antineuroinflammatory and neuroprotective effects of ...2018/07/20 ·...

Research ArticleAntineuroinflammatory and Neuroprotective Effects ofGyejibokryeong-Hwan in Lipopolysaccharide-StimulatedBV2 Microglia

Bo-Kyung Park1 Young Hwa Kim1 Yu Ri Kim1 Jeong June Choi 2 Changsop Yang 1

Ik-Soon Jang 3 and Mi Young Lee 1

1Clinical Medicine Division Korea Institute of Oriental Medicine Daejeon 34054 Republic of Korea2Laboratory of Molecular Medicine College of Korean Medicine Daejeon University Daejeon 34520 Republic of Korea3Division of Bioconvergence Analysis Korea Basic Science Institute Daejeon 34133 Republic of Korea

Correspondence should be addressed to Mi Young Lee myleekiomrekr

Received 20 July 2018 Revised 23 January 2019 Accepted 10 February 2019 Published 1 April 2019

Academic Editor Olumayokun A Olajide

Copyright copy 2019 Bo-Kyung Park et alThis 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

Microglia the central nervous systemrsquos innate immune cells mediate neuroinflammation and are implicated in a variety ofneuropathologies The present study investigated the antineuroinflammatory and neuroprotective effects of Gyejibokryeong-hwan(GBH) a traditional Korean medicine in lipopolysaccharide- (LPS-) stimulated murine BV2 microglia BV2 cells were pretreatedwith GBH fluoxetine (FXT) or amitriptyline (AMT) for 1 h and then stimulated with LPS (100 ngmL) The expression levels ofnitric oxide (NO) cytokines and chemokines were determined by the Griess method ELISA or real-time PCR Western blottingwas used to measure various transcription factors and mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)Akt activity GBH significantly reduced the levels of NO inducible nitric oxide synthase (iNOS) cyclooxygenase-(COX-) 2 tumor necrosis factor- (TNF-) 120572 interleukin- (IL-) 1120573 IL-6 macrophage inhibitory protein- (MIP-) 1120572 macrophagechemoattractant protein- (MCP-) 1 and IFN-120574 inducible protein- (IP-) 10 regulated upon activation normal T cell expressedsequence (RANTES) in a dose-dependent manner Expression of nuclear factor- (NF-) 120581B p65 was significantly decreased andphosphorylation of extracellular signal-regulated kinase (Erk) c-Jun NH2-terminal kinase (JNK) and PI3KAkt by GBH but notp38 MAPK was decreased Furthermore production of anti-inflammatory cytokine IL-10 was increased and Heme oxygenase-1 (HO-1) was upregulated via the nuclear factor-E2-related factor 2 (NRF2)cAMP response element-binding protein (CREB)pathway collectively indicating the neuroprotective effects of GBH We concluded that GBH may suppress neuroinflammatoryresponses by inhibiting NF-120581B activation and upregulating the neuroprotective factor HO-1 These results suggest that GBH haspotential as anti-inflammatory and neuroprotective agents against microglia-mediated neuroinflammatory disorders

1 Introduction

Microglia are the resident immune cells of the central nervoussystem (CNS) and act as an early defense system againstproinflammatory reactions in the brain and prevent someeffects of aging and repair damaged tissues [1 2] Howeverwhen microglia are activated by sustained stimulation theycan secrete a variety of inflammatory factors includingproinflammatory cytokines such as tumor necrosis factor-(TNF-) 120572 interleukin- (IL-) 1120573 and IL-6 chemokines includ-ing macrophage inhibitory protein- (MIP-) 1120572 monocyte

chemoattractant protein 1- (MCP-) 1 the interferon- (IFN-) 120574inducible protein- (IP-) 10 regulated upon activation normalT cell expressed sequence (RANTES) and reactive oxidants[2 3] When these inflammatory factors are chronicallysecreted they can cause neuroinflammation as in variousneurodegenerative disorders such as Alzheimerrsquos diseaseParkinsonrsquos disease depression and amyotrophic lateral scle-rosis (ALS) [4]

Lipopolysaccharide- (LPS-) stimulated microglia activateToll-like receptor (TLR) phosphorylate mitogen-activatedprotein kinase (MAPK) and translocate nuclear factor- (NF-)

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 7585896 14 pageshttpsdoiorg10115520197585896

2 Evidence-Based Complementary and Alternative Medicine

120581B p65 an inflammatory transcription factor into thenucleus via phosphatidylinositol 3-kinase (PI3K)Akt whereit increases the production of various proinflammatorycytokines and chemokines [5 6] In contrast an increasein neuroprotective factors such as nuclear factor erythroid2-related factor 2 (NRF2) cAMP response element-bindingprotein (CREB) and Heme oxygenase-1 (HO-1) can pro-duce the anti-inflammatory cytokine IL-10 and thus inhibitneuroinflammation Given this central role of microglia inneuroinflammation controlling the activation of microgliacells through upregulation of HO-1 may serve as a criticalpotential target mechanism for the treatment of neurologicaland neurodegenerative diseases [7 8]

Gyejibokryeong-hwan (GBH) is a traditional Koreanmedicine that is described in Donguibogam a textbook oftraditional Korean medicines [9] GBH has been extensivelyused throughout Asia in the treatment of blood stasis [1011] and is approved by the Ministry of Food and Drugsafety (MFDS) and the USA Food and Drug Administration(FDA) having been studied for safety and composition GBHcontains herbs including Cinnamomum cassia Presl Poriacocos Wolf Paeonia suffruticosa Andrews Paeonia lactifloraPallas and Prunus persica Batsch [12 13]

Cinnamomum cassia Presl a constitutive agent inGBH has been shown to exhibit anti-inflammatory effectsin murine BV2 microglia cells and Paeonia suffruticosaAndrews another element of GBH has been reported toinduce antioxidative stress effects in rat pheochromocytomaPC-12 cells [14 15] Moreover amygdalin a componentof Prunus persica Batsch induces neurotrophic effectsvia the activation of the extracellular signal-regulatedkinase (ERK)12 pathway in PC12 cells [16] paeoniflorin acomponent of Paeonia suffruticosa Andrews and Paeonialactiflora Pallas reduces proinflammatory cytokines inCNS via the activation of Akt-NF-120581B signaling pathway invivo and in vitro [17] and cinnamic acid a component ofCinnamomum cassia Presl upregulates the expression ofSOCS3 in glial cells via the CREB pathway [18]

In a previous study conducted by our group GBH wasshown to exhibit antineuroinflammation effects in the hip-pocampus of amousemodel of reserpine-induced depression[19] Several studies have reported that microglia regulatehippocampal neurogenesis in neurodegeneration [20 21]Despite these promising results the antineuroinflammationand neuroprotection effects of GBH inmurine BV2microgliahave yet to be investigated Therefore in the present study weconfirmed the antineuroinflammatory and neuroprotectiveeffects of GBH via several mechanisms in a proinflammatorymicroglia model where murine BV2 microglia were treatedwith LPS

2 Materials and Methods

21 Preparation of Gyejibokryeong-Hwan (GBH) GBH waspurchased from Hanpoong Pharm and Foods Co Ltd(Jeonju Korea) A voucher specimen of the herb sample wasdeposited in the Herbarium of Hanpoong Pharm and FoodsCo Ltd (voucher no 17248) The five-component herbs ofGBH (combined at a 11111 ratio total weight = 17 kg) were

boiled in water for 3 h to create an extract which was thenfiltered and concentrated via vacuum pressure The extractyield was approximately 2953 The extract was stored at -80∘C and dissolved in phosphate-buffered saline (PBS) beforeuse

22 Reagents and Liquid ChromatographyMass Spectrometry(LCMS) Analysis of Gyejibokryeong-Hwan

221 Amygdalin and Paeoniflorin Standards for amygdalin(PubChem CID 656516 purity 990) and paeoniflorin(PubChem CID 442534 purity 988) were purchasedfrom Sigma-Aldrich (St Louis MO USA) Solutions ofamygdalin and paeoniflorin were prepared in analyticalgrade methanol (Merck Darmstadt Germany) An opti-mized multiple reaction monitoring (MRM) method wasdeveloped using Ultra-Performance Liquid Chromatography(UPLC) coupled with tandem mass spectrometry (MSMS)A UPLC system (Acquity system Waters Milford USA) wascoupled to a Xevo TQ-S triple quadrupole mass spectrometer(Waters) Chromatographic separations were carried outusing revers phase hybrid column (Kinetex 26 120583m 50x 21 mm Phenomenex CA USA) maintained at 30∘CAmygdalin and paeoniflorin were separated using a gradientelution with a flow rate of 05 mLmin Mobile phase solventA was 50 mMAmmonium formate (Sigma-Aldrich) in water(pH 80) and solvent B was 50 mM Ammonium formatein acetonitrile The samples were eluted according to thefollowing linear gradient from 5 B buffer to 100 for10 mins Ions were generated in positive ionization modeusing electrospray ionization interface Tandem MS analysiswas performed using the multi-reaction-monitoring (MRM)mode by monitoring the transition pair of mz 47493 997888rarr32511 for amygdalin and mz 49793 997888rarr 17901 for paeoni-florin The gas flows of desolvation cone and nebulizer wereset at 650 LHr 150 LHr and 7 bar respectively

222 Cinnamic Acid Agilent 6410B Triple Quadrupole LCMS (Agilent Technologies Wilmington USA) equipped withan ESI source was employed for the analysis Cinnamic acid(PubChem CID 444539 purity 990) was purchased fromSigma-Aldrich and used as reference standard 1 g of eachsample was mixed with 10 mL of methanol and centrifugedAliquots of 5 120583L of the processed samples were injectedinto the HPLC system (1200 Series LC Agilent TechnologiesWilmington USA) fitted with Phenomenex Kinetex C18 26120583m 80 A 50 x 21 mm column maintained at 35∘C ESI wasoperating at +3000V and a source temperature of 380∘CCapillary voltage cone voltage and source offset were set at3kV 30kV and 30V respectively The gas flow of desolvationand the conewas set at 650LHr and 150 LHrwith a nebulizerpressure of 15 bar A mobile phase composed of 01 formicacid in distilled water (Buffer A) and 01 formic acid inacetonitrile (Buffer B) was used to separate the analysts andpumped into the ESI chamber at a flow rate of 05 mLminfor 20 min Fragmentor voltage and collision voltage wereset at 50V Detection of the ions was carried out in themultiple-reaction monitoring mode (MRM) by monitoringthe transition pairs of mz 1491 997888rarr131 (cinnamic acid) Data

Evidence-Based Complementary and Alternative Medicine 3

Table 1 The sequences of the real-time PCR primers

Gene Sequence

mouse IL-1120573 forward 5耠- GCTGAAAGCTCTCCACCTCA -3耠

reverse 5耠- AGGCCACAGGTATTTTGTCG -3耠

mouse IL-6 forward 5耠- GAGGATACCACTCCCAACAGACC-3耠

reverse 5耠- AAGTGCATCATCGTTGTTCATACA -3耠

mouse TNF-120572 forward 5耠- AGACCCTCACACTCAGATCATCTTC -3耠

reverse 5耠- CCACTTGGTGGTTTGCTACGA -3耠

mouseHO-1 forward 5耠- AGCCCCACCAAGTTCAAACA-3耠

reverse 5耠- CATCACCTGCAGCTCCTCAA -3耠

mouseMIP-1a forward 5耠- CCCAGCCAGGTGTCATTTTCC -3耠

reverse 5耠- GCATTCAGTTCCAGGTCAGTG -3耠

mouseMCP-1 forward 5耠- GCTCAGCCAGATGCAGTTAA -3耠

reverse 5耠- TCTTGAGCTTGGTGACAAAAACT -3耠

mouse IP-10 forward 5耠- GAATCCGGAATCTAAGACCATCAA -3耠

reverse 5耠- GTGCGTGGCTTCACTCCAGT -3耠

mouse RANTES forward 5耠- CAGCAGCAAGTGCTCCAATCTT -3耠

reverse 5耠- TTCTTGAACCCACTTCTTCTCTGG -3耠

mouse iNOS forward 5耠- GAATCTTGGAGCGAGTTGTGGA -3耠

reverse 5耠- GTGAGGGCTTGGCTGAGTGAG -3耠

mouse COX-2 forward 5耠- TGGGGTGATGAGCAACTATT-3reverse 5耠- 5-AAGGAGCTCTGGGTCAAACT-3

mouse GAPDH forward 5耠- AAGGTGGTGAAGCAGGCAT -3耠

reverse 5耠- GGTCCAGGGTTTCTTACTCCT -3耠

acquisition was performed with the MassHunter Software(Version B0400)

23 Reagents and Cell Culture LPS fluoxetine (FXT) andamitriptyline (AMT) were purchased from Sigma-AldrichTin protoporphyrin IX (SnPP HO-1 inhibitor) and cobaltprotoporphyrin IX (CoPP HO-1 inducer) were purchasedfromSantaCruz Biotechnology (SanDiego CA)MurineBV-2 microglial cells were obtained from Dr SW Chae (KoreaInstitute of Oriental Medicine) and cultured in DulbeccorsquosModified Eaglersquos medium (DMEM Lonza Walkersville MDUSA) supplemented with 10 fetal bovine serum (FBSGibco Thermo Fisher Scientific Carlsbad MA USA) and100 120583gmL penicillin-streptomycin (Gibco) at 37∘C in ahumidified atmosphere containing 5 CO2

24 Cell Viability Assay BV2 cells (5 times 105 mL) werecultured in the presence of GBH (125-800 120583gmL) for24 h and stained with 3-(45-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Hndashtetrazolium(MTS Promega USA) for 4 h at 37∘C Absorbance wasmeasured at 490 nm using a microplate reader (MolecularDevices Sunnyvale CA USA)

25 Nitric Oxide Determination BV2 cells (2 times 105 mL)were pretreated with GBH FXT or AMT for 1 h and thenstimulated with LPS (100 ngmL) for 24 h The levels ofnitric oxide in the culture supernatant were determined usinga nitric oxide (NO) detection kit (iNtRON BioTechnologyKorea) according to the manufacturerrsquos instructions

26 ELISA BV2 cells (2 times 105 mL) were pretreated withGBH FXT or AMT for 1 h and then stimulated withLPS (100 ngmL) for 16 h The levels of IL-6 IL-1120573 TNF-120572 and IL-10 in the culture supernatant were determinedusing a commercially available ELISA kit (RampD systemsMinneapolis MN USA) according to the manufacturerrsquosprotocols

27 Real-Time PCR Total RNA was isolated by using Trizol(Invitrogen) and cDNA synthesis was performed using thePrimeScript RT reagent kit (TaKaRa Shiga Japan) IL-1120573 IL-6 TNF-120572 MIP-1120572 MCP-1 IP-10 RANTES iNOSCOX-2 HO-1 and glyceraldehyde-3 phosphate dehydrogenase(GAPDH) mRNA were quantified using a QuantStudio 6Flex real-time polymerase chain reaction (real-time PCR)system (Applied Biosystems CA USA) with Power SYBRGreen PCR Master Mix (Applied Biosystems) [22] Primersequences are enumerated in Table 1

28 Western Blot Analyses Whole lysates were harvestedusing a Radioimmunoprecipitation Assay (RIPA) buffer con-taining protease inhibitor cocktail (Sigma-Aldrich) 1 mMphenylmethylsulfonyl fluoride and phosphatase inhibitorcocktail set III (CalbiochemCAUSA)Nuclear extractswereprepared using a nuclear extract kit (Active Motif CarlsbadCA USA) Equal amounts (20 120583g) of protein were separatedusing 10 sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) and transferred to polyvinylidenefluoride (PVDF) membranes (Amersham Biosciences Pis-cataway NJ USA) which were blocked with 5 skimmilk in


TBST (Tris buffered saline in 01 TWEEN 20) buffer for1 h The membranes were then treated overnight with anti-bodies specific to iNOS NF-120581B p65 HO-1 NRF2 phospho-CREB CREB phospho-p38 (Thr180Tyr182) p38 phospho-Erk (Thr202Tyr204) Erk phospho-JNK (Thr183Tyr185)JNK phospho-Akt (Ser473) and Akt (Cell Signaling Tech-nology Beverly MA) Blots were incubated with horseradishperoxidase- (HRP-) conjugated secondary antibody for 1 hat room temperature HRP was detected using a chemi-luminescent detection reagent (Amersham Biosciences) 120573-actin (Sigma-Aldrich) and proliferating cell nuclear antigen(PCNA Cell Signaling Technology) were used as load-ing controls Chemiluminescence was visualized using anLAS-3000 LuminoImage analyzer (Fujifilm Tokyo Japan)[23]

29 Statistical Analyses All data are expressed as meanplusmn standard deviation (SD) One-way analyses of variance(ANOVAs) were performed using GraphPad Prism ver-sion 7 (GraphPad Software Inc San Diego CA USA) toassess between-group differences Multiple group compar-isons were performed using one-way ANOVAs followed by apost-hocTukey testP valuelt005was considered statisticallysignificant

3 Results

31 LCMS Analysis of Gyejibokryeong-Hwan The composi-tion ofGBHwas verified using LCMSThis analysis providedchemical information which guaranteed the reproducibilityof our experiments in other batches of GBH Analyseswere performed in accordance with the chemical standardsdescribed by Kim et al [13] Quantitative and qualitativeanalyses of the three marker compounds (amygdalin paeoni-florin and cinnamic acid) in GBH were conducted using theLCMS method Each GBH component was identified usingMRMmode The retention times and amounts of each of thethree GBH components are shown in Figure 1

32 Gyejibokryeong-HwanInhibited LPS-Induced NO Produc-tion and iNOS and COX-2 Expression in BV2 Microglia Wefirst performed an MTS assay to determine the cytotoxicityand antineuroinflammatory and neuroprotective effects ofGBH The result of this assay demonstrated that GBH doesnot have cytotoxic effect at up to 800 120583gmL (Table 2) Toinvestigate the effects of GBH on NO production BV2 cellswere treated with LPS (100 ngmL) in presence or absence ofGBH The production of NO was increased up to seven-foldcompared over that of controls which treatment with GBH(50-400 120583gmL) significantly blocked in a dose-dependentmanner (Figure 2(a)) iNOS protein levels were also dramat-ically increased in LPS-treated controls and similar to NOtreatment with GBH (400 120583gmL) suppressed these to nearlycontrol levels (Figure 2(b)) Further as shown in Figures2(c) and 2(d) iNOS and COX-2 mRNA levels were greatlyincreased by LPS stimulation GBH (400 120583gmL) significantlysuppressed these iNOS and COX-2 gene expression increasesFurthermore FXT and AMT (10 120583M) positive controls usedin this study also had inhibitory effects on iNOS and NO

Table 2 Effects of Gyejibokryeong-hwan on cell viability

Concentration (120583gmL) Cell viability ()GBH

0 10115 plusmn 103125 10735 plusmn 06825 10782 plusmn 10250 10950 plusmn 145100 11005 plusmn 093200 10601 plusmn 183400 10765 plusmn 343800 10737 plusmn 296

production These data suggest that GBH may inhibit NOproduction through downregulation of iNOS and COX-2

BV2 cells were treated with various concentrations ofGBH for 24 h and then cell viability was measured byMTS assay The data represent the mean plusmn SD of triplicatedeterminations MTS 3-(45-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2Hndashtetrazolium

33 Gyejibokryeong-Hwan Inhibited LPS-Induced Proinflam-matory Cytokine and Chemokine Expression in BV2MicrogliaThe LPS-stimulated microglia produce high levels ofcytokines such as IL-6 IL-1120573 and TNF-120572 The mRNA levelsof IL-6 IL-1120573 and TNF-a which were dramatically increasedby LPS stimulation were significantly decreased by GBHin a dose-dependent manner (Figures 3(a)ndash3(c)) FXT andAMT (10 120583M) also showed inhibitory effect on mRNA levelsof IL-6 IL-1120573 and TNF-120572 Furthermore production of IL-6IL-1120573 and TNF-120572 was markedly increased in LPS-treatedcontrol however the pretreatment with GBH significantlyinhibited the cytokine levels in a dose-dependent manner(Figures 3(d)ndash3(f)) The mRNA levels of chemokines such asMIP-1120572 MCP-1 IP-10 and RANTES were greatly increasedin LPS-treated control but the presence of GBH significantlysuppressed the levels in a dose-dependent manner (Figures4(a)ndash4(d))These data suggest that GBHmight downregulateLPS-induced proinflammatory cytokines and chemokines

34 Gyejibokryeong-Hwan Inhibited LPS-Induced Activationof MAPK PI3KAkt and NF-120581B Inflammatory Pathways inBV2 Microglia MAPKs PI3KAkt and NF-120581B play impor-tant roles in the signaling pathways that induce a neu-roinflammatory response in microglia [24] Given this theeffects of GBH on MAPKs PI3KAkt and NF-120581B pathwayswere examined here As shown in Figure 5(a) the levels ofphosphorylated JNK in control cells were minimal whiletreatment with LPS dramatically increased these levels Addi-tionally GBH lowered phosphorylated JNK levels while totalJNK levels were unchanged Levels of phosphorylated Erkwere dramatically increased after LPS increases correctedby GBH treatment However levels of phosphorylated p-38 MAPK were unchanged by GBH Furthermore thephosphorylation of Akt was significantly increased by LPStreatment while total Akt was not affected With GBH


Amygdalin - STD

Amygdalin362

105855175106e6

Amygdalin - GBH

Amygdalin356

1071361107e4

F3MRM of 1 channelES+47493 gt 32511

1795e+005


1664e+007100

0250 500 750

min

100

0250 500 750

min

(a)

Paeoniflorin Paeoniflorin361

121163100121e6

Paeoniflorin - GBH

35419056984


1855e+007

191e5


3337e+006100

0250 500 750

min

100

0250 500 750

min

Paeoniflorin - STD

(b)

- Cinnamic acid

- Cinnamic acid

002040608

11214

005

115

225

335

4

19181716151413121110987654321Counts vs Acquisition Time (min)


times104

times103 +ESI MRM Frag=500V CID50 (14910000-gt 13100000) Geigi_1000Xd - GBH

+ESI MRM Frag=500V CID50 (14910000-gt 13100000) STD 1000ppbd -STD

(c)

Marker compound Retention time (min)

Amount (mgg) Sources

Amygdalin 36 1133 Prunus persica Batsch

Paeoniflorin 36 1593Paeonia lactiflora Pallas

Paeonia suffruticosa Andrews

Cinnamic acid 57 026 Cinnamomum cassia Presl

(d)

Figure 1 Chromatograms of Gyejibokryeong-hwan (GBH) samples based on liquid chromatography-mass spectrometry (LCMS) analysisLCMS chromatograms of a standard mixture and of the GBH sample revealed (a) amygdalin (b) paeoniflorin and (c) cinnamic acid (d)Identification of GBH components LCMS liquid chromatography-mass spectrometry

levels of phosphorylated Akt were significantly reduced NF-120581B is a key transcription factor that modulates iNOS andproinflammatory cytokine and chemokine gene expression inmicroglia Western blot analyses using nuclear extract fromtreated and control BV2 cells indicated that expression ofp65 a component of NF-120581B significantly increased by LPS

stimulation Pretreatment with GBH significantly inhibitedexpression of these factors in the nucleus (Figure 5(b))These data suggest that GBH may interfere with ErkJNK and Akt to facilitate altered NF-120581B pathway signalingand inhibit neuronal proinflammatory responses to LPSstimulation


0

2

4

6

8

Nitr

ite (u

M)

lowast

lowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

LPSGBH (gml) -

--+ ++++++

50 FXTAMT400200100

(a)

iNOS

-actin

0

2

4

6

8

10

iNO

S re

lativ

e int

ensit

y

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

GBH (μgml) - -

+ LPS

LPSGBH (gml) -

--

50 FXTAMT400200100

+ ++++++50 FXTAMT400200100

(b)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

lowastlowastlowast

lowastlowastlowast

(c)

0

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowast

lowastlowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

(d)

Figure 2 Effect of Gyejibokryeong-hwan on NO production and iNOS and COX-2 expression in BV2 cells BV2 cells were pretreated withGBH for 1 h and then stimulated with LPS (100 ngmL) for 24 h (a) The levels of NO in the cell culture supernatant were measured by NOdetection kit (b) The level of iNOS was determined by western blot analyses 120573-actin was used as a loading control The data represent threeindependent experiments BV2 cells were pretreated with GBH for 1 h and then stimulated with LPS (100 ngmL) for 6 h Levels of (c) iNOSand (d) COX-2 mRNA were determined by real-time PCR GAPDH was used as a loading control The data represent the mean plusmn SD oftriplicate determinations (one-way ANOVA p lt 0001 vs control lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 vs LPS-treated control)

35 Gyejibokryeong-Hwan Increased IL-10 and HO-1 Ex-pression via Upregulation of NRF2CREB Pathways in BV2Microglia IL-10 and HO-1 act as anti-inflammatory andantioxidant modulators via their upregulation of NRF2CREB pathways in microglia and other cells [25] Giventhis we examined the effect of GBH on IL-10 and HO-1expression in LPS-treated and control BV2 cells Levels of IL-10 in controls (137 pgmL) increased in a dose-dependent

fashion with GBH LPS-treated cells produced similar levelsof IL-10 as controls though cells treated with both LPS andGBH had increased IL-10 levels compared to those treatedwith GBH only (Figure 6(a)) Next we examined mRNA andprotein expression of HO-1 both of which were significantlyincreased in LPS-treated control cells over true control GBHand LPS treatment increased HO-1 expression over levelsin GBH control cells (Figures 6(b) and 6(c)) CoPP (20


(a)

(b)

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowastlowast lowastlowast

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

0

5

10

15

TNF-

re

lativ

e exp

ress

ion


lowastlowastlowast


(d)

(e)

(f)

0

25

50

75

100

IL-1

(pg

ml)

0

25

50

75

100

IL-6

(pg

ml)


lowastlowastlowastlowast



0

200

400

600

TNF-

(p

gm

l)

lowastlowastlowast

lowastlowastlowast


LPSGBH (gml)

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

Figure 3 Effect of Gyejibokryeong-hwan on mRNA expression of proinflammatory cytokines in BV2 cells BV2 cells were pretreated withGBH for 1 h and then stimulatedwith LPS (100 ngmL) for 6 h mRNA expression levels of (a) IL-6 (b) IL-1120573 and (c) TNF-120572were determinedby real-time PCR GAPDH was used as a loading control BV2 cells were pretreated with GBH for 1 h and then stimulated with LPS (100ngmL) for 16 h Levels of (d) IL-6 (e) IL-1120573 and (f) TNF-120572 in the cell culture supernatant were measured via ELISA The data representthe mean plusmn SD of triplicate determinations (one-way ANOVA p lt 0001 vs control lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 vs LPS-treatedcontrol)

120583M) was used as a western blot positive control to induceHO-1 expression (Figure 6(c)) Furthermore GBH increasednuclear translocation of NRF2 and phosphorylated CREBwhich acts as an upstream modulator of HO-1 expression(Figure 6(d)) These data suggest that GBHmight upregulateHO-1 via NRF2CREBpathways involved in the neuroprotec-tive effects in BV2 microglia

36 HO-1 Mediates the Effects of Gyejibokryeong-Hwanon NO Production and Proinflammatory Cytokine mRNAin BV2 Microglia To confirm the effects of GBH on

proinflammatory signaling pathways we examined whetherHO-1 mediates the effect of GBH on NO productionand proinflammatory cytokines gene expression This wasassessed by cotreating cells with SnPP an inhibitor ofHO-1 activity We found that GBH significantly reducedNO production and iNOS COX-2 and proinflammatorycytokine gene expression levels However SnPP did notreverse the inhibitory effects of GBH on NO produc-tion and iNOS COX-2 and proinflammatory cytokinemRNA levels (Figures 7(a)ndash7(f)) Collectively these resultssuggest that GBH act as an antineuroinflammatory and


0

5

10

15

20

MIP

-1

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(a)

0

5

10

15

MCP

-1 re

lativ

e exp

ress

ion

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(b)

0

50

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250

IP-1

0 re

lativ

e exp

ress

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lowastlowastlowast

lowast


+ + + + + +- 100 200 400 AMT FXT

LPSGBH (gml) -

-

(c)

0

20

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lowast lowastlowast

+ + + + + +

- 100 200 400 AMT FXTLPS

GBH (gml) --

RAN

TES

relat

ive e

xpre

ssio

n

(d)

Figure 4 Effect of Gyejibokryeong-hwan onmRNA expression of proinflammatory chemokines in BV2 cells BV2 cells were pretreated withGBH for 1 h and then stimulated with LPS (100 ngmL) for 6 h Levels of (a) MIP-1120572 (b) MCP-1 (c) IP-10 and (d) RANTES mRNA weredetermined by real-time PCR GAPDHwas used as a loading controlThe data represent themean plusmn SD of triplicate determinations (one-wayANOVA plt 0001 vs control lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 vs LPS-treated control)

neuroprotective agent in BV2 microglia via upregulation ofHO-1

4 Discussion

In adults microglia represent about 10 of the cells inthe brain and spinal cord [26] Microglia are involved ininnate immunity and regulate cytokines and inflammatoryprocesses in the brain [27] When activated by infectionor tissue damage microglia produce inflammatory factorsincluding proinflammatory cytokines chemokines and reac-tive oxidant species which can cause neuronal toxicity anddegeneration [28] Neuroprotective factors such as the anti-inflammatory cytokine IL-10 and HO-1 inhibit neuroinflam-mation and neuronal cell death [29]

Moreover in recent studies microglia have been sug-gested as an important factor leading to depression byregulating neuroimmune pathways affecting neuroplasticity[1 2] Various studies have been conducted to demonstratethis antidepressant effect using BV2 microglia cells [30 31]In a previous study by our group GBH was shown toinhibit neuroinflammation in the hippocampus of a mousemodel of reserpine-induced depression [19]Thus the present

in vitro study sought to understand the mechanisms ofthe antineuroinflammation effects of GBH observed in ourprevious study on in vivomodels of depression

Specifically antidepressants are known to inhibit theproduction of proinflammatory cytokines and suppress mi-croglial activation [32ndash35] FXT (selective serotonin reuptakeinhibitors SSRI) and AMT (tricyclic antidepressants TCA)have been reported to induce anti-inflammatory effects inactivated microglia and were therefore used as positivecontrols in the present study [36ndash38] In the experimentspresented here we demonstrated a molecular mechanismfor the antineuroinflammatory and neuroprotective effectsof GBH in LPS-activated microglia The proposed mech-anism underlying the effects of GBH is summarized inFigure 8

NO is a free radical that plays an important role invarious physiological and pathological processes Underphysiological conditions NO is involved in defense againsttumors parasites and bacteria [39] However when it isoverproduced NO acts as a toxic radical causing damage tocells and tissues [40]The expression of iNOS andCOX-2 keyenzymes inNOproduction has been reported to be increasedin activated glial cells [41] In the present study we found


p-p38

p38

-actin

p-Erk

Erk

p-JNK

JNK

00

05

10

15

20

25

p-ER

KER

K re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

0

50

100

150

p-p3

8p3

8 re

lativ

e int

ensit

y

p-Akt

Akt

GBH (μgml) -- 100

+ LPS

0

50

100

150

200

p-JN

KJN

K re

lativ

e int

ensit

y

-

lowastlowast

lowastlowastlowast


lowastlowastlowast

0

10

20

30

40

p-A

KTA

KT re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast

LPS -GBH (gml) - 100 FXTAMT400200

+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

FXTAMT400200

(a)

NF-B p65

PCNA

0

1

2

3

4

NF-

B

p65

relat

ive i

nten

sity

lowastlowastlowast

lowastlowast

lowastlowastlowast

GBH (μgml) --

+ LPS

100 FXTAMT400200

LPS - + +++++GBH (gml) -- 100 FXTAMT400200

(b)

Figure 5 Effect of Gyejibokryeong-hwan on the phosphorylation of MAPK and Akt and NF-120581B activity in BV2 cells (a) BV2 cells werepretreated with GBH for 1 h and then stimulated with LPS (100 ngmL) for 15 min Levels of p-JNK JNK p-Erk Erk p-p38 p38 p-Akt andAkt were determined by western blot analyses 120573-actin was used as a loading control (b) BV2 cells were pretreated with GBH for 1 h andthen stimulated with LPS (100 ngmL) for 1 h Nuclear extracts were analyzed by western blot analysis using an NF-120581B p65 antibody PCNAwas used as a loading control The data represent three independent experiments The data are expressed as the mean plusmn SD of triplicatedeterminations (one-way ANOVA p lt 0001 vs control lowastlowastplt 001 lowastlowastlowastp lt 0001 vs LPS-treated control)


0

20

40

60IL

-10

(pg

ml)

- - - - - -

lowastlowastlowast


lowastlowastlowast

LPS - + ++++++GBH (gml) - 50 - 50 FXTAMT400200100FXTAMT400200100

(a)

0

2

4

6

8

10

HO

-1 re

lativ

e exp

ress

ion

lowastlowastlowast

lowastlowastlowast

- - - - -LPS - + +++++

GBH (gml) - - FXTAMT400200100FXTAMT400200100

(b)

GBH (gml)HO-1

-actin

+ LPSAMT FXT100 400200 -- AMT FXT100 400200 CoPP

(c)

NRF2

PCNA

p-CREB

CREB

-actin

0

2

1

3

0

6

12

18

Rela

tive i

nten

sity

GBH (gml)

NRF2

p-CREBCREBB

GBH (gml) - 100 FXTAMT400200

100- FXTAMT400200

(d)

Figure 6 Effect of Gyejibokryeong-hwan on IL-10 production and HO-1 expression in BV2 cells (a) BV2 cells were pretreated with GBHfor 1 h and then stimulated with LPS (100 ngmL) for 16 h Levels of IL-10 in the cell culture supernatant were measured via ELISA (b) BV2cells were pretreated with GBH for 1 h and then stimulated with LPS (100 ngmL) for 6 h HO-1mRNA levels were determined by real-timePCR GAPDH was used as a loading control (c) BV2 cells were pretreated with GBH for 1 h and then stimulated with LPS (100ngmL) for12 h The level of HO-1 was determined by western blot 120573-actin was used as a loading control (d) BV2 cells were pretreated with GBH for 6h Nuclear extracts were analyzed by western blot using an NRF2 antibody PCNA was used as a loading control BV2 cells were pretreatedwith GBH for 30 min Levels of p-CREB and CREB were determined by western blot analyses 120573-actin was used as a loading control Thedata represent three independent experiments The data are expressed as the mean plusmn SD of triplicate determinations (one-way ANOVA plt 005 p lt 001 p lt 0001 vs control lowastlowastlowastp lt 0001 vs LPS-treated control)

that GBH inhibited NO production by suppressing iNOS andCOX-2 mRNA and protein levels

Proinflammatory cytokines play an important role in theimmune response and their excessive production has beenreported to exacerbate various inflammatory responses suchas neurodegenerative disorders multiple sclerosis [42] Basedon the results reported here mRNA expression levels ofTNF-120572 IL-1120573 and IL-6 were inhibited by GBH Further-more chemokines increase the infiltration of inflammatoryimmune cells in the brain [43] Such recruitment may con-tribute to neuropathologies including neuropathic pain after

nerve injury [44] Our data clearly demonstrated that BV2cell treatment with GBH significantly reduced mRNA levelsof IP-10 MIP-1120572 MCP-1 and RANTES in LPS-stimulatedmicroglia

Activation of Toll-like receptors (TLR) 4MyD88 byLPS stimulation of microglia triggers NF-120581B MAPKs andPI3KAktWith phosphorylation of IkB anNF-120581B inhibitoryprotein that masks the nuclear localization signal of NF-120581B the protein is ubiquitinated and degraded freeing it totranslocate into the nucleus and initiate the transcription ofinflammatory genes [45ndash47]Thepresent study demonstrated


0

2

4

6

8

Nitr

ite (u

M)

lowastlowastlowast

daggerdaggerdagger

SnPP (M) - - - 10 10 50 50GBH (400gml) - - + - + - +

LPS - + + + + + +

(a)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(b)

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

0

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

GBH (400gml) -SnPP (10M) - + - - + +

- - + - +LPS - - + + + +

(d)

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(e)

0

5

10

15

20

TNF-

re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(f)

Figure 7 HO-1mediates the effect of Gyejibokryeong-hwan onNO production andmRNA expression of proinflammatory cytokines in BV2cells (a) BV2 cells were pretreated with GBH for 1 h in the presence or absence of SnPP (10 120583M) and then stimulated with LPS (100 ngmL)for 24 h Levels of NO in the cell culture supernatant were measured by an NO detection kit BV2 cells were pretreated with GBH for 1 hin the presence or absence of SnPP (10 120583M) and then stimulated with LPS (100 ngmL) for 6 h Levels of (b) iNOS (c) COX-2 (d) IL-6 (e)IL-1120573 and (f) TNF-120572mRNA were determined by real-time PCR GAPDH was used as a loading control The data represent the mean plusmn SDof triplicate determinations (one-way ANOVA p lt 0001 vs control lowastlowastlowastp lt 0001 vs LPS-treated control daggerdaggerdaggerp lt 0001 vs LPS+GBH)

that GBH suppressed the activation of NF-120581B and thephosphorylation of JNK and Erk in specific MAPKs andAkt in LPS-stimulated BV2 cells Taken together those anti-inflammatory effects of GBHmight lead to diminished levelsof neuropathological factors such as NO proinflammatorycytokines and chemokines as also evidence here

Induction of HO-1 can serve as a potent protectoragainst inflammation and cell death via the generation of

its by-products including carbon monoxide biliverdin andiron [48] Recent studies have shown that NRF2 and CREBare important upstream contributors to HO-1 induction [25]In particular NRF2 has been reported to inhibit NF-120581Bactivation [49] In the present study we demonstrated thatGBH significantly increased HO-1 expression via increasednuclear translocation of NRF2 and CREB phosphorylationFurthermore levels of IL-10 a canonical anti-inflammatory


Inhibition by GBH Activation

Figure 8 The proposed mechanism underlying the antineuroinflammatory and neuroprotective effects of Gyejibokryeong-hwan inlipopolysaccharide-stimulated BV2 microglia

cytokine [50] were increased by GBH in both the presenceand absence of LPS Indeed the use of an HO-1 antagonistalso reversed the effect of GBH on NO production as well ason increased iNOS COX-2 and proinflammatory cytokinemRNA levels The regulation and balance of NF-120581B andNRF2 levels is critical to neuroinflammatory processes andto neuroprotection [49] Despite this understanding and themolecular markers implicated in in vitro work here furtherstudies are needed to fully elucidate the coregulation negativefeedback and competitive binding mechanisms by whichthese markers behave in vivo

5 Conclusions

Recently scientific work has begun to focus on the anti-inflammatory and antioxidant properties of natural medi-cines which may be effective in treating major neurode-generative autoimmune and cancerous disease states witha minimal risk of side effects [51] GBH one such activenatural compound has been reported to be safe at a doseof 500 mgkgday regardless of sex [52 53] Before the useof this compound can be fully realized in a clinical contexthowever studies must address its molecular mechanisms andactivity as we have endeavored to do here Taken togetherour results suggest that GBH may act as a neuroprotectiveagent by downregulating neuroinflammation in activatedcultured microglia GBH may thus have significant ther-apeutic potential for the treatment of depression associ-ated with microglial activation status without serious sideeffects

Data Availability

The datasets used andor analyzed in the current study areavailable from the corresponding author upon reasonablerequest The role of the funding body in the design of thestudy and collection analysis and interpretation of dataand in writing the manuscript should be declared in thisrequest

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by a grant (KSN1621060) from theKorea Institute of Oriental Medicine (KIOM) and a grantfrom the National Research Foundation of Korea (NRF)funded by the Korean government (MSIP) (grant numberNRF-2016R1A2B4009614)The funding bodies played no rolein the design of the study collection analyses interpretationof data or writing the manuscript

References

[1] R Yirmiya N Rimmerman and R Reshef ldquoDepression as amicroglial diseaserdquo Trends in Neurosciences vol 38 no 10 pp637ndash658 2015

[2] G Singhal and B T Baune ldquoMicroglia an interface betweenthe loss of neuroplasticity and depressionrdquo Frontiers in CellularNeuroscience vol 11 p 270 2017

[3] K Biber J Vinet andHWG M Boddeke ldquoNeuron-microgliasignaling chemokines as versatile messengersrdquo Journal of Neu-roimmunology vol 198 no 1-2 pp 69ndash74 2008

[4] R Fu Q Shen P Xu J J Luo and Y Tang ldquoPhagocytosis ofmicroglia in the central nervous system diseasesrdquo MolecularNeurobiology vol 49 pp 1422ndash1434 2014

[5] C Guo L Yang C-X Wan et al ldquoAnti-neuroinflammatoryeffect of Sophoraflavanone G from Sophora alopecuroidesin LPS-activated BV2 microglia by MAPK JAKSTAT andNrf2HO-1 signaling pathwaysrdquo Phytomedicine vol 23 no 13pp 1629ndash1637 2016

[6] M J Choi E J Lee J S Park et al ldquoAnti-inflammatory mech-anism of galangin in lipopolysaccharide-stimulated microgliacritical role of pp AR-Agamma signaling pathwayrdquoBiochemicalPharmacology vol 144 pp 120ndash131 2017

[7] B Li D S Lee H G Choi et al ldquoInvolvement of hemeoxygenase-1 induction in the cytoprotective and immunomod-ulatory activities of viola patrinii in murine hippocampal and


microglia cellsrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 128019 12 pages 2012

[8] G Y Sun Z Chen K J Jasmer et al ldquoQuercetin attenuatesinflammatory responses in BV-2microglial cells role ofMAPKson the Nrf2 pathway and induction of heme oxygenase-1rdquo PLoSONE vol 10 no 10 p e0141509 2015

[9] H JunDonguibogam Namsandang Seoul South Korea 1980[10] J S Park S Park C H Cheon et al ldquoEffects and safety

of gyejibongnyeong-hwan on dysmenorrhea caused by bloodstagnation a randomized controlled trialrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID424730 6 pages 2013

[11] S-J Jeong H-S Lim C-S Seo et al ldquoAnti-inflammatoryactions of herbal formula Gyejibokryeong-hwan regulated byinhibiting chemokine production and STAT1 activation inHaCaT cellsrdquo Biological amp Pharmaceutical Bulletin vol 38 no3 pp 425ndash434 2015

[12] X Lei J Chen C Liu J Lin J Lou and H Shang ldquoStatus andthoughts of Chinese patent medicines seeking approval in theUS marketrdquo Chinese Journal of Integrative Medicine vol 20 no6 pp 403ndash408 2014

[13] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds inGyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[14] S-H Hwang Y G Choi M-Y Jeong Y-M Hong J-H Leeand S Lim ldquoMicroarray analysis of gene expression profileby treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cellsrdquo Gene vol 443 no 1-2 pp 83ndash90 2009

[15] S Rho H-S Chung M Kang et al ldquoInhibition of productionof reactive oxygen species and gene expression profile by treat-ment of ethanol extract of Moutan Cortex Radicis in oxidativestressed PC12 cellsrdquo Biological amp Pharmaceutical Bulletin vol28 no 4 pp 661ndash666 2005

[16] C Yang J Zhao Y Cheng X Li and J Rong ldquoBioactivity-guided fractionation identifies amygdalin as a potent neu-rotrophic agent from herbal medicine semen persicae extractrdquoBioMed Research International vol 2014 Article ID 306857 10pages 2014

[17] B Hu G Xu X Zhang et al ldquoPaeoniflorin attenuates inflam-matory pain by inhibiting microglial activation and Akt-NF-120581Bsignaling in the central nervous systemrdquoCellularPhysiology andBiochemistry vol 47 no 2 pp 842ndash850 2018

[18] S Chakrabarti M Jana A Roy and K Pahan ldquoUpregulationof suppressor of cytokine signaling 3 in microglia by cinnamicacidrdquo Current Alzheimer Research vol 15 no 10 pp 894ndash9042018

[19] B-K Park Y R Kim Y H Kim et al ldquoAntidepressant-likeeffects of gyejibokryeong-hwan in a mouse model of reserpine-induced depressionrdquo BioMed Research International vol 2018no 59 Article ID 5845491 12 pages 2018

[20] C T Ekdahl Z Kokaia and O Lindvall ldquoBrain inflammationand adult neurogenesis the dual role of microgliardquo Neuro-science vol 158 no 3 pp 1021ndash1029 2009

[21] C De Lucia A Rinchon A Olmos-Alonso et al ldquoMicrogliaregulate hippocampal neurogenesis during chronic neurode-generationrdquoBrain Behavior and Immunity vol 55 pp 179ndash1902016

[22] B-K Park Y-C Park I C Jung et al ldquoGamisasangja-tang sup-presses pruritus and atopic skin inflammation in the NCNga

murine model of atopic dermatitisrdquo Journal of Ethnopharma-cology vol 165 pp 54ndash60 2015

[23] B-K Park S Park J-B Park M C Park T S Min and MJin ldquoOmega-3 fatty acids suppressTh2-associated cytokine geneexpressions and GATA transcription factors in mast cellsrdquoTheJournal of Nutritional Biochemistry vol 24 no 5 pp 868ndash8762013

[24] S Park J J Choi B-K Park S J Yoon J E Choi and M JinldquoPheophytin a and chlorophyll a suppress neuroinflammatoryresponses in lipopolysaccharide and interferon-120574-stimulatedBV2 microgliardquo Life Sciences vol 103 no 2 pp 59ndash67 2014

[25] E-J Lee H-M Ko Y-H Jeong E-M Park and H-S Kimldquo120573-Lapachone suppresses neuroinflammation by modulatingthe expression of cytokines and matrix metalloproteinases inactivated microgliardquo Journal of Neuroinflammation vol 12 no1 p 133 2015

[26] MW Salter andB Stevens ldquoMicroglia emerge as central playersin brain diseaserdquo Nature Medicine vol 23 no 9 pp 1018ndash10272017

[27] G J Harry and A D Kraft ldquoNeuroinflammation andmicrogliaconsiderations and approaches for neurotoxicity assessmentrdquoExpert Opinion on Drug Metabolism amp Toxicology vol 4 no10 pp 1265ndash1277 2008

[28] M B Graeber and W J Streit ldquoMicroglia biology and pathol-ogyrdquo Acta Neuropathologica vol 119 no 1 pp 89ndash105 2010

[29] D Lobo-Silva G M Carriche A G Castro S Roque and MSaraiva ldquoBalancing the immune response in the brain IL-10and its regulationrdquo Journal of Neuroinflammation vol 13 no1 article no 297 2016

[30] D-Y Lu Y-Y Tsao Y-M Leung and K-P Su ldquoDocosa-hexaenoic acid suppresses neuroinflammatory responses andinduces heme oxygenase-1 expression in BV-2microglia Impli-cations of antidepressant effects for omega-3 fatty acidsrdquo Neu-ropsychopharmacology vol 35 no 11 pp 2238ndash2248 2010

[31] M Dubovicky E Csaszar K Melichercıkova M Kuniakovaand L Rackova ldquoModulation of microglial function by theantidepressant drug venlafaxinerdquo Interdisciplinary Toxicologyvol 7 no 4 pp 201ndash207 2014

[32] S Hashioka A Klegeris A Monji et al ldquoAntidepressantsinhibit interferon-120574-inducedmicroglial production of IL-6 andnitric oxiderdquo Experimental Neurology vol 206 no 1 pp 33ndash422007

[33] A M Bielecka M Paul-Samojedny and E ObuchowiczldquoMoclobemide exerts anti-inflammatory effect in lipopolysac-charide-activated primary mixed glial cell culturerdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 382 no 5-6 pp409ndash417 2010

[34] F R Walker ldquoA critical review of the mechanism of actionfor the selective serotonin reuptake inhibitors Do these drugspossess anti-inflammatory properties andhow relevant is this inthe treatment of depressionrdquo Neuropharmacology vol 67 pp304ndash317 2013

[35] E Obuchowicz A M Bielecka M Paul-Samojedny APudełko and J Kowalski ldquoImipramine and fluoxetine inhibitLPS-induced activation and affect morphology of microglialcells in the rat glial culturerdquo Pharmacological Reports vol 66no 1 pp 34ndash43 2014

[36] E Ha K H Jung B-K Choe et al ldquoFluoxetine increases thenitric oxide production via nuclear factor kappa B-mediatedpathway in BV2 murine microglial cellsrdquo Neuroscience Lettersvol 397 no 3 pp 185ndash189 2006


[37] D Liu Z Wang S Liu F Wang S Zhao and A Hao ldquoAnti-inflammatory effects of fluoxetine in lipopolysaccharide(LPS)-stimulated microglial cellsrdquo Neuropharmacology vol 61 no 4pp 592ndash599 2011

[38] EObuchowicz J Kowalski K Labuzek R Krysiak J Pendzichand Z S Herman ldquoAmitriptyline and nortriptyline inhibitinterleukin-1 release by rat mixed glial and microglial cell cul-turesrdquo The International Journal of Neuropsychopharmacologyvol 9 no 1 pp 27ndash35 2006

[39] M A Panaro O Brandonisio A Acquafredda M Sistoand V Mitolo ldquoEvidences for iNOS expression and nitricoxide production in the human macrophagesrdquo Current DrugTargetsmdashImmune Endocrine amp Metabolic Disorders vol 3 no3 pp 210ndash221 2003

[40] H Scheiblich andG Bicker ldquoNitric oxide regulates antagonisti-cally phagocytic and neurite outgrowth inhibiting capacities ofmicrogliardquoDevelopmental Neurobiology vol 76 no 5 pp 566ndash584 2016

[41] T Filipovich-Rimon and S Fleisher-Berkovich ldquoGlial responseto lipopolysaccharide Possible role of endothelinsrdquo Peptidesvol 31 no 12 pp 2269ndash2275 2010

[42] E Ellwardt and F Zipp ldquoMolecular mechanisms linking neu-roinflammation and neurodegeneration in MSrdquo ExperimentalNeurology vol 262 Pt A pp 8ndash17 2014

[43] Y B Lee A Nagai and S U Kim ldquoCytokines chemokines andcytokine receptors in humanmicrogliardquo Journal of NeuroscienceResearch vol 69 no 1 pp 94ndash103 2002

[44] N Kiguchi Y Kobayashi and S Kishioka ldquoChemokines andcytokines in neuroinflammation leading to neuropathic painrdquoCurrent Opinion in Pharmacology vol 12 no 1 pp 55ndash61 2012

[45] H Shimizu K Mitomo T Watanabe S Okamoto and KYamamoto ldquoInvolvement of a NF-kappa B-like transcriptionfactor in the activation of the interleukin-6 gene by inflamma-tory lymphokinesrdquo Molecular and Cellular Biology vol 10 no2 pp 561ndash568 1990

[46] I O Han K W Kim J H Ryu and W K Kim ldquop38mitogen-activated protein kinase mediates lipopolysaccharidenot interferon-120574 -induced inducible nitric oxide synthaseexpression inmouse BV2microglial cellsrdquoNeuroscience Lettersvol 325 no 1 pp 9ndash12 2002

[47] Q Shen R Zhang and N R Bhat ldquoMAP kinase regulation ofIP10CXCL10 chemokine gene expression in microglial cellsrdquoBrain Research vol 1086 no 1 pp 9ndash16 2006

[48] N G Abraham J Alam and K Nath Heme Oxygenase inBiology and Medicine Kluwer AcademicPlenum PublishersNew York NY USA 2002

[49] J DWardyn A H Ponsford and CM Sanderson ldquoDissectingmolecular cross-talk between Nrf2 and NF-120581B response path-waysrdquo Biochemical Society Transactions vol 43 no 4 pp 621ndash626 2015

[50] I Bozic D Savic D Laketa et al ldquoBenfotiamine attenuatesinflammatory response in LPS stimulated BV-2 microgliardquoPLoS ONE vol 10 no 2 Article ID e0118372 2015

[51] M Rasool A Malik M S Qureshi et al ldquoRecent updatesin the treatment of neurodegenerative disorders using naturalcompoundsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2014 Article ID 979730 7 pages 2014

[52] Y Kobayashi S Minematsu S Maemura C Atkinson andM Mulhern ldquoA single oral dose toxicity study and a 13-week repeated dose study with a 4-week recovery periodof TSUMURA Keishi-bukuryo-gan (TJ-25) in ratsrdquo JapanesePharmacology andTherapeutics vol 23 no 7 pp 151ndash168 1995

[53] M Lee C Seo H Ha E Park J Kim and H Shin ldquoThegenotoxicity of an aqueous extract of Gyejibokryeong-hwanrdquoBMC Complementary and Alternative Medicine vol 18 no 1 p21 2018

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


120581B p65 an inflammatory transcription factor into thenucleus via phosphatidylinositol 3-kinase (PI3K)Akt whereit increases the production of various proinflammatorycytokines and chemokines [5 6] In contrast an increasein neuroprotective factors such as nuclear factor erythroid2-related factor 2 (NRF2) cAMP response element-bindingprotein (CREB) and Heme oxygenase-1 (HO-1) can pro-duce the anti-inflammatory cytokine IL-10 and thus inhibitneuroinflammation Given this central role of microglia inneuroinflammation controlling the activation of microgliacells through upregulation of HO-1 may serve as a criticalpotential target mechanism for the treatment of neurologicaland neurodegenerative diseases [7 8]

Gyejibokryeong-hwan (GBH) is a traditional Koreanmedicine that is described in Donguibogam a textbook oftraditional Korean medicines [9] GBH has been extensivelyused throughout Asia in the treatment of blood stasis [1011] and is approved by the Ministry of Food and Drugsafety (MFDS) and the USA Food and Drug Administration(FDA) having been studied for safety and composition GBHcontains herbs including Cinnamomum cassia Presl Poriacocos Wolf Paeonia suffruticosa Andrews Paeonia lactifloraPallas and Prunus persica Batsch [12 13]

Cinnamomum cassia Presl a constitutive agent inGBH has been shown to exhibit anti-inflammatory effectsin murine BV2 microglia cells and Paeonia suffruticosaAndrews another element of GBH has been reported toinduce antioxidative stress effects in rat pheochromocytomaPC-12 cells [14 15] Moreover amygdalin a componentof Prunus persica Batsch induces neurotrophic effectsvia the activation of the extracellular signal-regulatedkinase (ERK)12 pathway in PC12 cells [16] paeoniflorin acomponent of Paeonia suffruticosa Andrews and Paeonialactiflora Pallas reduces proinflammatory cytokines inCNS via the activation of Akt-NF-120581B signaling pathway invivo and in vitro [17] and cinnamic acid a component ofCinnamomum cassia Presl upregulates the expression ofSOCS3 in glial cells via the CREB pathway [18]

In a previous study conducted by our group GBH wasshown to exhibit antineuroinflammation effects in the hip-pocampus of amousemodel of reserpine-induced depression[19] Several studies have reported that microglia regulatehippocampal neurogenesis in neurodegeneration [20 21]Despite these promising results the antineuroinflammationand neuroprotection effects of GBH inmurine BV2microgliahave yet to be investigated Therefore in the present study weconfirmed the antineuroinflammatory and neuroprotectiveeffects of GBH via several mechanisms in a proinflammatorymicroglia model where murine BV2 microglia were treatedwith LPS

2 Materials and Methods

21 Preparation of Gyejibokryeong-Hwan (GBH) GBH waspurchased from Hanpoong Pharm and Foods Co Ltd(Jeonju Korea) A voucher specimen of the herb sample wasdeposited in the Herbarium of Hanpoong Pharm and FoodsCo Ltd (voucher no 17248) The five-component herbs ofGBH (combined at a 11111 ratio total weight = 17 kg) were

boiled in water for 3 h to create an extract which was thenfiltered and concentrated via vacuum pressure The extractyield was approximately 2953 The extract was stored at -80∘C and dissolved in phosphate-buffered saline (PBS) beforeuse

22 Reagents and Liquid ChromatographyMass Spectrometry(LCMS) Analysis of Gyejibokryeong-Hwan

221 Amygdalin and Paeoniflorin Standards for amygdalin(PubChem CID 656516 purity 990) and paeoniflorin(PubChem CID 442534 purity 988) were purchasedfrom Sigma-Aldrich (St Louis MO USA) Solutions ofamygdalin and paeoniflorin were prepared in analyticalgrade methanol (Merck Darmstadt Germany) An opti-mized multiple reaction monitoring (MRM) method wasdeveloped using Ultra-Performance Liquid Chromatography(UPLC) coupled with tandem mass spectrometry (MSMS)A UPLC system (Acquity system Waters Milford USA) wascoupled to a Xevo TQ-S triple quadrupole mass spectrometer(Waters) Chromatographic separations were carried outusing revers phase hybrid column (Kinetex 26 120583m 50x 21 mm Phenomenex CA USA) maintained at 30∘CAmygdalin and paeoniflorin were separated using a gradientelution with a flow rate of 05 mLmin Mobile phase solventA was 50 mMAmmonium formate (Sigma-Aldrich) in water(pH 80) and solvent B was 50 mM Ammonium formatein acetonitrile The samples were eluted according to thefollowing linear gradient from 5 B buffer to 100 for10 mins Ions were generated in positive ionization modeusing electrospray ionization interface Tandem MS analysiswas performed using the multi-reaction-monitoring (MRM)mode by monitoring the transition pair of mz 47493 997888rarr32511 for amygdalin and mz 49793 997888rarr 17901 for paeoni-florin The gas flows of desolvation cone and nebulizer wereset at 650 LHr 150 LHr and 7 bar respectively

222 Cinnamic Acid Agilent 6410B Triple Quadrupole LCMS (Agilent Technologies Wilmington USA) equipped withan ESI source was employed for the analysis Cinnamic acid(PubChem CID 444539 purity 990) was purchased fromSigma-Aldrich and used as reference standard 1 g of eachsample was mixed with 10 mL of methanol and centrifugedAliquots of 5 120583L of the processed samples were injectedinto the HPLC system (1200 Series LC Agilent TechnologiesWilmington USA) fitted with Phenomenex Kinetex C18 26120583m 80 A 50 x 21 mm column maintained at 35∘C ESI wasoperating at +3000V and a source temperature of 380∘CCapillary voltage cone voltage and source offset were set at3kV 30kV and 30V respectively The gas flow of desolvationand the conewas set at 650LHr and 150 LHrwith a nebulizerpressure of 15 bar A mobile phase composed of 01 formicacid in distilled water (Buffer A) and 01 formic acid inacetonitrile (Buffer B) was used to separate the analysts andpumped into the ESI chamber at a flow rate of 05 mLminfor 20 min Fragmentor voltage and collision voltage wereset at 50V Detection of the ions was carried out in themultiple-reaction monitoring mode (MRM) by monitoringthe transition pairs of mz 1491 997888rarr131 (cinnamic acid) Data



Gene Sequence
































3 Results











Amygdalin - STD

Amygdalin362

105855175106e6

Amygdalin - GBH

Amygdalin356

1071361107e4


1795e+005


1664e+007100

0250 500 750

min

100

0250 500 750

min

(a)


121163100121e6

Paeoniflorin - GBH

35419056984


1855e+007

191e5


3337e+006100

0250 500 750

min

100

0250 500 750

min

Paeoniflorin - STD

(b)

- Cinnamic acid

- Cinnamic acid

002040608

11214

005

115

225

335

4



times104



(c)







(d)





0

2

4

6

8

Nitr

ite (u

M)

lowast


lowastlowast

LPSGBH (gml) -

--+ ++++++

50 FXTAMT400200100

(a)

iNOS

-actin

0

2

4

6

8

10

iNO

S re

lativ

e int

ensit

y


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

GBH (μgml) - -

+ LPS

LPSGBH (gml) -

--

50 FXTAMT400200100

+ ++++++50 FXTAMT400200100

(b)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

lowastlowastlowast

lowastlowastlowast

(c)

0

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowast

lowastlowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

(d)





(a)

(b)

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n


0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n



0

5

10

15

TNF-

re

lativ

e exp

ress

ion


lowastlowastlowast


(d)

(e)

(f)

0

25

50

75

100

IL-1

(pg

ml)

0

25

50

75

100

IL-6

(pg

ml)





0

200

400

600

TNF-

(p

gm

l)

lowastlowastlowast

lowastlowastlowast


LPSGBH (gml)

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)






0

5

10

15

20

MIP

-1

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(a)

0

5

10

15

MCP

-1 re

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(b)

0

50

100

150

200

250

IP-1

0 re

lativ

e exp

ress

ion

lowastlowastlowast

lowast


+ + + + + +- 100 200 400 AMT FXT

LPSGBH (gml) -

-

(c)

0

20

40

60

80

lowast lowastlowast

+ + + + + +

- 100 200 400 AMT FXTLPS

GBH (gml) --

RAN

TES

relat

ive e

xpre

ssio

n

(d)



4 Discussion







p-p38

p38

-actin

p-Erk

Erk

p-JNK

JNK

00

05

10

15

20

25

p-ER

KER

K re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

0

50

100

150

p-p3

8p3

8 re

lativ

e int

ensit

y

p-Akt

Akt

GBH (μgml) -- 100

+ LPS

0

50

100

150

200

p-JN

KJN

K re

lativ

e int

ensit

y

-

lowastlowast

lowastlowastlowast


lowastlowastlowast

0

10

20

30

40

p-A

KTA

KT re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast


+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

FXTAMT400200

(a)

NF-B p65

PCNA

0

1

2

3

4

NF-

B

p65

relat

ive i

nten

sity

lowastlowastlowast

lowastlowast

lowastlowastlowast

GBH (μgml) --

+ LPS

100 FXTAMT400200


(b)



0

20

40

60IL

-10

(pg

ml)

- - - - - -

lowastlowastlowast


lowastlowastlowast


(a)

0

2

4

6

8

10

HO

-1 re

lativ

e exp

ress

ion

lowastlowastlowast

lowastlowastlowast

- - - - -LPS - + +++++


(b)

GBH (gml)HO-1

-actin


(c)

NRF2

PCNA

p-CREB

CREB

-actin

0

2

1

3

0

6

12

18

Rela

tive i

nten

sity

GBH (gml)

NRF2

p-CREBCREBB


100- FXTAMT400200

(d)







0

2

4

6

8

Nitr

ite (u

M)

lowastlowastlowast

daggerdaggerdagger

SnPP (M) - - - 10 10 50 50GBH (400gml) - - + - + - +

LPS - + + + + + +

(a)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(b)

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

0

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

GBH (400gml) -SnPP (10M) - + - - + +

- - + - +LPS - - + + + +

(d)

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(e)

0

5

10

15

20

TNF-

re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(f)









5 Conclusions


Data Availability




Acknowledgments


References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















Gene Sequence
































3 Results











Amygdalin - STD

Amygdalin362

105855175106e6

Amygdalin - GBH

Amygdalin356

1071361107e4


1795e+005


1664e+007100

0250 500 750

min

100

0250 500 750

min

(a)


121163100121e6

Paeoniflorin - GBH

35419056984


1855e+007

191e5


3337e+006100

0250 500 750

min

100

0250 500 750

min

Paeoniflorin - STD

(b)

- Cinnamic acid

- Cinnamic acid

002040608

11214

005

115

225

335

4



times104



(c)







(d)





0

2

4

6

8

Nitr

ite (u

M)

lowast


lowastlowast

LPSGBH (gml) -

--+ ++++++

50 FXTAMT400200100

(a)

iNOS

-actin

0

2

4

6

8

10

iNO

S re

lativ

e int

ensit

y


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

GBH (μgml) - -

+ LPS

LPSGBH (gml) -

--

50 FXTAMT400200100

+ ++++++50 FXTAMT400200100

(b)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

lowastlowastlowast

lowastlowastlowast

(c)

0

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowast

lowastlowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

(d)





(a)

(b)

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n


0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n



0

5

10

15

TNF-

re

lativ

e exp

ress

ion


lowastlowastlowast


(d)

(e)

(f)

0

25

50

75

100

IL-1

(pg

ml)

0

25

50

75

100

IL-6

(pg

ml)





0

200

400

600

TNF-

(p

gm

l)

lowastlowastlowast

lowastlowastlowast


LPSGBH (gml)

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)






0

5

10

15

20

MIP

-1

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(a)

0

5

10

15

MCP

-1 re

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(b)

0

50

100

150

200

250

IP-1

0 re

lativ

e exp

ress

ion

lowastlowastlowast

lowast


+ + + + + +- 100 200 400 AMT FXT

LPSGBH (gml) -

-

(c)

0

20

40

60

80

lowast lowastlowast

+ + + + + +

- 100 200 400 AMT FXTLPS

GBH (gml) --

RAN

TES

relat

ive e

xpre

ssio

n

(d)



4 Discussion







p-p38

p38

-actin

p-Erk

Erk

p-JNK

JNK

00

05

10

15

20

25

p-ER

KER

K re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

0

50

100

150

p-p3

8p3

8 re

lativ

e int

ensit

y

p-Akt

Akt

GBH (μgml) -- 100

+ LPS

0

50

100

150

200

p-JN

KJN

K re

lativ

e int

ensit

y

-

lowastlowast

lowastlowastlowast


lowastlowastlowast

0

10

20

30

40

p-A

KTA

KT re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast


+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

FXTAMT400200

(a)

NF-B p65

PCNA

0

1

2

3

4

NF-

B

p65

relat

ive i

nten

sity

lowastlowastlowast

lowastlowast

lowastlowastlowast

GBH (μgml) --

+ LPS

100 FXTAMT400200


(b)



0

20

40

60IL

-10

(pg

ml)

- - - - - -

lowastlowastlowast


lowastlowastlowast


(a)

0

2

4

6

8

10

HO

-1 re

lativ

e exp

ress

ion

lowastlowastlowast

lowastlowastlowast

- - - - -LPS - + +++++


(b)

GBH (gml)HO-1

-actin


(c)

NRF2

PCNA

p-CREB

CREB

-actin

0

2

1

3

0

6

12

18

Rela

tive i

nten

sity

GBH (gml)

NRF2

p-CREBCREBB


100- FXTAMT400200

(d)







0

2

4

6

8

Nitr

ite (u

M)

lowastlowastlowast

daggerdaggerdagger

SnPP (M) - - - 10 10 50 50GBH (400gml) - - + - + - +

LPS - + + + + + +

(a)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(b)

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

0

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

GBH (400gml) -SnPP (10M) - + - - + +

- - + - +LPS - - + + + +

(d)

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(e)

0

5

10

15

20

TNF-

re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(f)









5 Conclusions


Data Availability




Acknowledgments


References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















3 Results











Amygdalin - STD

Amygdalin362

105855175106e6

Amygdalin - GBH

Amygdalin356

1071361107e4


1795e+005


1664e+007100

0250 500 750

min

100

0250 500 750

min

(a)


121163100121e6

Paeoniflorin - GBH

35419056984


1855e+007

191e5


3337e+006100

0250 500 750

min

100

0250 500 750

min

Paeoniflorin - STD

(b)

- Cinnamic acid

- Cinnamic acid

002040608

11214

005

115

225

335

4



times104



(c)







(d)





0

2

4

6

8

Nitr

ite (u

M)

lowast


lowastlowast

LPSGBH (gml) -

--+ ++++++

50 FXTAMT400200100

(a)

iNOS

-actin

0

2

4

6

8

10

iNO

S re

lativ

e int

ensit

y


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

GBH (μgml) - -

+ LPS

LPSGBH (gml) -

--

50 FXTAMT400200100

+ ++++++50 FXTAMT400200100

(b)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

lowastlowastlowast

lowastlowastlowast

(c)

0

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowast

lowastlowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

(d)





(a)

(b)

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n


0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n



0

5

10

15

TNF-

re

lativ

e exp

ress

ion


lowastlowastlowast


(d)

(e)

(f)

0

25

50

75

100

IL-1

(pg

ml)

0

25

50

75

100

IL-6

(pg

ml)





0

200

400

600

TNF-

(p

gm

l)

lowastlowastlowast

lowastlowastlowast


LPSGBH (gml)

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)






0

5

10

15

20

MIP

-1

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(a)

0

5

10

15

MCP

-1 re

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(b)

0

50

100

150

200

250

IP-1

0 re

lativ

e exp

ress

ion

lowastlowastlowast

lowast


+ + + + + +- 100 200 400 AMT FXT

LPSGBH (gml) -

-

(c)

0

20

40

60

80

lowast lowastlowast

+ + + + + +

- 100 200 400 AMT FXTLPS

GBH (gml) --

RAN

TES

relat

ive e

xpre

ssio

n

(d)



4 Discussion







p-p38

p38

-actin

p-Erk

Erk

p-JNK

JNK

00

05

10

15

20

25

p-ER

KER

K re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

0

50

100

150

p-p3

8p3

8 re

lativ

e int

ensit

y

p-Akt

Akt

GBH (μgml) -- 100

+ LPS

0

50

100

150

200

p-JN

KJN

K re

lativ

e int

ensit

y

-

lowastlowast

lowastlowastlowast


lowastlowastlowast

0

10

20

30

40

p-A

KTA

KT re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast


+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

FXTAMT400200

(a)

NF-B p65

PCNA

0

1

2

3

4

NF-

B

p65

relat

ive i

nten

sity

lowastlowastlowast

lowastlowast

lowastlowastlowast

GBH (μgml) --

+ LPS

100 FXTAMT400200


(b)



0

20

40

60IL

-10

(pg

ml)

- - - - - -

lowastlowastlowast


lowastlowastlowast


(a)

0

2

4

6

8

10

HO

-1 re

lativ

e exp

ress

ion

lowastlowastlowast

lowastlowastlowast

- - - - -LPS - + +++++


(b)

GBH (gml)HO-1

-actin


(c)

NRF2

PCNA

p-CREB

CREB

-actin

0

2

1

3

0

6

12

18

Rela

tive i

nten

sity

GBH (gml)

NRF2

p-CREBCREBB


100- FXTAMT400200

(d)







0

2

4

6

8

Nitr

ite (u

M)

lowastlowastlowast

daggerdaggerdagger

SnPP (M) - - - 10 10 50 50GBH (400gml) - - + - + - +

LPS - + + + + + +

(a)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(b)

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

0

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

GBH (400gml) -SnPP (10M) - + - - + +

- - + - +LPS - - + + + +

(d)

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(e)

0

5

10

15

20

TNF-

re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(f)









5 Conclusions


Data Availability




Acknowledgments


References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Amygdalin - STD

Amygdalin362

105855175106e6

Amygdalin - GBH

Amygdalin356

1071361107e4


1795e+005


1664e+007100

0250 500 750

min

100

0250 500 750

min

(a)


121163100121e6

Paeoniflorin - GBH

35419056984


1855e+007

191e5


3337e+006100

0250 500 750

min

100

0250 500 750

min

Paeoniflorin - STD

(b)

- Cinnamic acid

- Cinnamic acid

002040608

11214

005

115

225

335

4



times104



(c)







(d)





0

2

4

6

8

Nitr

ite (u

M)

lowast


lowastlowast

LPSGBH (gml) -

--+ ++++++

50 FXTAMT400200100

(a)

iNOS

-actin

0

2

4

6

8

10

iNO

S re

lativ

e int

ensit

y


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

GBH (μgml) - -

+ LPS

LPSGBH (gml) -

--

50 FXTAMT400200100

+ ++++++50 FXTAMT400200100

(b)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

lowastlowastlowast

lowastlowastlowast

(c)

0

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowast

lowastlowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

(d)





(a)

(b)

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n


0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n



0

5

10

15

TNF-

re

lativ

e exp

ress

ion


lowastlowastlowast


(d)

(e)

(f)

0

25

50

75

100

IL-1

(pg

ml)

0

25

50

75

100

IL-6

(pg

ml)





0

200

400

600

TNF-

(p

gm

l)

lowastlowastlowast

lowastlowastlowast


LPSGBH (gml)

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)






0

5

10

15

20

MIP

-1

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(a)

0

5

10

15

MCP

-1 re

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(b)

0

50

100

150

200

250

IP-1

0 re

lativ

e exp

ress

ion

lowastlowastlowast

lowast


+ + + + + +- 100 200 400 AMT FXT

LPSGBH (gml) -

-

(c)

0

20

40

60

80

lowast lowastlowast

+ + + + + +

- 100 200 400 AMT FXTLPS

GBH (gml) --

RAN

TES

relat

ive e

xpre

ssio

n

(d)



4 Discussion







p-p38

p38

-actin

p-Erk

Erk

p-JNK

JNK

00

05

10

15

20

25

p-ER

KER

K re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

0

50

100

150

p-p3

8p3

8 re

lativ

e int

ensit

y

p-Akt

Akt

GBH (μgml) -- 100

+ LPS

0

50

100

150

200

p-JN

KJN

K re

lativ

e int

ensit

y

-

lowastlowast

lowastlowastlowast


lowastlowastlowast

0

10

20

30

40

p-A

KTA

KT re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast


+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

FXTAMT400200

(a)

NF-B p65

PCNA

0

1

2

3

4

NF-

B

p65

relat

ive i

nten

sity

lowastlowastlowast

lowastlowast

lowastlowastlowast

GBH (μgml) --

+ LPS

100 FXTAMT400200


(b)



0

20

40

60IL

-10

(pg

ml)

- - - - - -

lowastlowastlowast


lowastlowastlowast


(a)

0

2

4

6

8

10

HO

-1 re

lativ

e exp

ress

ion

lowastlowastlowast

lowastlowastlowast

- - - - -LPS - + +++++


(b)

GBH (gml)HO-1

-actin


(c)

NRF2

PCNA

p-CREB

CREB

-actin

0

2

1

3

0

6

12

18

Rela

tive i

nten

sity

GBH (gml)

NRF2

p-CREBCREBB


100- FXTAMT400200

(d)







0

2

4

6

8

Nitr

ite (u

M)

lowastlowastlowast

daggerdaggerdagger

SnPP (M) - - - 10 10 50 50GBH (400gml) - - + - + - +

LPS - + + + + + +

(a)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(b)

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

0

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

GBH (400gml) -SnPP (10M) - + - - + +

- - + - +LPS - - + + + +

(d)

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(e)

0

5

10

15

20

TNF-

re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(f)









5 Conclusions


Data Availability




Acknowledgments


References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

2

4

6

8

Nitr

ite (u

M)

lowast


lowastlowast

LPSGBH (gml) -

--+ ++++++

50 FXTAMT400200100

(a)

iNOS

-actin

0

2

4

6

8

10

iNO

S re

lativ

e int

ensit

y


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

GBH (μgml) - -

+ LPS

LPSGBH (gml) -

--

50 FXTAMT400200100

+ ++++++50 FXTAMT400200100

(b)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

lowastlowastlowast

lowastlowastlowast

(c)

0

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowast

lowastlowast

LPSGBH (gml) -

--+ +++++

100 FXTAMT400200

(d)





(a)

(b)

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n


0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n



0

5

10

15

TNF-

re

lativ

e exp

ress

ion


lowastlowastlowast


(d)

(e)

(f)

0

25

50

75

100

IL-1

(pg

ml)

0

25

50

75

100

IL-6

(pg

ml)





0

200

400

600

TNF-

(p

gm

l)

lowastlowastlowast

lowastlowastlowast


LPSGBH (gml)

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)






0

5

10

15

20

MIP

-1

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(a)

0

5

10

15

MCP

-1 re

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(b)

0

50

100

150

200

250

IP-1

0 re

lativ

e exp

ress

ion

lowastlowastlowast

lowast


+ + + + + +- 100 200 400 AMT FXT

LPSGBH (gml) -

-

(c)

0

20

40

60

80

lowast lowastlowast

+ + + + + +

- 100 200 400 AMT FXTLPS

GBH (gml) --

RAN

TES

relat

ive e

xpre

ssio

n

(d)



4 Discussion







p-p38

p38

-actin

p-Erk

Erk

p-JNK

JNK

00

05

10

15

20

25

p-ER

KER

K re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

0

50

100

150

p-p3

8p3

8 re

lativ

e int

ensit

y

p-Akt

Akt

GBH (μgml) -- 100

+ LPS

0

50

100

150

200

p-JN

KJN

K re

lativ

e int

ensit

y

-

lowastlowast

lowastlowastlowast


lowastlowastlowast

0

10

20

30

40

p-A

KTA

KT re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast


+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

FXTAMT400200

(a)

NF-B p65

PCNA

0

1

2

3

4

NF-

B

p65

relat

ive i

nten

sity

lowastlowastlowast

lowastlowast

lowastlowastlowast

GBH (μgml) --

+ LPS

100 FXTAMT400200


(b)



0

20

40

60IL

-10

(pg

ml)

- - - - - -

lowastlowastlowast


lowastlowastlowast


(a)

0

2

4

6

8

10

HO

-1 re

lativ

e exp

ress

ion

lowastlowastlowast

lowastlowastlowast

- - - - -LPS - + +++++


(b)

GBH (gml)HO-1

-actin


(c)

NRF2

PCNA

p-CREB

CREB

-actin

0

2

1

3

0

6

12

18

Rela

tive i

nten

sity

GBH (gml)

NRF2

p-CREBCREBB


100- FXTAMT400200

(d)







0

2

4

6

8

Nitr

ite (u

M)

lowastlowastlowast

daggerdaggerdagger

SnPP (M) - - - 10 10 50 50GBH (400gml) - - + - + - +

LPS - + + + + + +

(a)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(b)

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

0

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

GBH (400gml) -SnPP (10M) - + - - + +

- - + - +LPS - - + + + +

(d)

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(e)

0

5

10

15

20

TNF-

re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(f)









5 Conclusions


Data Availability




Acknowledgments


References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a)

(b)

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n


0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n



0

5

10

15

TNF-

re

lativ

e exp

ress

ion


lowastlowastlowast


(d)

(e)

(f)

0

25

50

75

100

IL-1

(pg

ml)

0

25

50

75

100

IL-6

(pg

ml)





0

200

400

600

TNF-

(p

gm

l)

lowastlowastlowast

lowastlowastlowast


LPSGBH (gml)

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT--

+ + + + + +

- 100 200 400 AMT FXT-- + + + + + +

- 100 200 400 AMT FXT--

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)

LPSGBH (gml)






0

5

10

15

20

MIP

-1

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(a)

0

5

10

15

MCP

-1 re

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(b)

0

50

100

150

200

250

IP-1

0 re

lativ

e exp

ress

ion

lowastlowastlowast

lowast


+ + + + + +- 100 200 400 AMT FXT

LPSGBH (gml) -

-

(c)

0

20

40

60

80

lowast lowastlowast

+ + + + + +

- 100 200 400 AMT FXTLPS

GBH (gml) --

RAN

TES

relat

ive e

xpre

ssio

n

(d)



4 Discussion







p-p38

p38

-actin

p-Erk

Erk

p-JNK

JNK

00

05

10

15

20

25

p-ER

KER

K re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

0

50

100

150

p-p3

8p3

8 re

lativ

e int

ensit

y

p-Akt

Akt

GBH (μgml) -- 100

+ LPS

0

50

100

150

200

p-JN

KJN

K re

lativ

e int

ensit

y

-

lowastlowast

lowastlowastlowast


lowastlowastlowast

0

10

20

30

40

p-A

KTA

KT re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast


+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

FXTAMT400200

(a)

NF-B p65

PCNA

0

1

2

3

4

NF-

B

p65

relat

ive i

nten

sity

lowastlowastlowast

lowastlowast

lowastlowastlowast

GBH (μgml) --

+ LPS

100 FXTAMT400200


(b)



0

20

40

60IL

-10

(pg

ml)

- - - - - -

lowastlowastlowast


lowastlowastlowast


(a)

0

2

4

6

8

10

HO

-1 re

lativ

e exp

ress

ion

lowastlowastlowast

lowastlowastlowast

- - - - -LPS - + +++++


(b)

GBH (gml)HO-1

-actin


(c)

NRF2

PCNA

p-CREB

CREB

-actin

0

2

1

3

0

6

12

18

Rela

tive i

nten

sity

GBH (gml)

NRF2

p-CREBCREBB


100- FXTAMT400200

(d)







0

2

4

6

8

Nitr

ite (u

M)

lowastlowastlowast

daggerdaggerdagger

SnPP (M) - - - 10 10 50 50GBH (400gml) - - + - + - +

LPS - + + + + + +

(a)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(b)

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

0

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

GBH (400gml) -SnPP (10M) - + - - + +

- - + - +LPS - - + + + +

(d)

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(e)

0

5

10

15

20

TNF-

re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(f)









5 Conclusions


Data Availability




Acknowledgments


References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

5

10

15

20

MIP

-1

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(a)

0

5

10

15

MCP

-1 re

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

LPS + + + + + +GBH (gml) --

-100 200 400 AMT FXT

(b)

0

50

100

150

200

250

IP-1

0 re

lativ

e exp

ress

ion

lowastlowastlowast

lowast


+ + + + + +- 100 200 400 AMT FXT

LPSGBH (gml) -

-

(c)

0

20

40

60

80

lowast lowastlowast

+ + + + + +

- 100 200 400 AMT FXTLPS

GBH (gml) --

RAN

TES

relat

ive e

xpre

ssio

n

(d)



4 Discussion







p-p38

p38

-actin

p-Erk

Erk

p-JNK

JNK

00

05

10

15

20

25

p-ER

KER

K re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

0

50

100

150

p-p3

8p3

8 re

lativ

e int

ensit

y

p-Akt

Akt

GBH (μgml) -- 100

+ LPS

0

50

100

150

200

p-JN

KJN

K re

lativ

e int

ensit

y

-

lowastlowast

lowastlowastlowast


lowastlowastlowast

0

10

20

30

40

p-A

KTA

KT re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast


+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

FXTAMT400200

(a)

NF-B p65

PCNA

0

1

2

3

4

NF-

B

p65

relat

ive i

nten

sity

lowastlowastlowast

lowastlowast

lowastlowastlowast

GBH (μgml) --

+ LPS

100 FXTAMT400200


(b)



0

20

40

60IL

-10

(pg

ml)

- - - - - -

lowastlowastlowast


lowastlowastlowast


(a)

0

2

4

6

8

10

HO

-1 re

lativ

e exp

ress

ion

lowastlowastlowast

lowastlowastlowast

- - - - -LPS - + +++++


(b)

GBH (gml)HO-1

-actin


(c)

NRF2

PCNA

p-CREB

CREB

-actin

0

2

1

3

0

6

12

18

Rela

tive i

nten

sity

GBH (gml)

NRF2

p-CREBCREBB


100- FXTAMT400200

(d)







0

2

4

6

8

Nitr

ite (u

M)

lowastlowastlowast

daggerdaggerdagger

SnPP (M) - - - 10 10 50 50GBH (400gml) - - + - + - +

LPS - + + + + + +

(a)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(b)

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

0

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

GBH (400gml) -SnPP (10M) - + - - + +

- - + - +LPS - - + + + +

(d)

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(e)

0

5

10

15

20

TNF-

re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(f)









5 Conclusions


Data Availability




Acknowledgments


References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















p-p38

p38

-actin

p-Erk

Erk

p-JNK

JNK

00

05

10

15

20

25

p-ER

KER

K re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

0

50

100

150

p-p3

8p3

8 re

lativ

e int

ensit

y

p-Akt

Akt

GBH (μgml) -- 100

+ LPS

0

50

100

150

200

p-JN

KJN

K re

lativ

e int

ensit

y

-

lowastlowast

lowastlowastlowast


lowastlowastlowast

0

10

20

30

40

p-A

KTA

KT re

lativ

e int

ensit

y

lowastlowastlowast


lowastlowastlowast


+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

-LPS -

GBH (gml) - 100 FXTAMT400200+++ + + +

FXTAMT400200

(a)

NF-B p65

PCNA

0

1

2

3

4

NF-

B

p65

relat

ive i

nten

sity

lowastlowastlowast

lowastlowast

lowastlowastlowast

GBH (μgml) --

+ LPS

100 FXTAMT400200


(b)



0

20

40

60IL

-10

(pg

ml)

- - - - - -

lowastlowastlowast


lowastlowastlowast


(a)

0

2

4

6

8

10

HO

-1 re

lativ

e exp

ress

ion

lowastlowastlowast

lowastlowastlowast

- - - - -LPS - + +++++


(b)

GBH (gml)HO-1

-actin


(c)

NRF2

PCNA

p-CREB

CREB

-actin

0

2

1

3

0

6

12

18

Rela

tive i

nten

sity

GBH (gml)

NRF2

p-CREBCREBB


100- FXTAMT400200

(d)







0

2

4

6

8

Nitr

ite (u

M)

lowastlowastlowast

daggerdaggerdagger

SnPP (M) - - - 10 10 50 50GBH (400gml) - - + - + - +

LPS - + + + + + +

(a)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(b)

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

0

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

GBH (400gml) -SnPP (10M) - + - - + +

- - + - +LPS - - + + + +

(d)

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(e)

0

5

10

15

20

TNF-

re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(f)









5 Conclusions


Data Availability




Acknowledgments


References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

20

40

60IL

-10

(pg

ml)

- - - - - -

lowastlowastlowast


lowastlowastlowast


(a)

0

2

4

6

8

10

HO

-1 re

lativ

e exp

ress

ion

lowastlowastlowast

lowastlowastlowast

- - - - -LPS - + +++++


(b)

GBH (gml)HO-1

-actin


(c)

NRF2

PCNA

p-CREB

CREB

-actin

0

2

1

3

0

6

12

18

Rela

tive i

nten

sity

GBH (gml)

NRF2

p-CREBCREBB


100- FXTAMT400200

(d)







0

2

4

6

8

Nitr

ite (u

M)

lowastlowastlowast

daggerdaggerdagger

SnPP (M) - - - 10 10 50 50GBH (400gml) - - + - + - +

LPS - + + + + + +

(a)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(b)

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

0

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

GBH (400gml) -SnPP (10M) - + - - + +

- - + - +LPS - - + + + +

(d)

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(e)

0

5

10

15

20

TNF-

re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(f)









5 Conclusions


Data Availability




Acknowledgments


References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

2

4

6

8

Nitr

ite (u

M)

lowastlowastlowast

daggerdaggerdagger

SnPP (M) - - - 10 10 50 50GBH (400gml) - - + - + - +

LPS - + + + + + +

(a)

0

200

400

600

800

1000

iNO

S re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(b)

5

10

15

COX-

2 re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

0

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + + + +

(c)

0

100

200

300

IL-6

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

GBH (400gml) -SnPP (10M) - + - - + +

- - + - +LPS - - + + + +

(d)

0

100

200

300

400

IL-1

relat

ive e

xpre

ssio

n

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(e)

0

5

10

15

20

TNF-

re

lativ

e exp

ress

ion

lowastlowastlowast

daggerdaggerdagger

SnPP (10M) - + - - + +GBH (400gml) - - - + - +

LPS - - + +++

(f)









5 Conclusions


Data Availability




Acknowledgments


References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























5 Conclusions


Data Availability




Acknowledgments


References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of









































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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