Study On The Mechanism Of Preventing Alcoholic Hepatic Injury By Various Pathways Of Vine Tea Based On Network Pharmacology

BackgroundThe morbidity increased in alcoholic liver disease(ALD)is a serious threat to human health.Recent surveys have shown that the death rate caused by alcohol abuse accounts for about 4% of the world’s total.Alcohol abusers are more than 90% of alcohol fatty liver(AFL)and eventually develop into cirrhosis,which leads to liver cancer.Previous studies have suggested that the pathogenesis of ALD is complicated,and oxidative stress,intestinal endotoxemia,immune response,genetic factors,and malnutrition are all involved in the development of ALD.In the recent years,more and more new targets have been researched and confirmed to be closely related to the ALD process,such as intestinal flora disturbance,cell autophagy,endoplasmic reticulum stress,IL-22,and endocannabinoid system.However,most of the drugs currently used for clinical treatment of ALD have a single target and the efficacy is not exact.At the same time,traditional Chinese medicine has great advantages and broad prospects in the prevention and control of ALD because of its multi-level and multi-target regulatory advantages.At present,it has been discovered that the leaves and stems of Ampelopsis grossedentata,a member of the Vitaceae family,are increasingly concerned by the fact that they are rich in flavonoids and have a wide range of pharmacological activities.This study evaluated the protective effect of vine tea extract on ALD and its mechanism of action through a mouse chronic alcoholic liver injury model to provide a theoretical basis for the development of safe and effective ALD intervention products.Methods1.The vine tea sample was prepared by 70% ethanol extraction,and the two hepatoprotective active ingredients of dihydromyricetin and myricetin in AGE were determined by HPLC.Using the TCMSP,the Pharm Mapper server based on reverse pharmacophore matching,the SEA online search tool,and the STITCH 4.0 database to excavated the potential targets of dihydromyricetin and myricetin which were the active ingredients in vine tea.Combining Cytoscape3.2.1 software to construct the active ingredient-target network and perform predictive GO enrichment analysis.Meanwhlie,we collected relevant targets of alcoholic liver disease,constructed a compound-action target-disease network,performed topological analysis and targets selection,and performed the Clue GO enrichment analysis on the core targets.2.In the present study,the C57BL/6 mice were ad libitum fed with the Lieber-De Carli diet containing alcohol or isocaloric maltose dextrin as control diet with or without AGE(150 and 300 mg/kg/d bw)for 6 weeks.Serum ALT,AST,LDH,and ALP levels,hepatic MDA and GSH levels,and histopathological changes were measured to assess the effects of AGE on chronic alcoholic liver injury.Meanwhile,serum TNF-α and hepatic IL-1β and IL-6 levels in the mice with chronic alcoholism were detected by ELISA,serum lipopolysaccharide(LPS)levels in mice were detected by Limulus Amebocyte Lysate assay.The protein expression of TLR4-related signaling pathways(CD14,TLR4,My D88)and intestinal tight junction proteins ZO-1 and Occudin were detected by Western blotting.Meanwhile,the ultrastructure and tight junction of small intestinal epithelial cells and the fluorescence localization of ZO-1 in small intestine were observed by transmission electron microscope(TEM)and immunofluorescence.The changes in composition of intestinal flora were detected by 16 S r DNA sequencing technology,and the sequencing results were analyzed by bioinformatics.3.In the present study,the C57BL/6 mice were ad libitum fed with the Lieber-De Carli diet containing alcohol or isocaloric maltose dextrin as control diet with or without DMY(75 and 150 mg/kg/d bw)for 6 weeks.The serum biomarkers,lipid peroxidation levels and histopathological changes were detected to evaluate the hepatoprotective effects of DMY which was the active substance in vine tea.Meanwhile,the hepatic inflammatory cytokines were detected by ELISA method.The expression of CYP2E1,Keap-1,and HO-1 and autophagy-related proteins p62,LC3 B,and Beclin 1 were detected by Western blotting.The autophagy phenomena in hepatocytes were observed by TEM.The nucleus expression of Nrf2 and NF-κB in the liver was detected by Western blotting and immunofluorescence.Results1.The contents of dihydromyricetin and myricetin in 70% ethanol extract of vine tea(AGE)were 628.11 mg/g and 44.22 mg/g,respectively.The results of GO enrichment analysis on the targets of the vine tea revealed that the protein targets may participate in the various biological processes that drug reaction,protein autophosphorylation,negative regulation of apoptosis,response to ethanol,response to hypoxia,inflammation reaction,lipopolysaccharide response.The CC analysis results showed that the predicted target protein is mostly distributed in the cytoplasm,exosomes,extracellular spaces,cytoplasmic membranes and other locations.The MF analysis results showed that the predicted protein targets may be closely related to molecular functions such as drug binding,protein kinase activity,ATP binding,protein tyrosine kinase activity,protein homodimerization activity,oxidoreductase activity,and hemoglobin binding.GEO analysis was performed on liver tissue samples of alcoholic liver patients to screened out 314 differential genes.Meanwhile,we added the Drug Bank,OMIM,GAD,TTD,and Goo LGe N databases and collected 418 related alcoholic liver diseases targets.And then we builded a Compound-Targets network and Disease-Targets network by Cytoscape 3.4.1 software.Furthermore,we adopted the Cyto NCA plug-in for topological analysis to screen out 255 core targets.The enrichment analysis of the core targets,it showed that the gene functions of the target components in vine tea mainly concentrated on the biological processes of peptide hormone response,negative regulatory defense response and response to lipopolysaccharide.2.Compared with the normal control group,serum ALT,AST,LDH,ALP levels and MDA in liver tissue were significantly increased in the alcohol control group,and the GSH level was significantly lower(P<0.01).Compared with alcohol control group,AGE(150mg/kg)group and AGE(300mg/kg)group can significantly reduce serum enzyme levels and liver MDA content,and increase hepatic GSH levels,showing a certain dose-effect relationship(P<0.01 or P<0.05).Pathological results showed that the AGE(150 mg/kg)and AGE(300 mg/kg)groups significantly improved liver steatosis and inflammation after chronic alcohol intake.Compared with the normal control group,serum LPS in the alcohol control group was significantly increased(P<0.01).Compared with the alcohol control group,AGE(150mg/kg)group,AGE(300mg/kg)group remarkedly reduced serum LPS levels.Moreover,compared with the normal control group,the protein expression of CD14,TLR4 and My D88 in the alcohol control group were significantly increased(P<0.01).Meanwile,compared with the alcohol control group,AGE(150mg/kg)group And AGE(300mg/kg)group significantly inhibited the expression of hepatic CD14,TLR4 and My D88(P<0.01 or P<0.05).After long-term alcohol ingestion,the tight junction proteins of ZO-1 and Occudin in the small intestine significantly decreased(P<0.01).Meanwile,AGE remarkly recovered the intestinal epithelial mucosal ZO-1 and Occudin expression(P<0.01 or P<0.05).The TEM results showed that the AGE administration impoved the destruction of ultrastructure and tight junction in the small intestine wall after chronic alcohol intake.Immunofluorescence results showed that the fluorescence intensity and distribution continuity of ZO-1 in the small intestine tissue were significantly increased after administration of AGE compared those in the alcohol group.It suggested that AGE significantly reduced the alcohol-induced intestinal mucosal wall damage and restored mucosal barrier function.In present experiment,we used 16 S r DNA sequencing to analyze the fecal microbiota diversity and composition in mice.The results of diversity show that chronic alcohol can reduce the diversity of intestinal flora to a certain extent.Although AGE intervention has the tendency to reverse the decline in bacterial diversity caused by alcohol,there is no significant difference between the groups.Compared to the alcohol control group,AGE administration significantly reduced the relative abundance of the proteobacteria phylum and the proteobacteria phylum in the intestinal flora of mice with chronic alcoholism,and significantly increased relative abundance(Verrucomicrobia)and the ratio of Bacteroidetes Phylum / Firmicutes Phylum.The results of the intestinal flora showed that chronic ethanol caused a significant reduction in the flora of the genus Alloprevotell(P<0.05)compared to the normal control group,AGE(150 mg/kg)Compared with alcohol control group,the AGE group signicantly increased the relative abundance of Prevotella,Parabacteroides and Alistipes relative abundance(P<0.01 or P<0.05)(see Fig.2-14.A).The results showed that long-term alcohol intake significantly increased relative abundance of opioid pathogens Oscillibacter and gram-negative bacteria Helicobacter(Proteobacteria)compared with the normal group(P<0.05).These changes in gut microbiota could be significantly reversed by AGE intervention(P<0.01 or P<0.05)(see Fig.2-14.A).At the same time,the study also found that chronic alcohol significantly reduced the relative abundance of beneficial bacteria(Akkermansia)in intestinal flora compared with normal control group(P<0.01),while AGE significantly increased the Akkermansia and producing short-chain fatty acid(SCFA)Intestinimonas due to chronic ethanol ingestion(P<0.01 or P<0.05)(see Fig.2-14-B.).3.Serum enzyme,pathological sections,and hepatic inflammatory factor detection results showed that the DMY(75 mg/kg)and DMY(150 mg/kg)groups significantly improved chronic alcohol-induced enzymology changes,hepatic fat deposition and inflammation.Compared with the normal control group,the expression of CYP2E1 in the liver of alcohol control group was significantly increased(P<0.01),and the expression of HO-1 was significantly decreased(P<0.01),but there was no significant difference in Keap-1 expression(P>0.05).Compared with the alcohol control group,DMY intervention significantly reversed the alcohol-induced CYP2E1 and HO-1 protein expression abnormalities in the liver(P<0.01 or P<0.05).Compared to the alcohol control group,the protein level of Keap-1 was significantly decreased after DMY intervention(P<0.05).Further studies found that Keap1 m RNA was not significantly different among all groups(P>0.05).It was suggested that the inhibition of Keap1 expression by DMY did not affect the Keap1 m RNA level.Compared with the normal control group,the level of hepatic autophagy-specific substrate p62 protein,relative LC3-II,and Beclin1 expression in the alcohol control group significantly increased(P<0.01 or P<0.05).The levels of autophagic marker proteins p62,LC3-II and expression with Beclin1 were further increased after DMY administration(P<0.05 or P <0.01).It is suggested that DMY can significantly activate the expression of key proteins autophagy pathway in the liver in mice with chronic alcoholism.TEM results showed that,the number of autophagic vacuoles in the liver of alcohol-treated mice did not increase compared with normal control mice,while the number of autolysosomes significantly increased after DMY intervention compared with the alcohol-treated group.And showed a certain dose-effect relationship.The results of Western Blot,EMSA and immunofluorescence showed that DMY could inhibit the activation of NF-κB induced by alcohol,and induce the nucleus expression of Nrf2 in hepatocytes to antagonize alcohol-induced oxidative stress and lipid peroxidation.ConclusionIn this study,we comprehensively evaluated the protection of vine extract(AGE)and its main active ingredient dihydromyricetin(DMY)against alcoholic liver injury by various methods such as network pharmacology,whole animal experiment,molecular biology and 16 S r DNA sequencing.And from the perspective of the "gut-liver axis" in body and anti-oxidant system to in-depth discuss the intervention mechanism of AGE,the specific conclusions are as follows:1.Using network pharmacology techniques analyzed the potential targets of vine tea prevention and treatment of ALD.The results showed that vine tea may regulate LPS-mediated TLR4 signaling pathway,hepatic lipid metabolism pathway and anti-oxidative stress to exert multiple-pathways and multiple-targets pharmacological effects against ALD.2.The chronic alcoholic liver injury mice experiments showed that the hepatoprotective mechanism of AGE may be closely related to the protection of the tight junction integrity of the intestinal mucosa,and it is closely related to the inhibition of hepatic LPS-mediated TLR4-related signaling pathway and the production of inflammatory factors.Further research found that AGE could regulate the intestinal flora,increase the abundance of beneficial microbes,while reduce the potential harmful bacteria and intestinal LPS production,so as to play a crucial role in the prevention and treatment of ALD.3.DMY serves as an antioxidant flavonoid probably activate Nrf2/Keap-1 pathway to protect aginst chronic alcoholic liver injury.Mechanistically,in this study we investigated the crosstalk among p62,Keap-1,Nrf2 and further observed p62 upregulation induced by DMY contributes to a positive feedback loop in the Keap-1/Nrf2 pathway.Noteworthily,DMY dramatically induced autophagy and possible provides beneficial and sufficient autophagy condition to confirm p62-mediated Keap-1 degradation.This study evaluated the protective effect of vine tea and its active ingredient dihydromyricetin on ALD and its action mechanism through a mouse model of chronic alcoholic liver injury.Our studies provide provide a theoretical basis for the development of safe and effective intervention products in ALD.