基于表观遗传学和代谢组学的野菊花活性部位抗乙肝病毒整体作用分子机制研究  

The overall action molecularmechanism of anti-hepatitis B active extracts in Flos chrysanthemi indici based on epigenetics and metabonomics

作　　者：张芳平 王云雨 程鑫涛 王东浩 李英梅 刘藤藤 李爽 郑一超[1] 符玲[1] 毕跃峰[1] ZHANG Fang-ping;WANG Yun-yu;CHENG Xin-tao;WANG Dong-hao;LI Ying-mei;LIU Teng-teng;LI Shuang;ZHENG Yi-chao;FU Ling;BI Yue-feng(School of Pharmaceutical Sciences,Zhengzhou University,Zhengzhou 450001,China)

机构地区：[1]郑州大学药学院,河南郑州450001

出　　处：《药学学报》2022年第8期2352-2363,共12页Acta Pharmaceutica Sinica

摘　　要：利用表观遗传学和代谢组学理念和方法,探讨阐明野菊花活性部位(Chrysanthemi indici C,CIC)抗乙肝病毒(hepatitis B virus,HBV)整体作用分子机制。CCK-8和乙肝抗原试剂盒检测CIC对HepG2.2.15细胞增殖和乙型肝炎病毒表面抗原(hepatitis B surface antigen,HBsAg)、乙型肝炎病毒e抗原(hepatitis B envelope antigen,HBeAg)、乙型肝炎病毒核酸(hepatitis B virus-deoxyribonucleic acid,HBV-DNA)的抑制作用;ELISA法检测CIC对DNA甲基转移酶(DNA methyltransferases,DNMTs)/去甲基转移酶2(ten-eleven-translocation-2,TET2)平衡关系的影响;利用Illumina 850K甲基化芯片、焦磷酸测序和qPCR技术,通过GO、KEGG等分析,确定CIC抗HBV的作用途径和靶点;80%甲醇提取细胞代谢物,LC-MS等代谢组学方法检测差异代谢物、差异代谢途径及细胞微环境的变化。结果表明,CIC对HepG2.2.15细胞增殖和HBsAg、HBeAg、HBV-DNA有明显的抑制作用,下调DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)、DNA甲基转移酶3a(DNA methyltransferase 3a,DNMT3a)、DNA甲基转移酶3b(DNA methyltransferase 3b,DNMT3b),上调TET2,恢复DNMTs/TET2平衡;DNA甲基化测序结果表明,CIC抗乙肝病毒的作用靶基因有磷脂酶C-γ2(phospholipase C gamma 2,PLCG2)、磷脂肌醇3激酶(phosphoinositide-3-kinase regulatory subunit 3,PIK3R3)、1酰基甘油3磷酸O酰基转移酶2(1-acylglycerol-3-phosphate O-acyltransferase 2,AGPAT2)、5-羟色胺受体2B(5-hydroxytryptamine receptor 2B,HTR2B)、神经生长因子(nerve growth factor,NGF),主要涉及脂质代谢、瞬时感受器电位(transient receptor potential,TRP)通道炎症介导调节、磷脂酶D信号、糖尿病并发症中晚期糖基化终产物-AGE受体(advanced glycation end product-receptor for AGE,AGE-RAGE)信号等通路;代谢组学研究表明,CIC可以显著影响脂肪酸代谢,同时对细胞微环境中酚酸、生物碱、脂质类代谢物影响较大。研究结果提示,野菊花活性部位抗乙肝病毒作用机制可能是通过调节表Using the concepts and methods of epigenetics and metabolomics,to investigate the overall action molecular mechanism of Chrysanthemi indici C(CIC),the anti-hepatitis B virus(HBV)active extracts from Flos chrysanthemi indici.The inhibitory effects of CIC on proliferation and hepatitis B surface antigen(HBsAg),hepatitis B envelope antigen(HBeAg)and HBV-DNA of HepG2.2.15 cells were detected by CCK-8 and antigen kit.The DNA methyltransferases(DNMTs)/ten-eleven-translocation-2(TET2)equilibrium was detected by ELISA.Illumina 850K methylation chip,pyrosequencing and qPCR were used to determine the action pathway and target of CIC by GO and KEGG analysis.Cell metabolites were extracted with 80%methanol,and the changes of differential metabolites,differential metabolic pathways and cell microenvironment were detected by LC-MS and other metabolomics methods.The results showed that CIC could inhibit the proliferation,HBsAg,HBeAg and HBV-DNA of HepG2.2.15 cells obviously,down-regulate DNA methyltransferase 1(DNMT1),DNA methyltransferase 3a(DNMT3a)and DNA methyltransferase 3b(DNMT3b),up-regulate TET2,and restore the balance of DNMTs/TET2.The action targets of CIC were phospholipase C gamma 2(PLCG2),phosphoinositide-3-kinase regulatory subunit 3(PIK3R3),1-acylglycerol-3-phosphate O-acyltransferase 2(AGPAT2),5-hydroxytryptamine receptor 2B(HTR2B),nerve growth factor(NGF),mainly involved in lipid metabolism,inflammation mediated regulation of transient receptor potential(TRP),phospholipase D signaling and advanced glycation end product-receptor for AGE(AGE-RAGE)signaling in diabetic complications pathways.CIC could significantly affect fatty acid metabolism and had great influence on phenolic acid,alkaloid and lipid metabolites in cell microenvironment.These results suggest that the action mechanism of CIC may be the synergistic action of multiple pathways and multiple targets,including related inflammatory pathways,immune pathways and lipid metabolism,through regulating epigenetic expression balance and restoring the balance of

关 键 词：野菊花 活性部位CIC 抗乙肝病毒 DNA甲基转移酶/去甲基转移酶2 表观遗传学 代谢组学 

分 类 号：R966[医药卫生—药理学]