protein interactions among proteins Brieflyinitial change of STAT3STAT5 or NF-120581B [5] caused byHDAC3i may further elicit later alterations of TLR sig-naling pathway and proinflammatory cytokines includingIL-6 and TNF-120572 The underlying mechanism for HDAC3in the regulation of STAT3STAT5 is unknown Some evi-dences from Pan-HDAC inhibitors indicated that promoter-associated histone acetylation of SOCS1 and SOCS3 caused byHDACi may further downregulated JAK2STAT3 signaling[32]

In conclusions we identified TLR signaling pathwaymicroglia activation and STAT35 pathway which wereinhibited by RGFP966 Identification of these changes mayprovide valuable clues for the future applications of selectiveHDAC3 inhibitor in the clinic

Competing Interests

The authors declare no conflict of interests

Neural Plasticity 11

DMSO RGFP966

DMSO + LPS RGFP966 + LPS

(a)

p-STAT3

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DMSO + LPS RGFP966 + LPS

STAT5

DMSO RGFP966 1h 2h 4h 1h 2h 4h

(b)

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Vehicle LPS

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1 2 3 8 12 240(h)

(e)

Figure 8 RGFP966 inhibited LPS-inducedmicroglia activation (a) Representativemicrophotographs of primarymicroglia cultures 24 hoursafter LPS and RGFP966 treatmentThe cultures were stained with CD1632 a marker of activatedmicroglia Scale bar = 10120583m (b) Expressionand phosphorylation of STAT3 and STAT5 in microglia pretreated with RGFP966 (c) Quantitative analysis of fluorescence intensity ofCD1632 All the photographs used for quantitative analysis were taken under the same exposure time and data were expressed as fluorescenceintensity per cell of randomly selected regions lowastlowast119901 lt 001119873 = 3 repeats Supernatant concentration of IL-6 (d) and TNF-120572 (e) detected atvarious stimulation times lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 5: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

Neural Plasticity 5

RSAD2_MOUSEIFIT2_MOUSEIL1B_MOUSE

SAP18_MOUSEIL1A_MOUSE

TMED5_MOUSERL30_MOUSE

MMP13_MOUSECKLF3_MOUSE

TRABD_MOUSETMCO1_MOUSE

NOS2_MOUSES15A3_MOUSERIPL2_MOUSE

COMD9_MOUSEHSC20_MOUSETIM50_MOUSEGMDS_MOUSEMECR_MOUSE

ADAP2_MOUSERM10_MOUSE

APOOL_MOUSECUTC_MOUSESTXB3_MOUSEGORS2_MOUSENNRD_MOUSECOG2_MOUSE

COASY_MOUSEH1BP3_MOUSEILVBL_MOUSE

PKHA2_MOUSEKHDR1_MOUSEMMP12_MOUSE

TOIP2_MOUSETGAP1_MOUSEPCAT1_MOUSEABCF2_MOUSE

LOX5_MOUSESCFD2_MOUSE

TAP2_MOUSEACSL4_MOUSE

CNNM3_MOUSEMD1L1_MOUSEBMP2K_MOUSEBICD2_MOUSE

EPS8_MOUSEVPS53_MOUSETLR3_MOUSENRP2_MOUSE

NAT10_MOUSEDIAP2_MOUSEAT8A1_MOUSEPI3R4_MOUSEU520_MOUSE

MCM7_MOUSECO3_MOUSE

SPCS2_MOUSE3BP1_MOUSE

AIMP1_MOUSETLR2_MOUSE

NUBP2_MOUSERM12_MOUSE

UN45A_MOUSERAB31_MOUSEC2D1B_MOUSETOR4A_MOUSERASL2_MOUSEVPS41_MOUSEPRAF3_MOUSE

STRUM_MOUSESWP70_MOUSE

ZPR1_MOUSECD47_MOUSE

SAR1A_MOUSEGTPB1_MOUSERBM25_MOUSENSUN2_MOUSE

LA_MOUSETHMS2_MOUSE

SH3L3_MOUSE2ABA_MOUSEPSB8_MOUSE

TWF2_MOUSECBL_MOUSE

FRIH_MOUSEUSO1_MOUSE

CASP8_MOUSECOMDA_MOUSE

KCMF1_MOUSEHOOK2_MOUSE

FKB15_MOUSEBAG1_MOUSE

ABRAL_MOUSEPDE12_MOUSESTING_MOUSE

SPB8_MOUSELYPA2_MOUSEPEX14_MOUSE

STAP1_MOUSECLC6A_MOUSE

ROA0_MOUSEPPP6_MOUSE

RBM47_MOUSEMA2C1_MOUSENUPL1_MOUSE

BPHL_MOUSEBRE_MOUSE

UFL1_MOUSETHOP1_MOUSEKANK2_MOUSE

SPS1_MOUSECCD93_MOUSESPAG7_MOUSEPON2_MOUSETR150_MOUSE

NCBP1_MOUSEMCCB_MOUSETTL12_MOUSE

IF2P_MOUSEASM_MOUSEIF4E_MOUSE

COPZ1_MOUSEPFD3_MOUSE

TBCA_MOUSERAGP1_MOUSECX6A1_MOUSEFMR1_MOUSEUMPS_MOUSE

GOSR1_MOUSEPPT2_MOUSEIMA7_MOUSEIMA3_MOUSEIMA4_MOUSE

VMA5A_MOUSEIF4H_MOUSE

ACADS_MOUSEKSYK_MOUSE

CELF2_MOUSETBCD_MOUSE

PIGS_MOUSEADAS_MOUSETITIN_MOUSECALM_MOUSEDC1I2_MOUSE

RL23_MOUSECOPG2_MOUSE

ILK_MOUSESF3B1_MOUSEISOC1_MOUSESGTA_MOUSE

HCFC1_MOUSEHMHA1_MOUSE

EMC8_MOUSEGSH0_MOUSE

MCTS1_MOUSEIF4B_MOUSEVIP2_MOUSEFXR1_MOUSEASNS_MOUSE

LC7L3_MOUSENIBAN_MOUSEMCM6_MOUSE

IRF3_MOUSEIF5A1_MOUSE

GNAQ_MOUSENU4M_MOUSE

GAK_MOUSESTK10_MOUSESTXB2_MOUSEVIGLN_MOUSE

EIF3B_MOUSEAGAL_MOUSE

SYIC_MOUSESCFD1_MOUSE

SCMC1_MOUSEVASP_MOUSE

EIF3D_MOUSEACSF2_MOUSEDNJA1_MOUSEAHSA1_MOUSE

APMAP_MOUSENDUAA_MOUSE

PGES2_MOUSESEPT9_MOUSE

CRK_MOUSEBAG6_MOUSEPIN1_MOUSE

DAZP1_MOUSEGTF2I_MOUSESDF2L_MOUSE

UB2V1_MOUSETPD54_MOUSE

SSRA_MOUSEGMFB_MOUSEPRP19_MOUSERCC1_MOUSE

NUP85_MOUSEDIAC_MOUSE

MARE2_MOUSEPDCL3_MOUSE

TM209_MOUSERCC2_MOUSE

NRDC_MOUSECLC7A_MOUSELPPRC_MOUSE

TTYH3_MOUSESORCN_MOUSE

PA1B2_MOUSESC31A_MOUSE

PCY1A_MOUSELDHB_MOUSE

TCEA1_MOUSEFYB_MOUSE

RL21_MOUSEAT1A1_MOUSE

ASC_MOUSERGS10_MOUSEVINC_MOUSESBP1_MOUSEGGH_MOUSE

CP062_MOUSERD23B_MOUSE

ACADV_MOUSESYHC_MOUSE

VDAC1_MOUSESMCA5_MOUSE

SIR2_MOUSEENASE_MOUSEFA98B_MOUSESRSF1_MOUSE

ASM3B_MOUSEPRDX3_MOUSEPLOD3_MOUSEMYG1_MOUSEARSK_MOUSEEI2BA_MOUSESYLC_MOUSE

FCGR3_MOUSEDOPD_MOUSE

RENT1_MOUSENPS3B_MOUSE

TOM22_MOUSEEIF2A_MOUSE

CAB39_MOUSEHINT1_MOUSEABCE1_MOUSEUCHL4_MOUSE

ACBP_MOUSEARHG7_MOUSE

PON3_MOUSEWASH7_MOUSE

STAB1_MOUSEPSME3_MOUSE

MANBA_MOUSEDYHC1_MOUSEETUD1_MOUSEPK3CG_MOUSETCRG1_MOUSEPARP1_MOUSEITIH2_MOUSE

NOMO1_MOUSECLCN5_MOUSEVPS52_MOUSE

EXOC5_MOUSEDP13B_MOUSESART3_MOUSE

PEX5_MOUSEPGM2L_MOUSEHP1B3_MOUSE

T2FA_MOUSEADPGK_MOUSE

DDB1_MOUSETMM43_MOUSE

TS101_MOUSESIAE_MOUSE

CSK22_MOUSELMAN1_MOUSEUCHL5_MOUSE

PGP_MOUSEDEOC_MOUSEPEX19_MOUSECTBP1_MOUSEAOAH_MOUSEKAP2_MOUSE

ETHE1_MOUSEADHX_MOUSEIF4E2_MOUSEDRG1_MOUSE

HNRPC_MOUSEDOCK2_MOUSENDUB8_MOUSEC1QBP_MOUSECNPY2_MOUSE

HDHD2_MOUSENFU1_MOUSE

NQO2_MOUSELKHA4_MOUSE

EDF1_MOUSENDUS8_MOUSERAB18_MOUSERS15A_MOUSE

RS21_MOUSE

DMSO + LPSversus DMSO

10

0

minus10

log10(ratio)

RGFP966 + LPS

DMSO + LPSversus

Figure 3 Heat map generated by MeV regarding proteins subjected to RGFP966 and LPS regulation Experimental groups were presentedon the horizontal axis and proteins were on the vertical axis Colors were consistent with protein expression levels red indicated upregulatedratio and green indicated downregulated ratio

6 Neural Plasticity

Biological process Cellular component Molecular function

0

1

2

3

4

5

6

7

Cel

lula

r pro

cess

Imm

une s

yste

m p

roce

ssEs

tabl

ishm

ent o

f loc

aliz

atio

nLo

caliz

atio

nM

ultio

rgan

ism p

roce

ssRe

spon

se to

stim

ulus

Met

abol

ic p

roce

ssD

eath

Loco

mot

ion

Cel

lula

r com

pone

nt o

rgan

izat

ion

Dev

elopm

enta

l pro

cess

Cel

lula

r com

pone

nt b

ioge

nesis

Biol

ogic

al ad

hesio

nRe

prod

uctiv

e pro

cess

Repr

oduc

tion

Mul

ticel

lula

r org

anism

al p

roce

ssBi

olog

ical

regu

latio

nO

rgan

elle

Mac

rom

olec

ular

com

plex

Org

anel

le p

art

Enve

lope

Cel

l par

tC

ell

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

part

Extr

acel

lula

r reg

ion

Bind

ing

Cata

lytic

activ

ityEn

zym

e reg

ulat

or ac

tivity

Stru

ctur

al m

olec

ule a

ctiv

ityEl

ectro

n ca

rrie

r act

ivity

Tran

spor

ter a

ctiv

ityTr

ansc

riptio

n re

gula

tor a

ctiv

ityM

olec

ular

tran

sduc

er ac

tivity

(a)

0

1

2

3

4

5

Biological process Cellular component Molecular function

Cel

lula

r pro

cess

Met

abol

ic p

roce

ssEs

tabl

ishm

ent o

f loc

aliz

atio

nLo

caliz

atio

n

Mul

tiorg

anism

pro

cess

Resp

onse

to st

imul

us

Dea

th

Loco

mot

ion

Cel

lula

r com

pone

nt o

rgan

izat

ion

Dev

elopm

enta

l pro

cess

Imm

une s

yste

m p

roce

ss

Cel

lula

r com

pone

nt b

ioge

nesis

Biol

ogic

al ad

hesio

n

Repr

oduc

tive p

roce

ssRe

prod

uctio

n

Mul

ticel

lula

r org

anism

al p

roce

ssBi

olog

ical

regu

latio

nO

rgan

elle

Mac

rom

olec

ular

com

plex

Org

anel

le p

art

Enve

lope

Cel

l par

tC

ell

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

part

Extr

acel

lula

r reg

ion

Bind

ing

Cata

lytic

activ

ity

Enzy

me r

egul

ator

activ

itySt

ruct

ural

mol

ecul

e act

ivity

Elec

tron

carr

ier a

ctiv

ity

Tran

spor

ter a

ctiv

ityTr

ansc

riptio

n re

gula

tor a

ctiv

ity

Mol

ecul

ar tr

ansd

ucer

activ

ity

(b)

Figure 4 The GO analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The GO analysis involvedbiological process cellular component and molecular function The vertical axis values equaled minus log(119901)

(272 plusmn 013 in DMSO + LPS versus 100 plusmn 007 in DMSO119901 lt 0001 145 plusmn 015 in RGFP966 + LPS versus 272 plusmn 013in DMSO + LPS 119901 lt 0001) were all significantly increasedat the stimulation time of 12 hours and decreased with thetreatment of RGFP966 In our study though there was nosignificant change seen in total MAPK p38 the protein levelof phospho-p38 (331 plusmn 040 in DMSO + LPS versus 100 plusmn023 in DMSO 119901 lt 0001 256 plusmn 058 in RGFP966 + LPSversus 331 plusmn 040 in DMSO + LPS 119901 = 0021) showed a

similar trend as the proteins mentioned above TLR3 (634 plusmn033 in DMSO + LPS versus 100 plusmn 115 in DMSO 119901 = 0003196 plusmn 093 in RGFP966 + LPS versus 634 plusmn 033 in DMSO+ LPS 119901 = 0011) an exceptional protein was significantlyaggregated after LPS stimulation and reduced by RGFP966 at24 hours

37 RGFP966 Inhibited LPS-Induced Microglia ActivationWe further verified the effect of HDAC3 inhibition on

Neural Plasticity 7

1

Pathways in cancerCalcium signaling pathway

Huntingtonrsquos diseaseInsulin signaling pathway

Cell cycleApoptosis

RIG-I-like receptor signaling pathwaySpliceosome

Cytosolic DNA-sensing pathwayAlzheimerrsquos disease

Toll-like receptor signaling pathway

15 20 05(a)

1 150 05Pathways in cancerAlzheimerrsquos disease

Chemokine signaling pathwayOxidative phosphorylation

Regulation of actin cytoskeletonLeukocyte transendothelial migration

Focal adhesionHuntingtonrsquos disease

RibosomeChronic myeloid leukemiaInsulin signaling pathway

Parkinsonrsquos diseaseSpliceosome

mTOR signaling pathwayFc gamma R-mediated phagocytosis

Aminoacyl-tRNA biosynthesis

(b)

Figure 5 The KEGG pathway analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The horizontalaxis values equaled minus log(119901)

functional microglia activation We first stained primarycultured microglia with CD1632 as shown in Figure 8(a)microglia demonstrated bipolar shape in intact status whilein LPS stimulation group microglia enlarged and grew lotsof branches The fluorescence intensity of CD1632 was sig-nificantly decreased in LPS + RGFP966-treated group (Fig-ure 8(c)) We then detected classical inflammatory cytokinesin the supernatants of cultured cells exposed to LPS for 1 h2 h 3 h 8 h 12 h and 24 h with or without RGFP966 Weobserved profound inhibition of IL-6 (Figure 8(d)) (24 h15230 plusmn 5213 pgmL in RGFP966 + LPS versus 25538 plusmn8741 pgmL in DMSO + LPS 119901 = 0012) and TNF-120572(Figure 8(e)) (12 h 5189 plusmn 1613 pgmL in RGFP966 + LPSversus 17241 plusmn 4716 pgmL in DMSO + LPS 119901 lt 000124 h 7332 plusmn 2436 pgmL in RGFP966 + LPS versus 20977 plusmn1091 pgmL in DMSO + LPS 119901 lt 0001) secretion byRGFP966 Additionally STAT3 and STAT5 were two funda-mental signaling pathways governing inflammatory responsein various neurological diseases [17] In order to found out themechanism responsible for its anti-inflammatory responses

we detected phosphorylation levels of STAT3 and STAT5As shown in Figure 8(b) phosphorylation of STAT3 andSTAT5 increased 2 h after LPS treatment and declined quickly4 h after LPS RGFP966 inhibited not only phosphorylationof STAT3 and STAT5 but also decreased STAT5 expressionlevel whichmay partially contribute to its anti-inflammatoryresponse

4 Discussion

In summary we analyzed the differentially expressed proteinsof LPS-stimulated primary microglia with the treatmentof RGFP966 by proteomics A total of 168 LPS-inducedproteins were significantly reduced by RGFP966 in thisstudy The altered proteins mainly concentrated on thefollowing categories Toll-like receptor signaling pathwayAlzheimerrsquos disease cytosolic DNA-sensing pathway andfunctioned in cellular process immune system process andorganelle For Alzheimerrsquos disease preliminary evidencesdemonstrated inhibition of HDAC3 enzyme in neurons

8 Neural Plasticity

DMSORGFP966

TLR2 mRNA

Vehicle LPS0

5

10

15lowastlowast

lowastlowast(fo

ld o

f con

trol)

Relat

ive m

RNA

leve

l

(a)

DMSORGFP966

TLR3 mRNA

Vehicle LPS

lowastlowast

lowastlowast

0

10

20

30

40

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(b)

DMSORGFP966

TLR6 mRNA

Vehicle LPS

lowastlowast

lowastlowast

00

05

10

15

20

25

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(c)

DMSORGFP966

CD36 mRNA

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(d)

Figure 6 RT-PCR analysis of TLR2 TLR3 TLR6 and CD36 in four experimental groups The mRNA levels of TLR2 (a) TLR3 (b) TLR6(c) and CD36 (d) were significantly reduced by RGFP966 at the stimulation time of 6 hours lowastlowast119901 lt 001119873 = 3 repeats

prevented amyloid-beta oligomer-induced synaptic plasticityimpairments [18] and enhanced memory process [15] ForDNA-sensing pathways HDAC3 was thought to participatein IRF3IFN-120573 signaling pathways [13] In this study wemainly analyzed the differentially expressed TLR relative pro-teins of LPS-stimulated primarymicroglia with the treatmentof RGFP966 Toll-like receptor signaling pathway played animportant role in the regulation of inflammation In thisregard we selected TLR2 TLR3 TLR6 CD36 and SYK forfurther validation and found that they were all significantlyupregulated after LPS stimulation and downregulated inthe presence of RGFP966 Additionally morphological andfunctional alterations of microglia exposure to LPS werealso relieved by RGFP966 Our data provided a hint thatHDAC3 inhibitor may be a potential therapeutic target

combating microglia activation and inflammatory responsein neurological diseases

TLRs are a family of type I transmembrane recep-tors which share the multiple leucine-rich repeats (LRRs)domains in the extracellular space and the similar cytosolicdomains of the interleukin-1 (IL-1) receptors [19] TLRsare considered to recognize the distinct pathogen-associatedmolecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) which then drivea cascade of inflammatory signaling and converge at tran-scription factors nuclear factor-120581B (NF-120581B) [20] MAPK[21 22] and JAKSTAT pathways [23] In addition to TLR236 TLR coreceptorCD36 and regulatory protein SYKwereall decreased by RGFP966 Spleen tyrosine kinase (SYK) anonreceptor tyrosine kinase is typically regarded as a vital

Neural Plasticity 9

GAPDH

TLR2

DMSO RGFP966DMSO+ LPS

RGFP966+ LPS DMSO RGFP966

DMSO+ LPS

RGFP966+ LPS

TLR3

GAPDH

TLR6

GAPDH

CD36

GAPDH

phospho-p38

GAPDH

p38

SYK

GAPDH

(a)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR2 Protein level

0

2

4

6

8

10

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

(b)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)TLR3 Protein level

0

2

4

6

8

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(c)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR6 Protein level

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(d)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

CD36 Protein level

0

1

2

3

4

5

DMSORGFP966

Vehicle LPS

lowastlowast

lowastlowast

(e)

Figure 7 Continued

10 Neural Plasticity

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)phospho-p38 Protein level

0

1

2

3

4

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(f)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

SYK Protein level

0

1

2

3

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

(g)

Figure 7Western blot analysis of TLR2 TLR3 TLR6MAPKp38 phospho-p38 SYK andCD36 in four experimental groups (a)The proteinlevels of TLR2 (b) TLR6 (d) SYK (g) phospho-p38 (f) and CD36 (e) were significantly altered by RGFP966 after 12 hoursrsquo stimulation of LPSwhile TLR3 was at 24 hours There was no significant difference of MAPK p38 protein expression among four groups lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

regulator in adaptive immunity [24] It consists of two tandemSH2 domains and a C-terminal tyrosine kinase domain SH2domains selectively bind to the phosphorylated immunore-ceptor tyrosine-based activation motifs (ITAMs) of immunereceptors such as MyD88 TRAF6 and TRIF transmittinginitiative signals to downstream pathways SYK is found to befundamental for Toll-like receptor signaling pathway and theinhibition of SYK suppresses the release of proinflammatorycytokines [25 26] In this study we detected alterations ofSYK in protein level but not mRNA level We surmisedthat posttranscriptional modification may also contributeto HDAC3rsquos functions Preliminary evidences demonstratethat HDAC family played a role in protein degradation[27] while inadequate time points of mRNA detection couldalso be an explanation for this discrepancy CD36 is a TLRcoreceptor playing a pivotal role in inflammatory responsesinitiated by TLRs [28] Previous researches reported thatCD36 contributed to the recognition of diacylglycerol ligandsby forming CD36-CD14-TLR2-TLR6 complex thus it con-trolled gram-positive bacterial infectionWhat ismore CD36was found to be involved in the formation of TLR4-TLR6heterodimers which induced the production of proinflam-matory cytokines nitric oxide and reactive oxygen speciesin response to endogenous ligands [29] Overall RGFP966demonstrated profound inhibitory effects on TLR signalingpathway and proinflammatory cytokines including IL-6 andTNF-120572

HDAC inhibitors have been reported to interfere withthe activation of the mitogen-activated protein kinasesSTAT1 AP-1 or NF-120581B signal transduction pathways [30

31] Here we observed transient STAT3 and STAT5 acti-vation at 2 hours after LPS stimulation which was aheadof the alteration of IL-6 and TNF-120572 Exposed to inflam-matory stimulation STAT35 was phosphorylated by JAKsleading to transcriptional activation Activation of STATselicited the expression of acute-phase proteins as well asa number of cytokines and chemokines including IL-6and TNF-120572 [17] Our study was in keeping with previousstudy inHDAC3 knockoutmacrophage which demonstratedthat HDAC3 knockout impaired STAT1STAT3STAT5 path-ways [13] Figure 9 presented a diagram showing theprotein and protein interactions among proteins Brieflyinitial change of STAT3STAT5 or NF-120581B [5] caused byHDAC3i may further elicit later alterations of TLR sig-naling pathway and proinflammatory cytokines includingIL-6 and TNF-120572 The underlying mechanism for HDAC3in the regulation of STAT3STAT5 is unknown Some evi-dences from Pan-HDAC inhibitors indicated that promoter-associated histone acetylation of SOCS1 and SOCS3 caused byHDACi may further downregulated JAK2STAT3 signaling[32]

In conclusions we identified TLR signaling pathwaymicroglia activation and STAT35 pathway which wereinhibited by RGFP966 Identification of these changes mayprovide valuable clues for the future applications of selectiveHDAC3 inhibitor in the clinic

Competing Interests

The authors declare no conflict of interests

Neural Plasticity 11

DMSO RGFP966

DMSO + LPS RGFP966 + LPS

(a)

p-STAT3

STAT3

p-STAT5

GAPDH

GAPDH

DMSO + LPS RGFP966 + LPS

STAT5

DMSO RGFP966 1h 2h 4h 1h 2h 4h

(b)

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

0

20000

40000

60000

80000

100000

Fluo

resc

ence

inte

nsity

of C

D16

32

(c)

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

0200400600800

100001500020000250003000035000 IL-6

LPSLPS + RGFP966

lowast

1 2 3 8 12 240(h)

(d)

TNF-120572

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

050

100

8000

3000

13000

18000

23000

LPSLPS + RGFP966

lowastlowastlowastlowast

1 2 3 8 12 240(h)

(e)

Figure 8 RGFP966 inhibited LPS-inducedmicroglia activation (a) Representativemicrophotographs of primarymicroglia cultures 24 hoursafter LPS and RGFP966 treatmentThe cultures were stained with CD1632 a marker of activatedmicroglia Scale bar = 10120583m (b) Expressionand phosphorylation of STAT3 and STAT5 in microglia pretreated with RGFP966 (c) Quantitative analysis of fluorescence intensity ofCD1632 All the photographs used for quantitative analysis were taken under the same exposure time and data were expressed as fluorescenceintensity per cell of randomly selected regions lowastlowast119901 lt 001119873 = 3 repeats Supernatant concentration of IL-6 (d) and TNF-120572 (e) detected atvarious stimulation times lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 6: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

6 Neural Plasticity

Biological process Cellular component Molecular function

0

1

2

3

4

5

6

7

Cel

lula

r pro

cess

Imm

une s

yste

m p

roce

ssEs

tabl

ishm

ent o

f loc

aliz

atio

nLo

caliz

atio

nM

ultio

rgan

ism p

roce

ssRe

spon

se to

stim

ulus

Met

abol

ic p

roce

ssD

eath

Loco

mot

ion

Cel

lula

r com

pone

nt o

rgan

izat

ion

Dev

elopm

enta

l pro

cess

Cel

lula

r com

pone

nt b

ioge

nesis

Biol

ogic

al ad

hesio

nRe

prod

uctiv

e pro

cess

Repr

oduc

tion

Mul

ticel

lula

r org

anism

al p

roce

ssBi

olog

ical

regu

latio

nO

rgan

elle

Mac

rom

olec

ular

com

plex

Org

anel

le p

art

Enve

lope

Cel

l par

tC

ell

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

part

Extr

acel

lula

r reg

ion

Bind

ing

Cata

lytic

activ

ityEn

zym

e reg

ulat

or ac

tivity

Stru

ctur

al m

olec

ule a

ctiv

ityEl

ectro

n ca

rrie

r act

ivity

Tran

spor

ter a

ctiv

ityTr

ansc

riptio

n re

gula

tor a

ctiv

ityM

olec

ular

tran

sduc

er ac

tivity

(a)

0

1

2

3

4

5

Biological process Cellular component Molecular function

Cel

lula

r pro

cess

Met

abol

ic p

roce

ssEs

tabl

ishm

ent o

f loc

aliz

atio

nLo

caliz

atio

n

Mul

tiorg

anism

pro

cess

Resp

onse

to st

imul

us

Dea

th

Loco

mot

ion

Cel

lula

r com

pone

nt o

rgan

izat

ion

Dev

elopm

enta

l pro

cess

Imm

une s

yste

m p

roce

ss

Cel

lula

r com

pone

nt b

ioge

nesis

Biol

ogic

al ad

hesio

n

Repr

oduc

tive p

roce

ssRe

prod

uctio

n

Mul

ticel

lula

r org

anism

al p

roce

ssBi

olog

ical

regu

latio

nO

rgan

elle

Mac

rom

olec

ular

com

plex

Org

anel

le p

art

Enve

lope

Cel

l par

tC

ell

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

part

Extr

acel

lula

r reg

ion

Bind

ing

Cata

lytic

activ

ity

Enzy

me r

egul

ator

activ

itySt

ruct

ural

mol

ecul

e act

ivity

Elec

tron

carr

ier a

ctiv

ity

Tran

spor

ter a

ctiv

ityTr

ansc

riptio

n re

gula

tor a

ctiv

ity

Mol

ecul

ar tr

ansd

ucer

activ

ity

(b)

Figure 4 The GO analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The GO analysis involvedbiological process cellular component and molecular function The vertical axis values equaled minus log(119901)

(272 plusmn 013 in DMSO + LPS versus 100 plusmn 007 in DMSO119901 lt 0001 145 plusmn 015 in RGFP966 + LPS versus 272 plusmn 013in DMSO + LPS 119901 lt 0001) were all significantly increasedat the stimulation time of 12 hours and decreased with thetreatment of RGFP966 In our study though there was nosignificant change seen in total MAPK p38 the protein levelof phospho-p38 (331 plusmn 040 in DMSO + LPS versus 100 plusmn023 in DMSO 119901 lt 0001 256 plusmn 058 in RGFP966 + LPSversus 331 plusmn 040 in DMSO + LPS 119901 = 0021) showed a

similar trend as the proteins mentioned above TLR3 (634 plusmn033 in DMSO + LPS versus 100 plusmn 115 in DMSO 119901 = 0003196 plusmn 093 in RGFP966 + LPS versus 634 plusmn 033 in DMSO+ LPS 119901 = 0011) an exceptional protein was significantlyaggregated after LPS stimulation and reduced by RGFP966 at24 hours

37 RGFP966 Inhibited LPS-Induced Microglia ActivationWe further verified the effect of HDAC3 inhibition on

Neural Plasticity 7

1

Pathways in cancerCalcium signaling pathway

Huntingtonrsquos diseaseInsulin signaling pathway

Cell cycleApoptosis

RIG-I-like receptor signaling pathwaySpliceosome

Cytosolic DNA-sensing pathwayAlzheimerrsquos disease

Toll-like receptor signaling pathway

15 20 05(a)

1 150 05Pathways in cancerAlzheimerrsquos disease

Chemokine signaling pathwayOxidative phosphorylation

Regulation of actin cytoskeletonLeukocyte transendothelial migration

Focal adhesionHuntingtonrsquos disease

RibosomeChronic myeloid leukemiaInsulin signaling pathway

Parkinsonrsquos diseaseSpliceosome

mTOR signaling pathwayFc gamma R-mediated phagocytosis

Aminoacyl-tRNA biosynthesis

(b)

Figure 5 The KEGG pathway analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The horizontalaxis values equaled minus log(119901)

functional microglia activation We first stained primarycultured microglia with CD1632 as shown in Figure 8(a)microglia demonstrated bipolar shape in intact status whilein LPS stimulation group microglia enlarged and grew lotsof branches The fluorescence intensity of CD1632 was sig-nificantly decreased in LPS + RGFP966-treated group (Fig-ure 8(c)) We then detected classical inflammatory cytokinesin the supernatants of cultured cells exposed to LPS for 1 h2 h 3 h 8 h 12 h and 24 h with or without RGFP966 Weobserved profound inhibition of IL-6 (Figure 8(d)) (24 h15230 plusmn 5213 pgmL in RGFP966 + LPS versus 25538 plusmn8741 pgmL in DMSO + LPS 119901 = 0012) and TNF-120572(Figure 8(e)) (12 h 5189 plusmn 1613 pgmL in RGFP966 + LPSversus 17241 plusmn 4716 pgmL in DMSO + LPS 119901 lt 000124 h 7332 plusmn 2436 pgmL in RGFP966 + LPS versus 20977 plusmn1091 pgmL in DMSO + LPS 119901 lt 0001) secretion byRGFP966 Additionally STAT3 and STAT5 were two funda-mental signaling pathways governing inflammatory responsein various neurological diseases [17] In order to found out themechanism responsible for its anti-inflammatory responses

we detected phosphorylation levels of STAT3 and STAT5As shown in Figure 8(b) phosphorylation of STAT3 andSTAT5 increased 2 h after LPS treatment and declined quickly4 h after LPS RGFP966 inhibited not only phosphorylationof STAT3 and STAT5 but also decreased STAT5 expressionlevel whichmay partially contribute to its anti-inflammatoryresponse

4 Discussion

In summary we analyzed the differentially expressed proteinsof LPS-stimulated primary microglia with the treatmentof RGFP966 by proteomics A total of 168 LPS-inducedproteins were significantly reduced by RGFP966 in thisstudy The altered proteins mainly concentrated on thefollowing categories Toll-like receptor signaling pathwayAlzheimerrsquos disease cytosolic DNA-sensing pathway andfunctioned in cellular process immune system process andorganelle For Alzheimerrsquos disease preliminary evidencesdemonstrated inhibition of HDAC3 enzyme in neurons

8 Neural Plasticity

DMSORGFP966

TLR2 mRNA

Vehicle LPS0

5

10

15lowastlowast

lowastlowast(fo

ld o

f con

trol)

Relat

ive m

RNA

leve

l

(a)

DMSORGFP966

TLR3 mRNA

Vehicle LPS

lowastlowast

lowastlowast

0

10

20

30

40

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(b)

DMSORGFP966

TLR6 mRNA

Vehicle LPS

lowastlowast

lowastlowast

00

05

10

15

20

25

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(c)

DMSORGFP966

CD36 mRNA

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(d)

Figure 6 RT-PCR analysis of TLR2 TLR3 TLR6 and CD36 in four experimental groups The mRNA levels of TLR2 (a) TLR3 (b) TLR6(c) and CD36 (d) were significantly reduced by RGFP966 at the stimulation time of 6 hours lowastlowast119901 lt 001119873 = 3 repeats

prevented amyloid-beta oligomer-induced synaptic plasticityimpairments [18] and enhanced memory process [15] ForDNA-sensing pathways HDAC3 was thought to participatein IRF3IFN-120573 signaling pathways [13] In this study wemainly analyzed the differentially expressed TLR relative pro-teins of LPS-stimulated primarymicroglia with the treatmentof RGFP966 Toll-like receptor signaling pathway played animportant role in the regulation of inflammation In thisregard we selected TLR2 TLR3 TLR6 CD36 and SYK forfurther validation and found that they were all significantlyupregulated after LPS stimulation and downregulated inthe presence of RGFP966 Additionally morphological andfunctional alterations of microglia exposure to LPS werealso relieved by RGFP966 Our data provided a hint thatHDAC3 inhibitor may be a potential therapeutic target

combating microglia activation and inflammatory responsein neurological diseases

TLRs are a family of type I transmembrane recep-tors which share the multiple leucine-rich repeats (LRRs)domains in the extracellular space and the similar cytosolicdomains of the interleukin-1 (IL-1) receptors [19] TLRsare considered to recognize the distinct pathogen-associatedmolecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) which then drivea cascade of inflammatory signaling and converge at tran-scription factors nuclear factor-120581B (NF-120581B) [20] MAPK[21 22] and JAKSTAT pathways [23] In addition to TLR236 TLR coreceptorCD36 and regulatory protein SYKwereall decreased by RGFP966 Spleen tyrosine kinase (SYK) anonreceptor tyrosine kinase is typically regarded as a vital

Neural Plasticity 9

GAPDH

TLR2

DMSO RGFP966DMSO+ LPS

RGFP966+ LPS DMSO RGFP966

DMSO+ LPS

RGFP966+ LPS

TLR3

GAPDH

TLR6

GAPDH

CD36

GAPDH

phospho-p38

GAPDH

p38

SYK

GAPDH

(a)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR2 Protein level

0

2

4

6

8

10

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

(b)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)TLR3 Protein level

0

2

4

6

8

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(c)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR6 Protein level

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(d)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

CD36 Protein level

0

1

2

3

4

5

DMSORGFP966

Vehicle LPS

lowastlowast

lowastlowast

(e)

Figure 7 Continued

10 Neural Plasticity

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)phospho-p38 Protein level

0

1

2

3

4

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(f)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

SYK Protein level

0

1

2

3

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

(g)

Figure 7Western blot analysis of TLR2 TLR3 TLR6MAPKp38 phospho-p38 SYK andCD36 in four experimental groups (a)The proteinlevels of TLR2 (b) TLR6 (d) SYK (g) phospho-p38 (f) and CD36 (e) were significantly altered by RGFP966 after 12 hoursrsquo stimulation of LPSwhile TLR3 was at 24 hours There was no significant difference of MAPK p38 protein expression among four groups lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

regulator in adaptive immunity [24] It consists of two tandemSH2 domains and a C-terminal tyrosine kinase domain SH2domains selectively bind to the phosphorylated immunore-ceptor tyrosine-based activation motifs (ITAMs) of immunereceptors such as MyD88 TRAF6 and TRIF transmittinginitiative signals to downstream pathways SYK is found to befundamental for Toll-like receptor signaling pathway and theinhibition of SYK suppresses the release of proinflammatorycytokines [25 26] In this study we detected alterations ofSYK in protein level but not mRNA level We surmisedthat posttranscriptional modification may also contributeto HDAC3rsquos functions Preliminary evidences demonstratethat HDAC family played a role in protein degradation[27] while inadequate time points of mRNA detection couldalso be an explanation for this discrepancy CD36 is a TLRcoreceptor playing a pivotal role in inflammatory responsesinitiated by TLRs [28] Previous researches reported thatCD36 contributed to the recognition of diacylglycerol ligandsby forming CD36-CD14-TLR2-TLR6 complex thus it con-trolled gram-positive bacterial infectionWhat ismore CD36was found to be involved in the formation of TLR4-TLR6heterodimers which induced the production of proinflam-matory cytokines nitric oxide and reactive oxygen speciesin response to endogenous ligands [29] Overall RGFP966demonstrated profound inhibitory effects on TLR signalingpathway and proinflammatory cytokines including IL-6 andTNF-120572

HDAC inhibitors have been reported to interfere withthe activation of the mitogen-activated protein kinasesSTAT1 AP-1 or NF-120581B signal transduction pathways [30

31] Here we observed transient STAT3 and STAT5 acti-vation at 2 hours after LPS stimulation which was aheadof the alteration of IL-6 and TNF-120572 Exposed to inflam-matory stimulation STAT35 was phosphorylated by JAKsleading to transcriptional activation Activation of STATselicited the expression of acute-phase proteins as well asa number of cytokines and chemokines including IL-6and TNF-120572 [17] Our study was in keeping with previousstudy inHDAC3 knockoutmacrophage which demonstratedthat HDAC3 knockout impaired STAT1STAT3STAT5 path-ways [13] Figure 9 presented a diagram showing theprotein and protein interactions among proteins Brieflyinitial change of STAT3STAT5 or NF-120581B [5] caused byHDAC3i may further elicit later alterations of TLR sig-naling pathway and proinflammatory cytokines includingIL-6 and TNF-120572 The underlying mechanism for HDAC3in the regulation of STAT3STAT5 is unknown Some evi-dences from Pan-HDAC inhibitors indicated that promoter-associated histone acetylation of SOCS1 and SOCS3 caused byHDACi may further downregulated JAK2STAT3 signaling[32]

In conclusions we identified TLR signaling pathwaymicroglia activation and STAT35 pathway which wereinhibited by RGFP966 Identification of these changes mayprovide valuable clues for the future applications of selectiveHDAC3 inhibitor in the clinic

Competing Interests

The authors declare no conflict of interests

Neural Plasticity 11

DMSO RGFP966

DMSO + LPS RGFP966 + LPS

(a)

p-STAT3

STAT3

p-STAT5

GAPDH

GAPDH

DMSO + LPS RGFP966 + LPS

STAT5

DMSO RGFP966 1h 2h 4h 1h 2h 4h

(b)

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

0

20000

40000

60000

80000

100000

Fluo

resc

ence

inte

nsity

of C

D16

32

(c)

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

0200400600800

100001500020000250003000035000 IL-6

LPSLPS + RGFP966

lowast

1 2 3 8 12 240(h)

(d)

TNF-120572

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

050

100

8000

3000

13000

18000

23000

LPSLPS + RGFP966

lowastlowastlowastlowast

1 2 3 8 12 240(h)

(e)

Figure 8 RGFP966 inhibited LPS-inducedmicroglia activation (a) Representativemicrophotographs of primarymicroglia cultures 24 hoursafter LPS and RGFP966 treatmentThe cultures were stained with CD1632 a marker of activatedmicroglia Scale bar = 10120583m (b) Expressionand phosphorylation of STAT3 and STAT5 in microglia pretreated with RGFP966 (c) Quantitative analysis of fluorescence intensity ofCD1632 All the photographs used for quantitative analysis were taken under the same exposure time and data were expressed as fluorescenceintensity per cell of randomly selected regions lowastlowast119901 lt 001119873 = 3 repeats Supernatant concentration of IL-6 (d) and TNF-120572 (e) detected atvarious stimulation times lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 7: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

Neural Plasticity 7

1

Pathways in cancerCalcium signaling pathway

Huntingtonrsquos diseaseInsulin signaling pathway

Cell cycleApoptosis

RIG-I-like receptor signaling pathwaySpliceosome

Cytosolic DNA-sensing pathwayAlzheimerrsquos disease

Toll-like receptor signaling pathway

15 20 05(a)

1 150 05Pathways in cancerAlzheimerrsquos disease

Chemokine signaling pathwayOxidative phosphorylation

Regulation of actin cytoskeletonLeukocyte transendothelial migration

Focal adhesionHuntingtonrsquos disease

RibosomeChronic myeloid leukemiaInsulin signaling pathway

Parkinsonrsquos diseaseSpliceosome

mTOR signaling pathwayFc gamma R-mediated phagocytosis

Aminoacyl-tRNA biosynthesis

(b)

Figure 5 The KEGG pathway analysis of differentially expressed proteins overlapped in Figure 2(a) (a) and Figure 2(b) (b) The horizontalaxis values equaled minus log(119901)

functional microglia activation We first stained primarycultured microglia with CD1632 as shown in Figure 8(a)microglia demonstrated bipolar shape in intact status whilein LPS stimulation group microglia enlarged and grew lotsof branches The fluorescence intensity of CD1632 was sig-nificantly decreased in LPS + RGFP966-treated group (Fig-ure 8(c)) We then detected classical inflammatory cytokinesin the supernatants of cultured cells exposed to LPS for 1 h2 h 3 h 8 h 12 h and 24 h with or without RGFP966 Weobserved profound inhibition of IL-6 (Figure 8(d)) (24 h15230 plusmn 5213 pgmL in RGFP966 + LPS versus 25538 plusmn8741 pgmL in DMSO + LPS 119901 = 0012) and TNF-120572(Figure 8(e)) (12 h 5189 plusmn 1613 pgmL in RGFP966 + LPSversus 17241 plusmn 4716 pgmL in DMSO + LPS 119901 lt 000124 h 7332 plusmn 2436 pgmL in RGFP966 + LPS versus 20977 plusmn1091 pgmL in DMSO + LPS 119901 lt 0001) secretion byRGFP966 Additionally STAT3 and STAT5 were two funda-mental signaling pathways governing inflammatory responsein various neurological diseases [17] In order to found out themechanism responsible for its anti-inflammatory responses

we detected phosphorylation levels of STAT3 and STAT5As shown in Figure 8(b) phosphorylation of STAT3 andSTAT5 increased 2 h after LPS treatment and declined quickly4 h after LPS RGFP966 inhibited not only phosphorylationof STAT3 and STAT5 but also decreased STAT5 expressionlevel whichmay partially contribute to its anti-inflammatoryresponse

4 Discussion

In summary we analyzed the differentially expressed proteinsof LPS-stimulated primary microglia with the treatmentof RGFP966 by proteomics A total of 168 LPS-inducedproteins were significantly reduced by RGFP966 in thisstudy The altered proteins mainly concentrated on thefollowing categories Toll-like receptor signaling pathwayAlzheimerrsquos disease cytosolic DNA-sensing pathway andfunctioned in cellular process immune system process andorganelle For Alzheimerrsquos disease preliminary evidencesdemonstrated inhibition of HDAC3 enzyme in neurons

8 Neural Plasticity

DMSORGFP966

TLR2 mRNA

Vehicle LPS0

5

10

15lowastlowast

lowastlowast(fo

ld o

f con

trol)

Relat

ive m

RNA

leve

l

(a)

DMSORGFP966

TLR3 mRNA

Vehicle LPS

lowastlowast

lowastlowast

0

10

20

30

40

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(b)

DMSORGFP966

TLR6 mRNA

Vehicle LPS

lowastlowast

lowastlowast

00

05

10

15

20

25

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(c)

DMSORGFP966

CD36 mRNA

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(d)

Figure 6 RT-PCR analysis of TLR2 TLR3 TLR6 and CD36 in four experimental groups The mRNA levels of TLR2 (a) TLR3 (b) TLR6(c) and CD36 (d) were significantly reduced by RGFP966 at the stimulation time of 6 hours lowastlowast119901 lt 001119873 = 3 repeats

prevented amyloid-beta oligomer-induced synaptic plasticityimpairments [18] and enhanced memory process [15] ForDNA-sensing pathways HDAC3 was thought to participatein IRF3IFN-120573 signaling pathways [13] In this study wemainly analyzed the differentially expressed TLR relative pro-teins of LPS-stimulated primarymicroglia with the treatmentof RGFP966 Toll-like receptor signaling pathway played animportant role in the regulation of inflammation In thisregard we selected TLR2 TLR3 TLR6 CD36 and SYK forfurther validation and found that they were all significantlyupregulated after LPS stimulation and downregulated inthe presence of RGFP966 Additionally morphological andfunctional alterations of microglia exposure to LPS werealso relieved by RGFP966 Our data provided a hint thatHDAC3 inhibitor may be a potential therapeutic target

combating microglia activation and inflammatory responsein neurological diseases

TLRs are a family of type I transmembrane recep-tors which share the multiple leucine-rich repeats (LRRs)domains in the extracellular space and the similar cytosolicdomains of the interleukin-1 (IL-1) receptors [19] TLRsare considered to recognize the distinct pathogen-associatedmolecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) which then drivea cascade of inflammatory signaling and converge at tran-scription factors nuclear factor-120581B (NF-120581B) [20] MAPK[21 22] and JAKSTAT pathways [23] In addition to TLR236 TLR coreceptorCD36 and regulatory protein SYKwereall decreased by RGFP966 Spleen tyrosine kinase (SYK) anonreceptor tyrosine kinase is typically regarded as a vital

Neural Plasticity 9

GAPDH

TLR2

DMSO RGFP966DMSO+ LPS

RGFP966+ LPS DMSO RGFP966

DMSO+ LPS

RGFP966+ LPS

TLR3

GAPDH

TLR6

GAPDH

CD36

GAPDH

phospho-p38

GAPDH

p38

SYK

GAPDH

(a)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR2 Protein level

0

2

4

6

8

10

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

(b)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)TLR3 Protein level

0

2

4

6

8

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(c)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR6 Protein level

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(d)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

CD36 Protein level

0

1

2

3

4

5

DMSORGFP966

Vehicle LPS

lowastlowast

lowastlowast

(e)

Figure 7 Continued

10 Neural Plasticity

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)phospho-p38 Protein level

0

1

2

3

4

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(f)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

SYK Protein level

0

1

2

3

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

(g)

Figure 7Western blot analysis of TLR2 TLR3 TLR6MAPKp38 phospho-p38 SYK andCD36 in four experimental groups (a)The proteinlevels of TLR2 (b) TLR6 (d) SYK (g) phospho-p38 (f) and CD36 (e) were significantly altered by RGFP966 after 12 hoursrsquo stimulation of LPSwhile TLR3 was at 24 hours There was no significant difference of MAPK p38 protein expression among four groups lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

regulator in adaptive immunity [24] It consists of two tandemSH2 domains and a C-terminal tyrosine kinase domain SH2domains selectively bind to the phosphorylated immunore-ceptor tyrosine-based activation motifs (ITAMs) of immunereceptors such as MyD88 TRAF6 and TRIF transmittinginitiative signals to downstream pathways SYK is found to befundamental for Toll-like receptor signaling pathway and theinhibition of SYK suppresses the release of proinflammatorycytokines [25 26] In this study we detected alterations ofSYK in protein level but not mRNA level We surmisedthat posttranscriptional modification may also contributeto HDAC3rsquos functions Preliminary evidences demonstratethat HDAC family played a role in protein degradation[27] while inadequate time points of mRNA detection couldalso be an explanation for this discrepancy CD36 is a TLRcoreceptor playing a pivotal role in inflammatory responsesinitiated by TLRs [28] Previous researches reported thatCD36 contributed to the recognition of diacylglycerol ligandsby forming CD36-CD14-TLR2-TLR6 complex thus it con-trolled gram-positive bacterial infectionWhat ismore CD36was found to be involved in the formation of TLR4-TLR6heterodimers which induced the production of proinflam-matory cytokines nitric oxide and reactive oxygen speciesin response to endogenous ligands [29] Overall RGFP966demonstrated profound inhibitory effects on TLR signalingpathway and proinflammatory cytokines including IL-6 andTNF-120572

HDAC inhibitors have been reported to interfere withthe activation of the mitogen-activated protein kinasesSTAT1 AP-1 or NF-120581B signal transduction pathways [30

31] Here we observed transient STAT3 and STAT5 acti-vation at 2 hours after LPS stimulation which was aheadof the alteration of IL-6 and TNF-120572 Exposed to inflam-matory stimulation STAT35 was phosphorylated by JAKsleading to transcriptional activation Activation of STATselicited the expression of acute-phase proteins as well asa number of cytokines and chemokines including IL-6and TNF-120572 [17] Our study was in keeping with previousstudy inHDAC3 knockoutmacrophage which demonstratedthat HDAC3 knockout impaired STAT1STAT3STAT5 path-ways [13] Figure 9 presented a diagram showing theprotein and protein interactions among proteins Brieflyinitial change of STAT3STAT5 or NF-120581B [5] caused byHDAC3i may further elicit later alterations of TLR sig-naling pathway and proinflammatory cytokines includingIL-6 and TNF-120572 The underlying mechanism for HDAC3in the regulation of STAT3STAT5 is unknown Some evi-dences from Pan-HDAC inhibitors indicated that promoter-associated histone acetylation of SOCS1 and SOCS3 caused byHDACi may further downregulated JAK2STAT3 signaling[32]

In conclusions we identified TLR signaling pathwaymicroglia activation and STAT35 pathway which wereinhibited by RGFP966 Identification of these changes mayprovide valuable clues for the future applications of selectiveHDAC3 inhibitor in the clinic

Competing Interests

The authors declare no conflict of interests

Neural Plasticity 11

DMSO RGFP966

DMSO + LPS RGFP966 + LPS

(a)

p-STAT3

STAT3

p-STAT5

GAPDH

GAPDH

DMSO + LPS RGFP966 + LPS

STAT5

DMSO RGFP966 1h 2h 4h 1h 2h 4h

(b)

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

0

20000

40000

60000

80000

100000

Fluo

resc

ence

inte

nsity

of C

D16

32

(c)

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

0200400600800

100001500020000250003000035000 IL-6

LPSLPS + RGFP966

lowast

1 2 3 8 12 240(h)

(d)

TNF-120572

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

050

100

8000

3000

13000

18000

23000

LPSLPS + RGFP966

lowastlowastlowastlowast

1 2 3 8 12 240(h)

(e)

Figure 8 RGFP966 inhibited LPS-inducedmicroglia activation (a) Representativemicrophotographs of primarymicroglia cultures 24 hoursafter LPS and RGFP966 treatmentThe cultures were stained with CD1632 a marker of activatedmicroglia Scale bar = 10120583m (b) Expressionand phosphorylation of STAT3 and STAT5 in microglia pretreated with RGFP966 (c) Quantitative analysis of fluorescence intensity ofCD1632 All the photographs used for quantitative analysis were taken under the same exposure time and data were expressed as fluorescenceintensity per cell of randomly selected regions lowastlowast119901 lt 001119873 = 3 repeats Supernatant concentration of IL-6 (d) and TNF-120572 (e) detected atvarious stimulation times lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 8: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

8 Neural Plasticity

DMSORGFP966

TLR2 mRNA

Vehicle LPS0

5

10

15lowastlowast

lowastlowast(fo

ld o

f con

trol)

Relat

ive m

RNA

leve

l

(a)

DMSORGFP966

TLR3 mRNA

Vehicle LPS

lowastlowast

lowastlowast

0

10

20

30

40

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(b)

DMSORGFP966

TLR6 mRNA

Vehicle LPS

lowastlowast

lowastlowast

00

05

10

15

20

25

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(c)

DMSORGFP966

CD36 mRNA

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(fold

of c

ontro

l)Re

lativ

e mRN

A le

vel

(d)

Figure 6 RT-PCR analysis of TLR2 TLR3 TLR6 and CD36 in four experimental groups The mRNA levels of TLR2 (a) TLR3 (b) TLR6(c) and CD36 (d) were significantly reduced by RGFP966 at the stimulation time of 6 hours lowastlowast119901 lt 001119873 = 3 repeats

prevented amyloid-beta oligomer-induced synaptic plasticityimpairments [18] and enhanced memory process [15] ForDNA-sensing pathways HDAC3 was thought to participatein IRF3IFN-120573 signaling pathways [13] In this study wemainly analyzed the differentially expressed TLR relative pro-teins of LPS-stimulated primarymicroglia with the treatmentof RGFP966 Toll-like receptor signaling pathway played animportant role in the regulation of inflammation In thisregard we selected TLR2 TLR3 TLR6 CD36 and SYK forfurther validation and found that they were all significantlyupregulated after LPS stimulation and downregulated inthe presence of RGFP966 Additionally morphological andfunctional alterations of microglia exposure to LPS werealso relieved by RGFP966 Our data provided a hint thatHDAC3 inhibitor may be a potential therapeutic target

combating microglia activation and inflammatory responsein neurological diseases

TLRs are a family of type I transmembrane recep-tors which share the multiple leucine-rich repeats (LRRs)domains in the extracellular space and the similar cytosolicdomains of the interleukin-1 (IL-1) receptors [19] TLRsare considered to recognize the distinct pathogen-associatedmolecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) which then drivea cascade of inflammatory signaling and converge at tran-scription factors nuclear factor-120581B (NF-120581B) [20] MAPK[21 22] and JAKSTAT pathways [23] In addition to TLR236 TLR coreceptorCD36 and regulatory protein SYKwereall decreased by RGFP966 Spleen tyrosine kinase (SYK) anonreceptor tyrosine kinase is typically regarded as a vital

Neural Plasticity 9

GAPDH

TLR2

DMSO RGFP966DMSO+ LPS

RGFP966+ LPS DMSO RGFP966

DMSO+ LPS

RGFP966+ LPS

TLR3

GAPDH

TLR6

GAPDH

CD36

GAPDH

phospho-p38

GAPDH

p38

SYK

GAPDH

(a)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR2 Protein level

0

2

4

6

8

10

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

(b)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)TLR3 Protein level

0

2

4

6

8

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(c)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR6 Protein level

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(d)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

CD36 Protein level

0

1

2

3

4

5

DMSORGFP966

Vehicle LPS

lowastlowast

lowastlowast

(e)

Figure 7 Continued

10 Neural Plasticity

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)phospho-p38 Protein level

0

1

2

3

4

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(f)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

SYK Protein level

0

1

2

3

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

(g)

Figure 7Western blot analysis of TLR2 TLR3 TLR6MAPKp38 phospho-p38 SYK andCD36 in four experimental groups (a)The proteinlevels of TLR2 (b) TLR6 (d) SYK (g) phospho-p38 (f) and CD36 (e) were significantly altered by RGFP966 after 12 hoursrsquo stimulation of LPSwhile TLR3 was at 24 hours There was no significant difference of MAPK p38 protein expression among four groups lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

regulator in adaptive immunity [24] It consists of two tandemSH2 domains and a C-terminal tyrosine kinase domain SH2domains selectively bind to the phosphorylated immunore-ceptor tyrosine-based activation motifs (ITAMs) of immunereceptors such as MyD88 TRAF6 and TRIF transmittinginitiative signals to downstream pathways SYK is found to befundamental for Toll-like receptor signaling pathway and theinhibition of SYK suppresses the release of proinflammatorycytokines [25 26] In this study we detected alterations ofSYK in protein level but not mRNA level We surmisedthat posttranscriptional modification may also contributeto HDAC3rsquos functions Preliminary evidences demonstratethat HDAC family played a role in protein degradation[27] while inadequate time points of mRNA detection couldalso be an explanation for this discrepancy CD36 is a TLRcoreceptor playing a pivotal role in inflammatory responsesinitiated by TLRs [28] Previous researches reported thatCD36 contributed to the recognition of diacylglycerol ligandsby forming CD36-CD14-TLR2-TLR6 complex thus it con-trolled gram-positive bacterial infectionWhat ismore CD36was found to be involved in the formation of TLR4-TLR6heterodimers which induced the production of proinflam-matory cytokines nitric oxide and reactive oxygen speciesin response to endogenous ligands [29] Overall RGFP966demonstrated profound inhibitory effects on TLR signalingpathway and proinflammatory cytokines including IL-6 andTNF-120572

HDAC inhibitors have been reported to interfere withthe activation of the mitogen-activated protein kinasesSTAT1 AP-1 or NF-120581B signal transduction pathways [30

31] Here we observed transient STAT3 and STAT5 acti-vation at 2 hours after LPS stimulation which was aheadof the alteration of IL-6 and TNF-120572 Exposed to inflam-matory stimulation STAT35 was phosphorylated by JAKsleading to transcriptional activation Activation of STATselicited the expression of acute-phase proteins as well asa number of cytokines and chemokines including IL-6and TNF-120572 [17] Our study was in keeping with previousstudy inHDAC3 knockoutmacrophage which demonstratedthat HDAC3 knockout impaired STAT1STAT3STAT5 path-ways [13] Figure 9 presented a diagram showing theprotein and protein interactions among proteins Brieflyinitial change of STAT3STAT5 or NF-120581B [5] caused byHDAC3i may further elicit later alterations of TLR sig-naling pathway and proinflammatory cytokines includingIL-6 and TNF-120572 The underlying mechanism for HDAC3in the regulation of STAT3STAT5 is unknown Some evi-dences from Pan-HDAC inhibitors indicated that promoter-associated histone acetylation of SOCS1 and SOCS3 caused byHDACi may further downregulated JAK2STAT3 signaling[32]

In conclusions we identified TLR signaling pathwaymicroglia activation and STAT35 pathway which wereinhibited by RGFP966 Identification of these changes mayprovide valuable clues for the future applications of selectiveHDAC3 inhibitor in the clinic

Competing Interests

The authors declare no conflict of interests

Neural Plasticity 11

DMSO RGFP966

DMSO + LPS RGFP966 + LPS

(a)

p-STAT3

STAT3

p-STAT5

GAPDH

GAPDH

DMSO + LPS RGFP966 + LPS

STAT5

DMSO RGFP966 1h 2h 4h 1h 2h 4h

(b)

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

0

20000

40000

60000

80000

100000

Fluo

resc

ence

inte

nsity

of C

D16

32

(c)

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

0200400600800

100001500020000250003000035000 IL-6

LPSLPS + RGFP966

lowast

1 2 3 8 12 240(h)

(d)

TNF-120572

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

050

100

8000

3000

13000

18000

23000

LPSLPS + RGFP966

lowastlowastlowastlowast

1 2 3 8 12 240(h)

(e)

Figure 8 RGFP966 inhibited LPS-inducedmicroglia activation (a) Representativemicrophotographs of primarymicroglia cultures 24 hoursafter LPS and RGFP966 treatmentThe cultures were stained with CD1632 a marker of activatedmicroglia Scale bar = 10120583m (b) Expressionand phosphorylation of STAT3 and STAT5 in microglia pretreated with RGFP966 (c) Quantitative analysis of fluorescence intensity ofCD1632 All the photographs used for quantitative analysis were taken under the same exposure time and data were expressed as fluorescenceintensity per cell of randomly selected regions lowastlowast119901 lt 001119873 = 3 repeats Supernatant concentration of IL-6 (d) and TNF-120572 (e) detected atvarious stimulation times lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 9: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

Neural Plasticity 9

GAPDH

TLR2

DMSO RGFP966DMSO+ LPS

RGFP966+ LPS DMSO RGFP966

DMSO+ LPS

RGFP966+ LPS

TLR3

GAPDH

TLR6

GAPDH

CD36

GAPDH

phospho-p38

GAPDH

p38

SYK

GAPDH

(a)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR2 Protein level

0

2

4

6

8

10

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

(b)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)TLR3 Protein level

0

2

4

6

8

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(c)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

TLR6 Protein level

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

0

5

10

15

(d)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

CD36 Protein level

0

1

2

3

4

5

DMSORGFP966

Vehicle LPS

lowastlowast

lowastlowast

(e)

Figure 7 Continued

10 Neural Plasticity

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)phospho-p38 Protein level

0

1

2

3

4

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(f)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

SYK Protein level

0

1

2

3

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

(g)

Figure 7Western blot analysis of TLR2 TLR3 TLR6MAPKp38 phospho-p38 SYK andCD36 in four experimental groups (a)The proteinlevels of TLR2 (b) TLR6 (d) SYK (g) phospho-p38 (f) and CD36 (e) were significantly altered by RGFP966 after 12 hoursrsquo stimulation of LPSwhile TLR3 was at 24 hours There was no significant difference of MAPK p38 protein expression among four groups lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

regulator in adaptive immunity [24] It consists of two tandemSH2 domains and a C-terminal tyrosine kinase domain SH2domains selectively bind to the phosphorylated immunore-ceptor tyrosine-based activation motifs (ITAMs) of immunereceptors such as MyD88 TRAF6 and TRIF transmittinginitiative signals to downstream pathways SYK is found to befundamental for Toll-like receptor signaling pathway and theinhibition of SYK suppresses the release of proinflammatorycytokines [25 26] In this study we detected alterations ofSYK in protein level but not mRNA level We surmisedthat posttranscriptional modification may also contributeto HDAC3rsquos functions Preliminary evidences demonstratethat HDAC family played a role in protein degradation[27] while inadequate time points of mRNA detection couldalso be an explanation for this discrepancy CD36 is a TLRcoreceptor playing a pivotal role in inflammatory responsesinitiated by TLRs [28] Previous researches reported thatCD36 contributed to the recognition of diacylglycerol ligandsby forming CD36-CD14-TLR2-TLR6 complex thus it con-trolled gram-positive bacterial infectionWhat ismore CD36was found to be involved in the formation of TLR4-TLR6heterodimers which induced the production of proinflam-matory cytokines nitric oxide and reactive oxygen speciesin response to endogenous ligands [29] Overall RGFP966demonstrated profound inhibitory effects on TLR signalingpathway and proinflammatory cytokines including IL-6 andTNF-120572

HDAC inhibitors have been reported to interfere withthe activation of the mitogen-activated protein kinasesSTAT1 AP-1 or NF-120581B signal transduction pathways [30

31] Here we observed transient STAT3 and STAT5 acti-vation at 2 hours after LPS stimulation which was aheadof the alteration of IL-6 and TNF-120572 Exposed to inflam-matory stimulation STAT35 was phosphorylated by JAKsleading to transcriptional activation Activation of STATselicited the expression of acute-phase proteins as well asa number of cytokines and chemokines including IL-6and TNF-120572 [17] Our study was in keeping with previousstudy inHDAC3 knockoutmacrophage which demonstratedthat HDAC3 knockout impaired STAT1STAT3STAT5 path-ways [13] Figure 9 presented a diagram showing theprotein and protein interactions among proteins Brieflyinitial change of STAT3STAT5 or NF-120581B [5] caused byHDAC3i may further elicit later alterations of TLR sig-naling pathway and proinflammatory cytokines includingIL-6 and TNF-120572 The underlying mechanism for HDAC3in the regulation of STAT3STAT5 is unknown Some evi-dences from Pan-HDAC inhibitors indicated that promoter-associated histone acetylation of SOCS1 and SOCS3 caused byHDACi may further downregulated JAK2STAT3 signaling[32]

In conclusions we identified TLR signaling pathwaymicroglia activation and STAT35 pathway which wereinhibited by RGFP966 Identification of these changes mayprovide valuable clues for the future applications of selectiveHDAC3 inhibitor in the clinic

Competing Interests

The authors declare no conflict of interests

Neural Plasticity 11

DMSO RGFP966

DMSO + LPS RGFP966 + LPS

(a)

p-STAT3

STAT3

p-STAT5

GAPDH

GAPDH

DMSO + LPS RGFP966 + LPS

STAT5

DMSO RGFP966 1h 2h 4h 1h 2h 4h

(b)

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

0

20000

40000

60000

80000

100000

Fluo

resc

ence

inte

nsity

of C

D16

32

(c)

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

0200400600800

100001500020000250003000035000 IL-6

LPSLPS + RGFP966

lowast

1 2 3 8 12 240(h)

(d)

TNF-120572

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

050

100

8000

3000

13000

18000

23000

LPSLPS + RGFP966

lowastlowastlowastlowast

1 2 3 8 12 240(h)

(e)

Figure 8 RGFP966 inhibited LPS-inducedmicroglia activation (a) Representativemicrophotographs of primarymicroglia cultures 24 hoursafter LPS and RGFP966 treatmentThe cultures were stained with CD1632 a marker of activatedmicroglia Scale bar = 10120583m (b) Expressionand phosphorylation of STAT3 and STAT5 in microglia pretreated with RGFP966 (c) Quantitative analysis of fluorescence intensity ofCD1632 All the photographs used for quantitative analysis were taken under the same exposure time and data were expressed as fluorescenceintensity per cell of randomly selected regions lowastlowast119901 lt 001119873 = 3 repeats Supernatant concentration of IL-6 (d) and TNF-120572 (e) detected atvarious stimulation times lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 10: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

10 Neural Plasticity

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)phospho-p38 Protein level

0

1

2

3

4

DMSORGFP966

Vehicle LPS

lowastlowastlowast

(f)

Relat

ive p

rote

in b

and

inte

nsity

(fold

of c

ontro

l)

SYK Protein level

0

1

2

3

DMSORGFP966

Vehicle LPS

lowastlowastlowastlowast

(g)

Figure 7Western blot analysis of TLR2 TLR3 TLR6MAPKp38 phospho-p38 SYK andCD36 in four experimental groups (a)The proteinlevels of TLR2 (b) TLR6 (d) SYK (g) phospho-p38 (f) and CD36 (e) were significantly altered by RGFP966 after 12 hoursrsquo stimulation of LPSwhile TLR3 was at 24 hours There was no significant difference of MAPK p38 protein expression among four groups lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

regulator in adaptive immunity [24] It consists of two tandemSH2 domains and a C-terminal tyrosine kinase domain SH2domains selectively bind to the phosphorylated immunore-ceptor tyrosine-based activation motifs (ITAMs) of immunereceptors such as MyD88 TRAF6 and TRIF transmittinginitiative signals to downstream pathways SYK is found to befundamental for Toll-like receptor signaling pathway and theinhibition of SYK suppresses the release of proinflammatorycytokines [25 26] In this study we detected alterations ofSYK in protein level but not mRNA level We surmisedthat posttranscriptional modification may also contributeto HDAC3rsquos functions Preliminary evidences demonstratethat HDAC family played a role in protein degradation[27] while inadequate time points of mRNA detection couldalso be an explanation for this discrepancy CD36 is a TLRcoreceptor playing a pivotal role in inflammatory responsesinitiated by TLRs [28] Previous researches reported thatCD36 contributed to the recognition of diacylglycerol ligandsby forming CD36-CD14-TLR2-TLR6 complex thus it con-trolled gram-positive bacterial infectionWhat ismore CD36was found to be involved in the formation of TLR4-TLR6heterodimers which induced the production of proinflam-matory cytokines nitric oxide and reactive oxygen speciesin response to endogenous ligands [29] Overall RGFP966demonstrated profound inhibitory effects on TLR signalingpathway and proinflammatory cytokines including IL-6 andTNF-120572

HDAC inhibitors have been reported to interfere withthe activation of the mitogen-activated protein kinasesSTAT1 AP-1 or NF-120581B signal transduction pathways [30

31] Here we observed transient STAT3 and STAT5 acti-vation at 2 hours after LPS stimulation which was aheadof the alteration of IL-6 and TNF-120572 Exposed to inflam-matory stimulation STAT35 was phosphorylated by JAKsleading to transcriptional activation Activation of STATselicited the expression of acute-phase proteins as well asa number of cytokines and chemokines including IL-6and TNF-120572 [17] Our study was in keeping with previousstudy inHDAC3 knockoutmacrophage which demonstratedthat HDAC3 knockout impaired STAT1STAT3STAT5 path-ways [13] Figure 9 presented a diagram showing theprotein and protein interactions among proteins Brieflyinitial change of STAT3STAT5 or NF-120581B [5] caused byHDAC3i may further elicit later alterations of TLR sig-naling pathway and proinflammatory cytokines includingIL-6 and TNF-120572 The underlying mechanism for HDAC3in the regulation of STAT3STAT5 is unknown Some evi-dences from Pan-HDAC inhibitors indicated that promoter-associated histone acetylation of SOCS1 and SOCS3 caused byHDACi may further downregulated JAK2STAT3 signaling[32]

In conclusions we identified TLR signaling pathwaymicroglia activation and STAT35 pathway which wereinhibited by RGFP966 Identification of these changes mayprovide valuable clues for the future applications of selectiveHDAC3 inhibitor in the clinic

Competing Interests

The authors declare no conflict of interests

Neural Plasticity 11

DMSO RGFP966

DMSO + LPS RGFP966 + LPS

(a)

p-STAT3

STAT3

p-STAT5

GAPDH

GAPDH

DMSO + LPS RGFP966 + LPS

STAT5

DMSO RGFP966 1h 2h 4h 1h 2h 4h

(b)

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

0

20000

40000

60000

80000

100000

Fluo

resc

ence

inte

nsity

of C

D16

32

(c)

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

0200400600800

100001500020000250003000035000 IL-6

LPSLPS + RGFP966

lowast

1 2 3 8 12 240(h)

(d)

TNF-120572

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

050

100

8000

3000

13000

18000

23000

LPSLPS + RGFP966

lowastlowastlowastlowast

1 2 3 8 12 240(h)

(e)

Figure 8 RGFP966 inhibited LPS-inducedmicroglia activation (a) Representativemicrophotographs of primarymicroglia cultures 24 hoursafter LPS and RGFP966 treatmentThe cultures were stained with CD1632 a marker of activatedmicroglia Scale bar = 10120583m (b) Expressionand phosphorylation of STAT3 and STAT5 in microglia pretreated with RGFP966 (c) Quantitative analysis of fluorescence intensity ofCD1632 All the photographs used for quantitative analysis were taken under the same exposure time and data were expressed as fluorescenceintensity per cell of randomly selected regions lowastlowast119901 lt 001119873 = 3 repeats Supernatant concentration of IL-6 (d) and TNF-120572 (e) detected atvarious stimulation times lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 11: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

Neural Plasticity 11

DMSO RGFP966

DMSO + LPS RGFP966 + LPS

(a)

p-STAT3

STAT3

p-STAT5

GAPDH

GAPDH

DMSO + LPS RGFP966 + LPS

STAT5

DMSO RGFP966 1h 2h 4h 1h 2h 4h

(b)

DMSORGFP966

Vehicle LPS

lowastlowast lowastlowast

0

20000

40000

60000

80000

100000

Fluo

resc

ence

inte

nsity

of C

D16

32

(c)

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

0200400600800

100001500020000250003000035000 IL-6

LPSLPS + RGFP966

lowast

1 2 3 8 12 240(h)

(d)

TNF-120572

Supe

rnat

ant c

once

ntra

tion

(pg

mL)

050

100

8000

3000

13000

18000

23000

LPSLPS + RGFP966

lowastlowastlowastlowast

1 2 3 8 12 240(h)

(e)

Figure 8 RGFP966 inhibited LPS-inducedmicroglia activation (a) Representativemicrophotographs of primarymicroglia cultures 24 hoursafter LPS and RGFP966 treatmentThe cultures were stained with CD1632 a marker of activatedmicroglia Scale bar = 10120583m (b) Expressionand phosphorylation of STAT3 and STAT5 in microglia pretreated with RGFP966 (c) Quantitative analysis of fluorescence intensity ofCD1632 All the photographs used for quantitative analysis were taken under the same exposure time and data were expressed as fluorescenceintensity per cell of randomly selected regions lowastlowast119901 lt 001119873 = 3 repeats Supernatant concentration of IL-6 (d) and TNF-120572 (e) detected atvarious stimulation times lowast119901 lt 005 lowastlowast119901 lt 001119873 = 3 repeats

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 12: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

12 Neural Plasticity

IL-6

STAT5

STAT3

SYK

TNF-120572

TLR2

p38

TLR6

CD36

HDAC3

TLR3

From curated databasesExperimentally determined

TextminingProtein homology

Figure 9 Protein-protein interaction network analysis Each color of string represented interaction type respectively

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81571135 81400971) and Nanjingoutstanding youth foundation (JQX15003) The project issponsored by ldquoYoung Talent Support Programrdquo for ChinaStroke Association from China Association for Science andTechnology

References

[1] N Cartier C-A Lewis R Zhang and F M V Rossi ldquoThe roleof microglia in human disease therapeutic tool or targetrdquo ActaNeuropathologica vol 128 no 3 pp 363ndash380 2014

[2] J Slusarczyk E Trojan K Głombik et al ldquoFractalkine attenu-atesmicroglial cell activation induced by prenatal stressrdquoNeuralPlasticity vol 2016 Article ID 7258201 11 pages 2016

[3] Y Ren andWYoung ldquoManaging inflammation after spinal cordinjury through manipulation of macrophage functionrdquo NeuralPlasticity vol 2013 Article ID 945034 9 pages 2013

[4] H Jia J Pallos V Jacques et al ldquoHistone deacetylase(HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate

polyglutamine-elicited phenotypes in model systems of Hunt-ingtonrsquos diseaserdquoNeurobiology of Disease vol 46 no 2 pp 351ndash361 2012

[5] N G J Leus M R H Zwinderman and F J Dekker ldquoHistonedeacetylase 3 (HDAC 3) as emerging drug target in NF-120581B-mediated inflammationrdquo Current Opinion in Chemical Biologyvol 33 pp 160ndash168 2016

[6] K A Bode and A H Dalpke ldquoHDAC inhibitors block innateimmunityrdquo Blood vol 117 no 4 pp 1102ndash1103 2011

[7] T Roger J Lugrin D Le Roy et al ldquoHistone deacetylaseinhibitors impair innate immune responses to Toll-like receptoragonists and to infectionrdquo Blood vol 117 no 4 pp 1205ndash12172011

[8] AMGrabiec S KrauszWDe Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoidarthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[9] I Gojo A Jiemjit J B Trepel et al ldquoPhase 1 and pharmacologicstudy of MS-275 a histone deacetylase inhibitor in adults withrefractory and relapsed acute leukemiasrdquo Blood vol 109 no 7pp 2781ndash2790 2007

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 13: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

Neural Plasticity 13

[10] R S Broide J M Redwine N Aftahi W Young F E Bloomand C J Winrow ldquoDistribution of histone deacetylases 1-11 inthe rat brainrdquo Journal of Molecular Neuroscience vol 31 no 1pp 47ndash58 2007

[11] A Venkatraman Y-S Hu A Didonna et al ldquoThe histonedeacetylase HDAC3 is essential for Purkinje cell functionpotentially complicating the use of HDAC inhibitors in SCA1rdquoHuman Molecular Genetics vol 23 no 14 pp 3733ndash3745 2014

[12] G A Rogge H Singh R Dang and M A Wood ldquoHDAC3is a negative regulator of cocaine-context-associated memoryformationrdquo Journal of Neuroscience vol 33 no 15 pp 6623ndash6632 2013

[13] X Chen I Barozzi A Termanini et al ldquoRequirement for thehistone deacetylase Hdac3 for the inflammatory gene expres-sion program in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no42 pp E2865ndashE2874 2012

[14] S E Mullican C A Gaddis T Alenghat et al ldquoHistonedeacetylase 3 is an epigenomic brake in macrophage alternativeactivationrdquo Genes and Development vol 25 no 23 pp 2480ndash2488 2011

[15] M Malvaez S C McQuown G A Rogge et al ldquoHDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent mannerrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no7 pp 2647ndash2652 2013

[16] C E Wells S Bhaskara K R Stengel et al ldquoInhibition ofhistone deacetylase 3 causes replication stress in cutaneous Tcell lymphomardquo PLoS ONE vol 8 no 7 Article ID e68915 2013

[17] H Yu D Pardoll and R Jove ldquoSTATs in cancer inflammationand immunity a leading role for STAT3rdquo Nature ReviewsCancer vol 9 no 11 pp 798ndash809 2009

[18] K Krishna T Behnisch and S Sajikumar ldquoInhibition of histonedeacetylase 3 restores amyloid-120573 oligomer-induced plasticitydeficit in hippocampal CA1 pyramidal neuronsrdquo Journal ofAlzheimerrsquos Disease vol 51 no 3 pp 783ndash791 2016

[19] J Brown H Wang G N Hajishengallis and M MartinldquoTLR-signaling networks an integration of adaptor moleculeskinases and cross-talkrdquo Journal of Dental Research vol 90 no4 pp 417ndash427 2011

[20] J-Y Sun D-L Li Y Dong et al ldquoBaicalin inhibits toll-likereceptor 24 expression and downstream signaling in rat experi-mental periodontitisrdquo International Immunopharmacology vol36 pp 86ndash93 2016

[21] M Shu D-D Huang Z-A Hung X-R Hu and S ZhangldquoInhibition of MAPK and NF-120581B signaling pathways alleviatecarbon tetrachloride (CCl4)-induced liver fibrosis in Toll-likereceptor 5 (TLR5) deficiencymicerdquo Biochemical and BiophysicalResearch Communications vol 471 no 1 pp 233ndash239 2016

[22] D Awasthi S Nagarkoti A Kumar et al ldquoOxidized LDLinduced extracellular trap formation in human neutrophils viaTLR-PKC-IRAK-MAPK and NADPH-oxidase activationrdquo FreeRadical Biology and Medicine vol 93 pp 190ndash203 2016

[23] K Luu C J Greenhill A Majoros T Decker B J Jenkins andA Mansell ldquoSTAT1 plays a role in TLR signal transduction andinflammatory responsesrdquo Immunology and Cell Biology vol 92no 9 pp 761ndash769 2014

[24] E Kim Y-J Son Y Yang et al ldquo1-(23-dibenzimidazol-2-ylpropyl)-2-methoxybenzene is a syk inhibitor with anti-inflammatory propertiesrdquo Molecules vol 21 no 4 article 5082016

[25] B Aouar D Kovarova S Letard et al ldquoDual role of thetyrosine kinase Syk in regulation of Toll-like receptor signalingin plasmacytoid dendritic cellsrdquo PLoSONE vol 11 no 6 ArticleID e0156063 2016

[26] H Yin H Zhou Y Kang et al ldquoSyk negatively regulates TLR4-mediated IFN120573 and IL-10 production and promotes inflam-matory responses in dendritic cellsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1860 no 3 pp 588ndash598 2016

[27] C Zou M J Synan J Li et al ldquoLPS impairs oxygen utilizationin epithelia by triggering degradation of the mitochondrialenzyme Alcat1rdquo Journal of Cell Science vol 129 no 1 pp 51ndash642016

[28] M Di Gioia and I Zanoni ldquoToll-like receptor co-receptorsas master regulators of the immune responserdquo MolecularImmunology vol 63 no 2 pp 143ndash152 2015

[29] C R Stewart L M Stuart K Wilkinson et al ldquoCD36 ligandspromote sterile inflammation through assembly of a Toll-likereceptor 4 and 6 heterodimerrdquo Nature Immunology vol 11 no2 pp 155ndash161 2010

[30] G Faraco M Pittelli L Cavone et al ldquoHistone deacetylase(HDAC) inhibitors reduce the glial inflammatory response invitro and in vivordquo Neurobiology of Disease vol 36 no 2 pp269ndash279 2009

[31] E A Stronach A Alfraidi N Rama et al ldquoHDAC4-regulatedSTAT1 activation mediates platinum resistance in ovarian can-cerrdquo Cancer Research vol 71 no 13 pp 4412ndash4422 2011

[32] H Xiong W Du Y-J Zhang et al ldquoTrichostatin A a his-tone deacetylase inhibitor suppresses JAK2STAT3 signalingvia inducing the promoter-associated histone acetylation ofSOCS1 and SOCS3 in human colorectal cancer cellsrdquoMolecularCarcinogenesis vol 51 no 2 pp 174ndash184 2012

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 14: Proteomic Analysis of HDAC3 Selective Inhibitor in the ...ysis, western blotting, CBA, and Q-PCR at indicated time points. 2.2.ProteomicAnalysis. Proteomicanalysiswasperformed by AB

Submit your manuscripts athttpswwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


miR-326-HistoneDeacetylase-3FeedbackLoopRegulates ... · 2014-09-26 · Background: The role of HDAC3 in anti-cancer drug resistance remains unknown. Results: HDAC3 forms a negative

Nrf2 status affects tumor growth, HDAC3 gene promoter

Fluorescent Western Blotting - Abcamdocs.abcam.com/pdf/events/Fluorescent-Western-Blotting-webinar.pdf · western blotting detection system ... Challenges or pitfalls in fluorescent

HDAC3 promotes meiotic apparatus assembly in mouse …RESEARCH ARTICLE HDAC3 promotes meiotic apparatus assembly in mouse oocytes by modulating tubulin acetylation Xiaoyan Li1, Xiaohui

Cancer Discovery - Selective Inhibition of HDAC3 Targets ......440 | CANCER DISCOVERY MARCH 2020 AACRJournals.org Selective Inhibition of HDAC3 Targets Synthetic Vulnerabilities and

Blotting techniques1

223-F · (Southern blotting) (Eastern blotting) (Northern blotting) (f (Western blotting) (t ( PBR322 Cosmid (t Acidophile - microaerophilic Acidophile - aerotolerant (t Episome (f

Protein Blotting Guide - Bio-Rad · Protein Blotting Workflow 6 7 Protein Blotting Guide Theory and Products Transfer The first phase of protein blotting is the transfer step, which

CBX4 Suppresses Metastasis via Recruitment of HDAC3 to the ... · CBX4 Suppresses Metastasis via Recruitment of HDAC3 to the Runx2 Promoter in Colorectal Carcinoma Xin Wang, Liping

Southern blotting

Protein Blotting Guide - Wolfson Centre Home Pagewolfson.huji.ac.il/.../BIORAD_ProtBlottingGuide.pdf · Protein Blotting Guide Theory and Products ... western blotting (immunoblotting),

MeCP2 co-ordinates liver lipid metabolism with the NCoR1/HDAC3

Northern blotting

The dual role of LSD1 and HDAC3 in STAT5-dependent ... · The dual role of LSD1 and HDAC3 in STAT5-dependent transcription is determined by protein interactions, binding affinities,

Western Blotting - SERVA GmbH Western Blotting Ju… · BlueBlot Semi-Dry Blotter Additional Instruments for Western Blotting 6 7 Blotting of proteins onto a membrane after separation

226-F · (Western blotting) (Y (Northern blotting) (Southern blotting) (f (Eastern blotting) (Y cAMP PA ,(photoreactivation) Schick test (f Dick test (Y DNA Antony test (Y ASO test

Blotting Techniques

THIS IS YOUR PRESENTATION TITLE - fac.ksu.edu.sa · Disadvantage of Northern blotting. 3. WESTERN BLOTTING . Western blotting, also known as immunoblotting“ or protein blotting,

ECL Western Blotting Detection Substrate. - Bimakefile.biotool.com/.../ECL-Western-Blotting-Detection-Substrate.pdf · ECL Western Blotting Detection Substrate Description ... The

Blotting Methods

Protein Blotting

Western Blotting

Blotting Techniques 10.pdf · 2017. 1. 10. · blotting, northern (RNA) blotting, and immunoblotting (for protein; also known as western blotting). In this chapter, a brief introduction

Macromolecule Blotting - e-PGPathshalaepgp.inflibnet.ac.in/epgpdata/uploads/epgp_content/S000002BI/P... · Macromolecule Blotting Blotting Southern Northern Western. 5 Macromolecule

Kelompok 6 Kimia B (Southern Blotting dan Northern Blotting)

CRISPR screen identifies the NCOR/HDAC3 complex as a major ... · CRISPR screen identifies the NCOR/HDAC3 complex as a major suppressor of differentiation in rhabdomyosarcoma Michael