Objective:To explore the molecular bases of Chinese medicine(CM)syndrome classification in chronic hepatitis B(CHB)patients in terms of DNA methylation,transcription and cytokines.Methods:Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients(DNA methylation:15 cases;serum cytokines:62 cases)with different CM syndromes,including dampness and heat of Gan(Liver)and gallbladder(CHB1,DNA methylation:5 cases,serum cytokines:15 cases),Gan stagnation and Pi(Spleen)deficiency(CHB2,DNA methylation:5 cases,serum cytokines:15 cases),Gan and Shen(Kidney)yin deficiency(CHB3,DNA methylation:5 cases,serum cytokines:16 cases),CHB with hidden symptoms(HS,serum cytokines:16 cases)and healthy controls(DNA methylation:6 cases).DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples.Genome-wide DNA methylation was detected using Human Methylation 450K Assay and further verified using pyrosequencing.Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay(ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction(RT-qPCR),respectively.Results:Totally 28,667 loci,covering 18,403 genes were differently methylated among CHB1,CHB2 and CHB3(P＜0.05 and| △ β value)＞0.17).Further validation showed that compared with HS,the hg19 CHR6:29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1(P＜0.05).However,they remained unaltered in CHB2(P＞0.05).Levels of Interleukin(IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups(P＜0.05).Levels of macrophage inflammatory protein(MIP)-1 αand MIP-1 β were higher in CHB3 than other groups and leukemia inhibitory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups(P＜0.05).IL-12,MIP-1 α and MIP-1 β concentrations were positively correlated with human leukocyte antigen F(HLA-F)mRNA expression(R2=0.238,P＜0.05;R2=0.224,P＜0.05;R2=0.447,P＜0.01;respectively).Furthermore,combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1,CHB2 and HS.Conclusions:Demethylation of CpG loci in 5'UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12,MIP-1 α and MIP-1 β levels,indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB.(Registration No.ChiCTR-RCS-13004001)

Research Center for Traditional Chinese Medicine Complexity System,Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai(201203),China;Department of Medical Oncology,Shuguang Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai(201203),China;Academy of Integrative Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai(201203),China;Department of Vascular Disease,Shanghai Traditional Chinese Medicine-Integrated Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai(201203),China;Department of Vascular Disease,Shanghai Traditional Chinese Medicine-Integrated Institute of Vascular Disease,Shanghai(201203),China;Institute of Liver Disease,Shuguang Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai(201203),China

[1]HU Xue-qing;ZHOU Yuan;CHEN Jian;LU Yi-yu;CHEN Qi-long;HU Yi-yang;SU Shi-bing-.DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B)[J].中国结合医学杂志（英文版）,2022(06):501-508

Relate,CHB1,CHB2,CHB3,450K,CHR6,demethylated,Demethylation

Methylation,Transcription,HLA,Serum,Cytokines,Chinese,Medicine,Syndrome,Classification,Patients,Chronic,Hepatitis,Objective,explore,molecular,bases,medicine,CM,classification,chronic,hepatitis,patients,terms,transcription,cytokines,Methods,Genome,wide,serum,were,detected,cases,syndromes,including,dampness,heat,Gan,Liver,gallbladder,stagnation,Pi,Spleen,deficiency,Shen,Kidney,yin,hidden,symptoms,HS,healthy,controls,critical,was,further,validated,its,enlarged,samples,using,Human,Assay,verified,pyrosequencing,evaluated,multiplex,biometric,enzyme,linked,immunosorbent,immunoassay,reverse,quantitative,polymerase,chain,reaction,qPCR,respectively,Results,Totally,loci,covering,genes,differently,among,value,validation,showed,that,compared,hg19,closely,surrounded,CpG,expressions,significantly,regulated,However,they,remained,unaltered,Levels,Interleukin,higher,than,groups,macrophage,inflammatory,protein,MIP,other,leukemia,inhibitory,concentrations,positively,correlated,human,leukocyte,antigen,Furthermore,combination,differential,greatly,improved,differentiating,accuracy,Conclusions,UTR,may,associated,levels,indicating,might,jointly,involve,Registration,No,ChiCTR,RCS

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