Study On The Mechanism Of Anti HBV Active Sites Of Flos Chrysanthemi Indici Based On Epigenetics

Chronic Hepatitis B is an infectious disease caused by continuous replication of Hepatitis B virus?HBV?,which not only poses a serious threat to human life and health,but also causes tremendous social harm.In the early stage,through the in-depth and systematic study of flos chrysanthemi indici,it was found that the CIC of anti-HBV active sites in flos chrysanthemi indici had multi-pathway effects such as anti-HBV,liver protection and immune regulation.Based on the previous research,the Hep G 2.2.15 cell model was used to study the anti-HBV effects of CIC,CIT and CIF in vitro,and explore the effect of CIC on cellular DNA methylation level for the first time to clarify the mechanism of CIC anti-HBV.This paper can be distributed into two parts.Part 1:Study on the direct inhibitory effects of CIC,CIT and CIF on HBV in vitro.First,the cytotoxic effects of CIC,CIT and CIF were detected by CCK-8 kit,and half of the toxic concentration(TC₅₀)and the effective nontoxic concentration were screened out.Then,ELISA and fluorescence quantitative PCR methods were used to measure the effects of CIC,CIT and CIF on HBs Ag,HBe Ag and HBV-DNA secreted by cells.The results stated that CIC?CIT and CIF had cytotoxic effects on Hep G 2.2.15 cells,and the cytotoxic effects increased with the increase of drug concentration.The cytotoxic effects of the three active sites were CIT>CIC>CIF,with TC₅₀ values of 16.86?g/m L,26.27?g/m L and 35.50?g/m L,and the effective nontoxic concentration was 12.5?g/m L,17.5?g/m L and 25?g/m L,respectively.CIC,CIT,and CIF had a good inhibitory effect on HBs Ag,HBe Ag,and HBV-DNA secreted by Hep G 2.2.15 cells at the effective nontoxic concentration.Among them,CIC and CIT had stronger inhibitory effects on HBs Ag than HBe Ag,and CIF inhibited HBe Ag better than HBs Ag.The results showed that all three active sites had good anti-HBV effects in vitro.Part 2:Study on the anti-HBV mechanism of CIC.?1?Illumina 850K was used to screen differential methylation sites in CIC group and control group,and the overall methylation differences between the two groups were compared.The GO functional analysis and KEGG functional analysis were performed on the genes with significant methylation differences to screen candidate differential methylation genes.The results indicated that the overall methylation level of CIC was slightly higher than that of control group.When P<0.05,|beta.difference|>0.1,there were 94genome-wide significantly different methylation sites,distributed on 81 genes,46genes with higher methylation degree and 35 genes with lower methylation degree.GO and KEGG functional analysis demonstrated that the gene functions of differential methylation mainly focused on lipid metabolism and biosynthesis processes such as glycerophospholipids and glycerides,regulation of MAP kinase activity.The major inflammatory pathways involved in differential methylation genes were Inflammatory mediator regulation of TRP channels,Fc Epsilon RI signaling pathways and Erb B signaling pathways,and the major immunomodulatory pathways were B cell receptor signaling pathway and Natural killer cell mediated cytotoxicity.The abnormally hypermethylated genes related to that were mainly NGF,PIK3R3,PLCG2 and AGPAT2,and the abnormally hypomethylation genes were HTR2B.?2?The m RNA levels of these genes were measured by Real-time PCR technology.The results proved that the expression levels of NGF,PIK3R3 and AGPAT2 genes in the CIC group decreased and had significant differences?P<0.05?,PLCG2 gene expression levels was increased and had significantly difference?P<0.05?,however,there was no significant difference in HTR2B gene.?3?Then the differential methylation sites of differential genes were verified by pyrosequencing.The results manifested that the methylation level of NGF gene in CIC group was increased and had significant differences?P<0.01?,there were no significant difference in the methylation levels of PIK3R3 and AGPAT2 genes between the two groups.The results illustrated that CIC may inhibit HBV through multiple pathways and targets,such as reduce virus load,anti-inflammatory,regulation of immunity and lipid metabolism,and achieve anti-HBV effect by regulating DNA methylation level.In summary,the effects of flos chrysanthemi indici anti-HBV active sites CIC,CIT and CIF on direct inhibition of HBV were studied in this paper,and the mechanism of multi-path and multi-target anti-HBV of CIC was explored from the microscopic and the molecular level for the first time,which provided a certain scientific reference value for the research of the role of traditional Chinese medicine in anti-HBV.