research article effects of wutou decoction on dna...

Research ArticleEffects of Wutou Decoction on DNA Methylation and HistoneModifications in Rats with Collagen-Induced Arthritis

Ya-Fei Liu12 Cai-Yu-Zhu Wen3 Zhe Chen1 Yu Wang1 Ying Huang1

Yong-Hong Hu1 and Sheng-Hao Tu1

1 Institute of Integrated Traditional Chinese and Western Medicine Tongji Hospital Tongji Medical CollegeHuazhong University of Science and Technology 1095 Jiefang Avenue Wuhan Hubei 430030 China2Department of Nephrology The First Affiliated Hospital of Zhengzhou University 1 Jianshe East Road ZhengzhouHenan 450052 China3Hubei University of Chinese Medicine 1 Huangjiahu West Road Wuhan Hubei 430065 China

Correspondence should be addressed to Sheng-Hao Tu shtutjhtjmueducn

Received 16 October 2015 Accepted 11 February 2016

Academic Editor Musa T Yakubu

Copyright copy 2016 Ya-Fei Liu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

BackgroundWutou decoction (WTD) has beenwildly applied in the treatment of rheumatoid arthritis and experimental arthritis inrats formany years Epigenetic deregulation is associated with the aetiology of rheumatoid arthritis however the effects ofWTDonepigenetic changes are unclearThis study is set to explore the effects ofWTDonDNAmethylation and histonemodifications in ratswith collagen-induced arthritis (CIA) Methods The CIA model was established by the stimulation of collagen and adjuvant Theknee synovium was stained with hematoxylin and eosin The DNA methyltransferase 1 (DNMT1) and methylated CpG bindingdomain 2 (MBD2) expression of peripheral blood mononuclear cells (PBMCs) were determined by Real-Time PCR The globalDNA histone H3-K4H3-K27 methylation and total histones H3 and H4 acetylation of PBMCs were detected Results Our datademonstrated that the DNMT1 mRNA expression was significantly lowered in group WTD compared to that in group CIA (119875 lt005) The DNA methylation level was significantly reduced in group WTD compared to that in group CIA (119875 lt 005) MoreoverH3 acetylation of PBMCs was overexpressed in WTD compared with CIA (119875 lt 005) Conclusions WTD may modulate DNAmethylation and histone modifications functioning as anti-inflammatory potential

1 Introduction

Rheumatoid arthritis (RA) is a systemic autoimmune diseaseof unknown aetiology which is characterized by swellingpain stiffness and deformity of peripheral joints [1] Envi-ronmental factors and epigenetic deregulation are associatedwith the etiopathology of RA [2] Epigenetics is defined asstable and heritable changes in gene expression which occurwithout a change in DNA sequence [3] The predominantepigenetic mechanisms are DNA methylation histone mod-ification and chromatin remodeling It has been extensivelydemonstrated that epigenetics plays an important role in thepathogenesis of RA [4ndash8]

Moreover various TCM-based herbal formulas and theextracts such as Wutou decoction (WTD) [9] Xinfengcapsule [10] and Tripterygium wilfordii Hook F [11] have

been employed in ameliorating articular and extra-articularmanifestations of RA WTD is constituted of six individualherbs which are prepared as seen in Table 1 Clinical studiesdemonstrated that WTD which was described in a famousTCM monograph Synopsis of Prescriptions of the GoldenChamber in Han Dynasty of China has been wildly appliedfor the treatment of RA [12] sciatica [13] and scapulohumeralperiarthritis [14] Meanwhile in vivo animal experimentsalso validated that WTD or its derivatives significantlyalleviate swelling arthritis index and hyperaemia in rats withadjuvant-induced arthritis [15ndash18]

In traditional Chinese medicine (TCM) RA falls intothe category of arthromyodynia caused by wind cold anddampness which was described in the Yellow EmperorrsquosClassic of Internal Medicine WTD is capable of dispellingdampness by warming the channels and alleviating pain by

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 5836879 9 pageshttpdxdoiorg10115520165836879

2 Evidence-Based Complementary and Alternative Medicine

Table 1 The composition of herbal formula WTD

Crude herbs Content Main componentsEphedra (Herbal Ephedrae) 9 Ephedrine D-pseudoephedrineRed Peony Root (Radix Paeoniae Rubra) 45 PaeoniflorinWhite Peony Root (Radix Paeoniae Alba) 45 PaeoniflorinRoot of Membranous Milkvetch (Radix Astragali) 9 AstragalosidePrepared Liquorice Root (Radix Glycyrrhizae Preparata) 9 Glycyrrhizic acidPrepared Monkshood Mother Root (Radix Aconiti Preparata) 6 Aconitine mesaconitineWTD Wutou decoction

dispersing coldness which is employed in the treatment ofRA And yet the impacts of WTD on epigenetic changesare unknown The study aims to explore the effect of WTDon DNA methylation and histone modifications in rats ofcollagen-induced arthritis (CIA)

2 Materials and Methods

21 Reagents and Main Devices Bovine type II collagen(catalog 20022) and incomplete Freundrsquos adjuvant (catalog7002) were purchased from Amyjet Scientific Inc ChinaThe components of WTD except Herbal Ephedrae (Chi-nese Herbal Powder Beijing Tcmages Pharmaceutical CoLtd China) and Herbal Ephedrae were provided by TongjiHospital TRIzol Reagent (Invitrogen 15596026) DNaseI (Fermentas EN0521) RevertAid Reverse Transcriptase(Fermentas EP0442) dNTP (Fermentas R0191) RiboLockRNase Inhibitor (Fermentas E00381) All-in-Onetrade qPCRMaster Mix (GeneCopoeiaTM AOPR-1200) DNA Extrac-tion Kit (Aidlab Biotechnologies Co Ltd China catalogDN07) and Lymphocyte Separation Medium (Tian Jin HaoYang Biological Manufacture Co Ltd) were ordered fromWuhan Boster Company China MethylFlashtrade MethylatedDNA Quantification Kit (catalog P-1034) EpiQuiktrade TotalHistone Extraction Kit (catalog OP-0006) EpiQuiktrade GlobalPan-Methyl Histone H3-K4 Quantification Kit (catalog P-3028) EpiQuiktrade Global Pan-Methyl Histone H3-K27 Quan-tification Kit (catalog P-3044) EpiQuiktrade Total HistoneH3 Acetylation Detection Fast Kit (catalog P-4030) andEpiQuiktrade Total Histone H4 Acetylation Detection Fast Kit(catalog P-4032) were purchased fromAmyjet Scientific IncChina

Electric Homogenizer (Glas-Col USA) Ice Machine(AF-100AS Scotsman USA) Centrifuge (2-16K SigmaUSA) Nucleic AcidProtein Analyzer (DU730 BeckmanCoulter Inc California USA) Microplate Reader (BioTekSynergy2 Vermont USA) Heated Circulating Bath (DKB-600B Keelrein instrument Co Ltd China) MiliporeRiOs 5Water Purification Systems (MilliporeUSA) Applied Biosys-tems StepOne Real-Time PCR System (Applied BiosystemsCalifornia USA) and Esco Airstream Class II BiologicalSafety Cabinet (Beijing China) were used

22 Animals and Rearing Conditions Five-week-old weight-ed 130ndash150 g female Wistar rats (119899 = 45) SPF grade wereprovided by the Center for Disease Control and Prevention ofHubei province (the animal certificate SCXK no 2008-0005)

and fed in the barrier system according to The Guidelinesfor the Care and Use of Animals in Research enforced byHubei Municipal Science and Technology Commission Allprotocols were approved by the Institutional Animal Careand Ethics Committee of Tongji Medical College HuazhongUniversity of Science and Technology Food and water weregiven ad libitum throughout the experiment The rats werecaged in a standard barrier system with a 12 h lightdarkcycle

23 Grouping and Treatment After 7 days of acclimation theanimals were randomly divided into three groups normalgroup (N) CIA group and WTD group (42 gkgd) with15 rats in each group The rat doses of treatment groupwere converted from human doses (Chinese Pharmacopeia2010) based on body surface areas The final concentrationof treatment group was 042 gmL for WTD group Oralgavage was performed once a day from day 28 after the initialimmunization for four weeks Rats in groups N and CIAwereorally administered with the same volume of distilled waterThe rats were fasted for 12 h but permitted water ad libitumbefore blood collection

24 CIA Model Establishment and Arthritis Index AssessmentCIA model was established according to the manufacturerrsquosstandard protocols and previous research [19] In addition togroup N CIAmodel was established in the other two groupsBovine type II collagen was emulsified in an equal volume ofincomplete Freund adjuvant and 02mL of the emulsion wasinjected subcutaneously about 3 cm distal from the base ofthe tail to induce arthritis To ensure a high CIA incidencea booster immunization (administer 01mL of the emulsionsubcutaneously in the tail) was performed twoweeks after theinitial immunization about 15 cm distal from the base of thetail

As shown in Figure 1 the arthritis severity was evaluatedby applying a previous published scoring system [20] Oncearthritis occurred the arthritis index (AI) was evaluatedtwice a week for arthritis developmentThe arthritis score foreach rat was the sum of the scores for two hind paws

25 Sampling At sixty minutes after the last intragastricadministration the rats were narcotized with 10 chloralhydrate by intraperitoneal injection Blood was extractedfrom aorta abdominalis The synovium isolated from kneejoints was fixed in 4 paraformaldehyde for 12 h and embed-ded in paraffin Paraffin-embedded synovial tissues were

Evidence-Based Complementary and Alternative Medicine 3

(a) (b) (c)

(d) (e) (f)

Figure 1 Arthritis index assessment (a) 0 (b) 1 (c) 2 (d) 3 (e) 4 (f) right hind paw which is unable to bear weight

Table 2 Primer sequence

Gene Primer Sequence

120573-actin Forward 51015840-CGTTGACATCCGTAAAGACCTC-31015840

Reverse 51015840-TAGGAGCCAGGGCAGTAATCT-31015840

DNMT1 Forward 51015840-CGCTCATTGGCTTTTCTACCG-31015840

Reverse 51015840-AGAACTCGACCACAATCTT-31015840

MBD2 Forward 51015840-AATGATGAGACCCTTCTGTCTGCC-31015840

Reverse 51015840-TCCTCTAGTTTCTTTCGGACTTGTTG-31015840

DNMT1 DNA methyltransferase 1 MBD2 methyl CpG binding domain 2

cut in 5 120583m thick sections and stained with hematoxylinand eosin The sections were evaluated by two indepen-dent pathologists The peripheral blood mononuclear cells(PBMCs) were separated by Lymphocyte SeparationMediumusing density gradient centrifugation method

26 Real-Time PCR for PBMCs DNA Methyltransferase 1(DNMT1) and Methylated CpG Binding Domain 2 (MBD2)Expression Total RNA was extracted from PBMCs withTRIzol Reagent consistent with the manufacturerrsquos instruc-tions RNA purity and concentration were measured by18 agarose gel electrophoresis and Nucleic AcidProteinAnalyzer cDNA was synthesized in accordance with themanufacturerrsquos instructions The cDNA was stored at minus20∘Cprior to PCR amplification Real-Time PCR reactions wereperformed applying StepOne Real-Time PCR System byfollowing the manufacturerrsquos instructions Thermal cyclerprotocol stage 1 Reps 1 95∘C 10min stage 2 Reps 40 95∘C

10 s 60∘C 20 s and 72∘C 20 s The data were analyzed byemploying 2minusΔΔct method The primers were designed inaccordance with published sequences (Table 2)

27 Determination of Global DNA Methylation of PBMCsTotal DNAwas extracted from PBMCs with DNA ExtractionKit consistent with the manufacturerrsquos instructions DNApurity and concentration were measured by 1 agarosegel electrophoresis and Nucleic AcidProtein Analyzer TheglobalDNAmethylation of PBMCswas detected according tothe manufacturerrsquos instructions The amount and percentageof methylated DNA in total DNA were calculated usingthe following formulas 5-mC (ng) = (Sample OD minus ME3OD)(Slope times 2) or 5-mC= (5-mCAmount (ng)S) times 100

28 Determination of Global Pan-Methyl Histones H3-K4and H3-K27 of PBMCs Total histones were extracted from


(a) (b) (c)

(d) (e) (f)

Figure 2 The hematoxylin and eosin staining of synovium corresponding to arthritis index score (a) (b) (c) (d) and (e) represent thearthritis index scores 0 1 2 3 and 4 respectively (f) The process of isolating knee synovium

PBMCs with Histone Extraction Kit according to the manu-facturerrsquos instructions Histone concentration was measuredby BCA protein assay kit The global histones H3-K4 andH3-K27 methylation of PBMCs was detected according tothe manufacturerrsquos instructions The amount and percentageof mono- di- and trimethylated H3-K4 and H3-K27 werecalculated applying the following formulas Methylation= OD (treated (tested) sample minus blank)OD (untreated(control) sampleminus blank)times 100orAmount (ngmg protein)= (OD (sample minus blank)Protein (120583g) times slope) times 1000

29 Determination of Total Histones H3 and H4 Acetylationof PBMCs Total histones were extracted from PBMCs withhistone extraction kit in accordance with the manufacturerrsquosinstructions Histone concentration was measured by BCAprotein assay kit The total histones H3 and H4 acetylation ofPBMCswas detected according to themanufacturerrsquos instruc-tions The amount and percentage of acetyls H3 and H4 werecalculated using the following formulas Acetylation = OD(treated (tested) sample minus blank)OD (untreated (control)sample minus blank) times 100 or Amount (ngmg protein) = (OD(sample minus blank)Protein (120583g) times slope) times 1000

210 Statistical Analysis All data with a normal distributionwere presented as mean plusmn standard deviation and analyzedwith aid of SPSS170 statistical software Statistical significancewas determined by one-way analysis of variance (ANOVA)For data with equal variances assumed ANOVA followedby LSD test was applied For data with equal variances

not assumed ANOVA followed by Dunnettrsquos 1198793 test wasadopted A probability of less than 005 was considered to bestatistically significant

3 Results

31 The Hematoxylin and Eosin Staining of Synovium Corre-sponding to AI Score The histopathological characteristicsof synovial membrane corresponding to AI score werepresented as follows 0 (AI score) no alteration 1 (AIscore) the synovial lining cells form 2-3 layers 2 (AI score)the synovial lining cells form 4-5 layers (multinucleatedcells might occur) 3 (AI score) the synovial lining cellsform 5ndash8 layers hyperplastic synovial stroma and pannusformation 4 (AI score) the synovial lining cells form morethan 8 layers numerous inflammatory cell infiltration andneovascularization (Figure 2)

32 Expression of PBMCs DNMT1 and MBD2 mRNA Com-pared with group N the DNMT1 mRNA expression wassignificantly elevated in group CIA (119875 lt 005) The DNMT1mRNA expression was significantly lowered in group WTDcompared to that in group CIA (119875 lt 005) (Figure 3(a))Compared with group N the MBD2 mRNA expression wasincreasing in group CIA however there was no signifi-cant difference between the two groups Compared withgroup CIA there was a reduction in group WTD howeverthere was no significant difference between the two groups(Figure 3(b))


Relat

ive D

NM

T1m

RNA

0

2

4

6

WTDN CIA

998779

(a)

0

5

10

15

Rela

tive M

BD2

mRN

A

WTDN CIA(b)

Figure 3 (a) Expression of PBMCs DNMT1mRNA and (b) expression of PBMCsMBD2mRNA Values are mean plusmn SD 119875 lt 005 comparedwith group N and 119875 lt 005 compared with group CIA N normal CIA collagen-induced arthritis WTD Wutou decoction PBMCsperipheral blood mononuclear cells DNMT1 DNA methyltransferase 1 MBD2 methyl CpG binding domain 2

0

1

2

3

4

5

DN

A m

ethy

latio

n (n

g)

CIA WTDN

998779

Figure 4 Expression of global DNA methylation level in PBMCsValues are mean plusmn SD 119875 lt 005 compared with group CIA Nnormal CIA collagen-induced arthritis WTD Wutou decoctionPBMCs peripheral blood mononuclear cells

33 Expression of Global DNA Methylation Level in PBMCsCompared with group N the global DNA methylationlevel was enhancing in group CIA however there was nosignificant difference between the two groups Comparedwith group CIA the global DNA methylation level wassignificantly reduced in group WTD (119875 lt 005) (Figure 4)

34 Expression of Global Pan-Methyl Histones H3-K4 and H3-K27 in PBMCs Comparedwith groupN themono- di- andtrimethylated H3-K4 and H3-K27 were increasing in groupCIA however there was no significant difference between thetwo groups Compared with group CIA the mono- di- andtrimethylated H3-K4 and H3-K27 were enhancing in groupWTD however there was no significant difference betweenthe two groups (Figures 5(a) and 5(b))

35 Expression of Total Histones H3 and H4 Acetylationin PBMCs Compared with group N the total histone H3

acetylation in group CIA was no significant alteration Thetotal histone H3 acetylation in group WTD was significantlyelevated compared to that in group CIA (119875 lt 005)(Figure 6(a)) Compared with group N the total histoneH4 acetylation level was enhancing in group CIA howeverthere was no significant difference between the two groupsCompared with group CIA the total histone H4 acetylationlevel was reduced in group WTD however there was nosignificant difference between the two groups (Figure 6(b))

4 Discussion

Based on the character of herbs WTD has been wildlyemployed in the treatment of RA However the mechanismby whichWTD acts on RA is unclear Systems biology-basedinvestigation indicated that the predicted effect or moleculesof WTD were significantly enhanced in neuroactive ligand-receptor interaction and calcium signaling pathway [9] Asthe primary component of WTD the methanol extracts ofAconitum roots have shown inhibition of hind paw edemaproduced by carrageenin in mice [21] Seventy-four compo-nents of WTD have been identified by applying an ultra per-formance liquid chromatography coupled with quadrupoletime-of-flight mass spectrometry method [22] The studyfocuses on investigating the potential epigenetic mechanismsof WTD in rats with CIA

CIA is a typical experimental autoimmune disease thatis widely used as a model of RA CIA model was firstlyestablished by Trentham and colleagues [23] There aresubstantial differences in histopathological characteristics ofsynovium even in two knees of a rat with CIA Consequentlyit is difficult to compare the pathological difference amongthe groups For the reasons outlined above we only elucidatedthe histopathological characteristics of synovial membranecorresponding to AI score

DNA methylation the most characterized epigeneticmark occurs by the covalent addition of a methyl group atthe 5-carbon of the cytosine ring by a family of DNMTs with


times104

60

80

100

120

Pan-

Met

hyl H3

-K4

(ng

mg

prot

ein)

Dimethyl TrimethylMonomethyl

NCIAWTD

(a)

times104

60

80

100

120

Pan-

Met

hyl H3

-K27

(ng

mg

prot

ein)

Monomethyl TrimethylDimethyl

NCIAWTD

(b)

Figure 5 Expression of Global Pan-Methyl HistonesH3-K4 andH3-K27 in PBMCs Values aremeanplusmn SDN normal CIA collagen-inducedarthritis WTD Wutou decoction PBMCs peripheral blood mononuclear cells

998779

times104

50

60

70

80

90

H3

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(a)

times104

0

20

40

60

H4

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(b)

Figure 6 Expression of total histones H3 and H4 acetylation in PBMCs Values are mean plusmn SD 119875 lt 005 compared with group CIA Nnormal CIA collagen-induced arthritis WTD Wutou decoction PBMCs peripheral blood mononuclear cells

S-adenosyl-methionine as the methyl source resulting in 5-methylcytosine DNA methylation is catalyzed by DNMTspredominantly including DNMT1 DNMT3a and DNMT3bDNMT1 is the most abundant DNMT in mammalian cellsand is considered to be the keymaintenance DNMT inmam-mals DNMT1 principally methylates hemimethylated CpGdinucleotides in the mammalian genome DNA methylationsuppresses the expression of genes at transcriptional level[24]

Our results were consistent with the findings of Liu etal concerning the higher expression of DNMT1 mRNA ingroup CIA than that in N [25] Compared with group CIAthe DNMT1 mRNA expression was significantly loweredin group WTD The global DNA methylation level wassignificantly reduced in group WTD compared to that in

group CIA which may result from the lower expression ofDNMT1 mRNA in group WTD However tumor necrosisfactor 120572 (TNF120572) blocker has no impact on DNMT1 mRNAexpression in RA patients [25] The different mechanismsof WTD and TNF120572 blocker in the treatment of RA maycontribute to the differences

Human MBDs are a family of methyl CpG bindingdomain proteins consisted of methyl CpG binding protein2 (MECP2) MBD1 MBD2 MBD3 and MBD4 Apart fromMBD3 each of these proteins is capable of binding specif-ically to methylated DNA MECP2 MBD1 and MBD2 canalso bind to histone deacetylases (HDACs) functioning astranscription repressors [24] MBD2 selectively binds tomethylated DNA and may function as a mediator of the bio-logical consequences of the methylation signal [26]


The MBD2 mRNA expression was increasing in groupCIA compared to that in N however there was no sig-nificant difference between the two groups Clinical studyalso demonstrated that the MBD2 mRNA expression washigher in RA patients [25] However the MBD2 mRNAexpression was decreasing in group WTD compared withgroup CIA although there was no significant differencebetween the two groups Anti-TNF120572 biological agents do notseem to affect mRNA expression of MBD2 in RA patients[25] These suggest that anti-TNF120572 biologics and WTDcould not influence MBD2 mRNA expression in treatingRA

Current researches also showed that DNA hypomethyla-tion exists in synovial fibroblasts T cell and PBMCs in RApatients [25 27 28] indicating that DNA hypomethylation isassociated with the pathogenesis of RA MBD2 participatesin silencing of methylated genes [29] and MBD2 activatesgene demethylation [30] It was assumed that group CIAhad a higher expression of DNMT1 mRNA with the suc-ceeding global hypermethylation of DNA and then led toa higher expression of MBD2 mRNA through a feedbackmechanism

Histone methylation and histone acetylation two post-translational modifications of histones are investigatedintensively for their critical roles in modulating gene tran-scription Histone methylation marks can be correlated withtranscriptional activation or silencing dependent on theposition and the degree of methylation [31] The trimethylmark onH3-K4 (H3-K4me3) was often associatedwith activetranscription [32] Enhancer of zeste homologue 2 (EZH2)generated the trimethyl mark on H3-K27 (H3-K27me3)which correlated with gene silencing [32 33]

Our experiments suggested that the mono- di- andtrimethylatedH3-K4 andH3-K27 had no significant increasein group WTD compared to that in group CIA EZH2 ahistone methyltransferase enhancer was overexpressed inRA synovial fibroblasts (SF) compared with osteoarthri-tis (OA) SF [34] However the studies regarding histonesH3-K4 and H3-K27 methylation are rare Further studiesare needed to clarify the role of histone methylation inRA

Histone acetylation is regulated by the opposite activityof two enzyme families histone acetyl transferases (HATs)and HDACs Histone acetylation is catalyzed by HATsenhancing the rate of gene transcription On the otherhand histone deacetylation is catalyzed by HDACs leadingto transcriptional silence of certain genes The imbalancebetween histone acetylation and deacetylation regulates thetranscription rates of various genes and has been indicated tobe related to disease states [35]

Our data indicated that the total histone H3 acetylationin group WTD was significantly elevated compared to thatin group CIA HDAC1 was overexpressed in RASF comparedto OASF supporting cell proliferation and survival of RASF[36] Meanwhile HDAC2 also played a crucial role in cellproliferation and apoptosis of RASF [36] In addition nuclearHDAC activity and expression of HDAC1 were significantlyelevated in RA compared to those in OA synovial tissues[37] A wide range of HDAC inhibitors (HDACis) showed

protective effects in prophylactic and therapeutic modelsof RA [38] Trichostatin A (TSA) a nonselective HDACicould potently inhibit the lipopolysaccharide (LPS)-inducedproduction of TNF and interleukin-6 (IL6) in both RA andhealthy PBMCs [39] The HDAC-3-selective inhibitor MI192inhibited TNF production at high concentrations and dose-dependently reduced IL6 in RA PBMCs but not healthyPBMCs [39] Class III HDACi TSA as well as class III HDACnicotinamide blocked the TNF120572-stimulated expression ofIL6 and the LPS-induced expression of IL6 and TNF120572 inmacrophages of RA patients [38] TSA time-dependentlyincreased the acetylation of H3 and H4 in macrophages[38] However incubation ofmacrophageswith nicotinamidefailed to induce detectable acetylation of H3 or H4 [38]WTDmay function as a nonselectiveHDACi resulting in theacetylation of H3

5 Conclusions

In recent years DNA methylation and histone modificationswere involved in the pathogenesis of RA Moreover numer-ous epigenetics-based drugs were employed in attenuatingthe inflammatory activity of RASF ormacrophages especiallyvarious selective or nonselective HDACis WTD may serveas a traditional drug in alleviating disease activity of RA viaDNAmethylation and histonemodificationsThemethylatedloci and other epigenetic changes of RA are needed to beinvestigated to confirm this

Conflict of Interests

The authors have no competing interests

Authorsrsquo Contribution

Ya-Fei Liu and Cai-Yu-Zhu Wen carried out all the assaysand drafted the paper Ya-Fei Liu and Cai-Yu-Zhu Wencontributed equally to this work and should be consideredco-first authors Ya-Fei Liu Zhe Chen Yu Wang and YingHuang participated in the design of the study and carriedout the statistical analysis Yong-HongHu and Sheng-Hao Tuconceived of the study andwere responsible for its design andhelped in revising the paper All authors read and approvedthe final paper

Acknowledgments

The authors thank Professor Ming Xiang for helpful sug-gestions in modeling This work was supported by Grantsfrom the National Natural Science Foundation of China (no81341085)

References

[1] D M Lee and M E Weinblatt ldquoRheumatoid arthritisrdquo TheLancet vol 358 no 9285 pp 903ndash911 2001

[2] B M Javierre H Hernando and E Ballestar ldquoEnvironmentaltriggers and epigenetic deregulation in autoimmune diseaserdquoDiscovery Medicine vol 12 no 67 pp 535ndash545 2011


[3] J P Hamilton ldquoEpigenetics principles and practicerdquo DigestiveDiseases vol 29 no 2 pp 130ndash135 2011

[4] T T Glant K Mikecz and T A Rauch ldquoEpigenetics in thepathogenesis of rheumatoid arthritisrdquo BMC Medicine vol 12article 35 2014

[5] K Klein C Ospelt and S Gay ldquoEpigenetic contributions inthe development of rheumatoid arthritisrdquoArthritis Research andTherapy vol 14 no 6 article 227 2012

[6] E Karouzakis R E Gay S Gay and M Neidhart ldquoEpigeneticderegulation in rheumatoid arthritisrdquoAdvances in ExperimentalMedicine and Biology vol 711 pp 137ndash149 2011

[7] S Strietholt B Maurer M A Peters T Pap and S GayldquoEpigenetic modifications in rheumatoid arthritisrdquo ArthritisResearch andTherapy vol 10 no 5 article 219 2008

[8] M TrenkmannM Brock C Ospelt and S Gay ldquoEpigenetics inrheumatoid arthritisrdquo Clinical Reviews in Allergy and Immunol-ogy vol 39 no 1 pp 10ndash19 2010

[9] Y Zhang D Wang S Tan H Xu C Liu and N Lin ldquoAsystems biology-based investigation into the pharmacologicalmechanisms of wu tou tang acting on rheumatoid arthritis byintegrating network analysisrdquo Evidence-Based Complementaryand Alternative Medicine vol 2013 Article ID 548498 12 pages2013

[10] J Liu and R-L Liu ldquoThe potential role of Chinese medicinein ameliorating extra-articular manifestations of rheumatoidarthritisrdquo Chinese Journal of Integrative Medicine vol 17 no 10pp 735ndash737 2011

[11] Y F Liu S H TuWN Gao et al ldquoExtracts ofTripterygiumwil-fordiihookF in the treatment of rheumatoid arthritis a systemicreview and meta-analysis of randomised controlled trialsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 410793 11 pages 2013

[12] S L Dong and L X Zhang ldquoWutoudecoction in treating 146cases of rheumatoid arthritisrdquo Zhejiang Journal of TraditionalChinese Medicine vol 27 no 10 pp 471ndash472 1992

[13] J Y Li ldquoModified wutou decoction in treating 54 cases ofsciaticardquoHenan Traditional Chinese Medicine vol 29 no 11 pp1055ndash1056 2009

[14] PWang ldquoWutou decoction in the treatment of scapulohumeralperiarthritisrdquo Shanxi Journal of Traditional Chinese Medicinevol 22 no 10 p 629 2001

[15] M Li J He L-L Jiang et al ldquoThe anti-arthritic effects of Aconi-tum vilmorinianum a folk herbal medicine in SouthwesternChinardquo Journal of Ethnopharmacology vol 147 no 1 pp 122ndash127 2013

[16] M Kubo T Moriura and H Matsuda ldquoPharmacological studyon aconiti tuber I Effect of water extract from aconiti tuber onadjuvant-induced arthritisrdquo Yakugaku Zasshi vol 110 no 1 pp16ndash26 1990

[17] L Xue H L Zhang L Qin X C Wang and L Wang ldquoEffectof chuanwu and baishao used separately or in combination onadjuvant arthritis in ratsrdquo China Journal of Chinese MateriaMedica vol 25 no 3 pp 175ndash178 2000

[18] F-C Zhao L Fu C Wu et al ldquoResearch on the effect ofAconitum soongaricum and its processed products on AA ratrdquoJournal of Chinese Medicinal Materials vol 35 no 10 pp 1572ndash1576 2012

[19] Z Chen S H Tu Y H Hu Y Wang Y K Xia and Y JiangldquoPrediction of response of collagen-induced arthritis rats tomethotrexate an 1H-NMR-based urine metabolomic analysisrdquoJournal of Huazhong University of Science and Technology[Medical Sciences] vol 32 no 3 pp 438ndash443 2012

[20] E F Rosloniec M Cremer A H Kang L K Myers andD D Brand ldquoCollagen-induced arthritisrdquo Current Protocols inImmunology chapter 15Unit 1551-25 2010

[21] H Hikino C Konno H Takata et al ldquoAntiinflammatory prin-ciples of aconitum rootsrdquo Journal of Pharmacobio-Dynamicsvol 3 no 10 pp 514ndash525 1980

[22] Y Qi S Li Z Pi et al ldquoChemical profiling ofWu-tou decoctionby UPLC-Q-TOF-MSrdquo Talanta vol 118 pp 21ndash29 2014

[23] D E Trentham A S Townes and A H Kang ldquoAutoimmunityto type II collagen an experimental model of arthritisrdquo Journalof Experimental Medicine vol 146 no 3 pp 857ndash868 1977

[24] G Redei J Birchler K Kurachi and M Roberts EpigeneticsPrinciples Protocols and Practices Shanghai Science amp Technol-ogy Press Shanghai China 1st edition 2006

[25] C-C Liu T-J Fang T-T Ou et al ldquoGlobal DNA methylationDNMT1 and MBD2 in patients with rheumatoid arthritisrdquoImmunology Letters vol 135 no 1-2 pp 96ndash99 2011

[26] B Hendrich and A Bird ldquoIdentification and characterizationof a family of mammalian methyl-CpG binding proteinsrdquoMolecular and Cellular Biology vol 18 no 11 pp 6538ndash65471998

[27] E Karouzakis R E Gay B A Michel S Gay and M Neid-hart ldquoDNA hypomethylation in rheumatoid arthritis synovialfibroblastsrdquo Arthritis and Rheumatism vol 60 no 12 pp 3613ndash3622 2009

[28] B Richardson L Scheinbart J Strahler L Gross S Hanashand M Johnson ldquoEvidence for impaired T cell DNA methyla-tion in systemic lupus erythematosus and rheumatoid arthritisrdquoArthritis and Rheumatism vol 33 no 11 pp 1665ndash1673 1990

[29] H-H Ng Y Zhang B Hendrich et al ldquoMBD2 is a transcrip-tional repressor belonging to the MeCP1 histone deacetylasecomplexrdquo Nature Genetics vol 23 no 1 pp 58ndash61 1999

[30] N Detich J Theberge and M Szyf ldquoPromoter-specific activa-tion and demethylation by MBD2demethylaserdquoThe Journal ofBiological Chemistry vol 277 no 39 pp 35791ndash35794 2002

[31] K Klein and S Gay ldquoEpigenetic modifications in rheumatoidarthritis a reviewrdquo Current Opinion in Pharmacology vol 13no 3 pp 420ndash425 2013

[32] R Margueron and D Reinberg ldquoThe Polycomb complex PRC2and itsmark in liferdquoNature vol 469 no 7330 pp 343ndash349 2011

[33] A Barski S Cuddapah K Cui et al ldquoHigh-resolution profilingof histone methylations in the human genomerdquo Cell vol 129no 4 pp 823ndash837 2007

[34] M Trenkmann M Brock R E Gay et al ldquoExpression andfunction of EZH2 in synovial fibroblasts epigenetic repressionof the Wnt inhibitor SFRP1 in rheumatoid arthritisrdquo Annals ofthe Rheumatic Diseases vol 70 no 8 pp 1482ndash1488 2011

[35] C-G Miao Y-Y Yang X He and J Li ldquoNew advances of DNAmethylation and histone modifications in rheumatoid arthritiswith special emphasis on MeCP2rdquo Cellular Signalling vol 25no 4 pp 875ndash882 2013

[36] M Horiuchi A Morinobu T Chin Y Sakai M Kurosaka andS Kumagai ldquoExpression and function of histone deacetylases inrheumatoid arthritis synovial fibroblastsrdquo Journal of Rheumatol-ogy vol 36 no 8 pp 1580ndash1589 2009

[37] T Kawabata K Nishida K Takasugi et al ldquoIncreased activityand expression of histone deacetylase 1 in relation to tumornecrosis factor-alpha in synovial tissue of rheumatoid arthritisrdquoArthritis Research andTherapy vol 12 no 4 article R133 2010

[38] AM Grabiec S KrauszW de Jager et al ldquoHistone deacetylaseinhibitors suppress inflammatory activation of rheumatoid


arthritis patient synovial macrophages and tissuerdquo Journal ofImmunology vol 184 no 5 pp 2718ndash2728 2010

[39] J Gillespie S Savic C Wong et al ldquoHistone deacetylasesare dysregulated in rheumatoid arthritis and a novel histonedeacetylase 3ndashselective inhibitor reduces interleukin-6 produc-tion by peripheral blood mononuclear cells from rheumatoidarthritis patientsrdquo Arthritis and Rheumatism vol 64 no 2 pp418ndash422 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 The composition of herbal formula WTD

Crude herbs Content Main componentsEphedra (Herbal Ephedrae) 9 Ephedrine D-pseudoephedrineRed Peony Root (Radix Paeoniae Rubra) 45 PaeoniflorinWhite Peony Root (Radix Paeoniae Alba) 45 PaeoniflorinRoot of Membranous Milkvetch (Radix Astragali) 9 AstragalosidePrepared Liquorice Root (Radix Glycyrrhizae Preparata) 9 Glycyrrhizic acidPrepared Monkshood Mother Root (Radix Aconiti Preparata) 6 Aconitine mesaconitineWTD Wutou decoction

dispersing coldness which is employed in the treatment ofRA And yet the impacts of WTD on epigenetic changesare unknown The study aims to explore the effect of WTDon DNA methylation and histone modifications in rats ofcollagen-induced arthritis (CIA)

2 Materials and Methods

21 Reagents and Main Devices Bovine type II collagen(catalog 20022) and incomplete Freundrsquos adjuvant (catalog7002) were purchased from Amyjet Scientific Inc ChinaThe components of WTD except Herbal Ephedrae (Chi-nese Herbal Powder Beijing Tcmages Pharmaceutical CoLtd China) and Herbal Ephedrae were provided by TongjiHospital TRIzol Reagent (Invitrogen 15596026) DNaseI (Fermentas EN0521) RevertAid Reverse Transcriptase(Fermentas EP0442) dNTP (Fermentas R0191) RiboLockRNase Inhibitor (Fermentas E00381) All-in-Onetrade qPCRMaster Mix (GeneCopoeiaTM AOPR-1200) DNA Extrac-tion Kit (Aidlab Biotechnologies Co Ltd China catalogDN07) and Lymphocyte Separation Medium (Tian Jin HaoYang Biological Manufacture Co Ltd) were ordered fromWuhan Boster Company China MethylFlashtrade MethylatedDNA Quantification Kit (catalog P-1034) EpiQuiktrade TotalHistone Extraction Kit (catalog OP-0006) EpiQuiktrade GlobalPan-Methyl Histone H3-K4 Quantification Kit (catalog P-3028) EpiQuiktrade Global Pan-Methyl Histone H3-K27 Quan-tification Kit (catalog P-3044) EpiQuiktrade Total HistoneH3 Acetylation Detection Fast Kit (catalog P-4030) andEpiQuiktrade Total Histone H4 Acetylation Detection Fast Kit(catalog P-4032) were purchased fromAmyjet Scientific IncChina

Electric Homogenizer (Glas-Col USA) Ice Machine(AF-100AS Scotsman USA) Centrifuge (2-16K SigmaUSA) Nucleic AcidProtein Analyzer (DU730 BeckmanCoulter Inc California USA) Microplate Reader (BioTekSynergy2 Vermont USA) Heated Circulating Bath (DKB-600B Keelrein instrument Co Ltd China) MiliporeRiOs 5Water Purification Systems (MilliporeUSA) Applied Biosys-tems StepOne Real-Time PCR System (Applied BiosystemsCalifornia USA) and Esco Airstream Class II BiologicalSafety Cabinet (Beijing China) were used

22 Animals and Rearing Conditions Five-week-old weight-ed 130ndash150 g female Wistar rats (119899 = 45) SPF grade wereprovided by the Center for Disease Control and Prevention ofHubei province (the animal certificate SCXK no 2008-0005)

and fed in the barrier system according to The Guidelinesfor the Care and Use of Animals in Research enforced byHubei Municipal Science and Technology Commission Allprotocols were approved by the Institutional Animal Careand Ethics Committee of Tongji Medical College HuazhongUniversity of Science and Technology Food and water weregiven ad libitum throughout the experiment The rats werecaged in a standard barrier system with a 12 h lightdarkcycle

23 Grouping and Treatment After 7 days of acclimation theanimals were randomly divided into three groups normalgroup (N) CIA group and WTD group (42 gkgd) with15 rats in each group The rat doses of treatment groupwere converted from human doses (Chinese Pharmacopeia2010) based on body surface areas The final concentrationof treatment group was 042 gmL for WTD group Oralgavage was performed once a day from day 28 after the initialimmunization for four weeks Rats in groups N and CIAwereorally administered with the same volume of distilled waterThe rats were fasted for 12 h but permitted water ad libitumbefore blood collection

24 CIA Model Establishment and Arthritis Index AssessmentCIA model was established according to the manufacturerrsquosstandard protocols and previous research [19] In addition togroup N CIAmodel was established in the other two groupsBovine type II collagen was emulsified in an equal volume ofincomplete Freund adjuvant and 02mL of the emulsion wasinjected subcutaneously about 3 cm distal from the base ofthe tail to induce arthritis To ensure a high CIA incidencea booster immunization (administer 01mL of the emulsionsubcutaneously in the tail) was performed twoweeks after theinitial immunization about 15 cm distal from the base of thetail

As shown in Figure 1 the arthritis severity was evaluatedby applying a previous published scoring system [20] Oncearthritis occurred the arthritis index (AI) was evaluatedtwice a week for arthritis developmentThe arthritis score foreach rat was the sum of the scores for two hind paws

25 Sampling At sixty minutes after the last intragastricadministration the rats were narcotized with 10 chloralhydrate by intraperitoneal injection Blood was extractedfrom aorta abdominalis The synovium isolated from kneejoints was fixed in 4 paraformaldehyde for 12 h and embed-ded in paraffin Paraffin-embedded synovial tissues were


(a) (b) (c)

(d) (e) (f)

















(a) (b) (c)

(d) (e) (f)






3 Results




Relat

ive D

NM

T1m

RNA

0

2

4

6

WTDN CIA

998779

(a)

0

5

10

15

Rela

tive M

BD2

mRN

A

WTDN CIA(b)


0

1

2

3

4

5

DN

A m

ethy

latio

n (n

g)

CIA WTDN

998779






4 Discussion





times104

60

80

100

120

Pan-

Met

hyl H3

-K4

(ng

mg

prot

ein)


NCIAWTD

(a)

times104

60

80

100

120

Pan-

Met

hyl H3

-K27

(ng

mg

prot

ein)


NCIAWTD

(b)


998779

times104

50

60

70

80

90

H3

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(a)

times104

0

20

40

60

H4

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(b)














5 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)

(d) (e) (f)

















(a) (b) (c)

(d) (e) (f)






3 Results




Relat

ive D

NM

T1m

RNA

0

2

4

6

WTDN CIA

998779

(a)

0

5

10

15

Rela

tive M

BD2

mRN

A

WTDN CIA(b)


0

1

2

3

4

5

DN

A m

ethy

latio

n (n

g)

CIA WTDN

998779






4 Discussion





times104

60

80

100

120

Pan-

Met

hyl H3

-K4

(ng

mg

prot

ein)


NCIAWTD

(a)

times104

60

80

100

120

Pan-

Met

hyl H3

-K27

(ng

mg

prot

ein)


NCIAWTD

(b)


998779

times104

50

60

70

80

90

H3

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(a)

times104

0

20

40

60

H4

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(b)














5 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)

(d) (e) (f)






3 Results




Relat

ive D

NM

T1m

RNA

0

2

4

6

WTDN CIA

998779

(a)

0

5

10

15

Rela

tive M

BD2

mRN

A

WTDN CIA(b)


0

1

2

3

4

5

DN

A m

ethy

latio

n (n

g)

CIA WTDN

998779






4 Discussion





times104

60

80

100

120

Pan-

Met

hyl H3

-K4

(ng

mg

prot

ein)


NCIAWTD

(a)

times104

60

80

100

120

Pan-

Met

hyl H3

-K27

(ng

mg

prot

ein)


NCIAWTD

(b)


998779

times104

50

60

70

80

90

H3

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(a)

times104

0

20

40

60

H4

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(b)














5 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Relat

ive D

NM

T1m

RNA

0

2

4

6

WTDN CIA

998779

(a)

0

5

10

15

Rela

tive M

BD2

mRN

A

WTDN CIA(b)


0

1

2

3

4

5

DN

A m

ethy

latio

n (n

g)

CIA WTDN

998779






4 Discussion





times104

60

80

100

120

Pan-

Met

hyl H3

-K4

(ng

mg

prot

ein)


NCIAWTD

(a)

times104

60

80

100

120

Pan-

Met

hyl H3

-K27

(ng

mg

prot

ein)


NCIAWTD

(b)


998779

times104

50

60

70

80

90

H3

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(a)

times104

0

20

40

60

H4

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(b)














5 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















times104

60

80

100

120

Pan-

Met

hyl H3

-K4

(ng

mg

prot

ein)


NCIAWTD

(a)

times104

60

80

100

120

Pan-

Met

hyl H3

-K27

(ng

mg

prot

ein)


NCIAWTD

(b)


998779

times104

50

60

70

80

90

H3

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(a)

times104

0

20

40

60

H4

acet

ylat

ion

(ng

mg

prot

ein)

CIA WTDN(b)














5 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























5 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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