Based On Network Pharmacology And Microbiology To Explore The Role And Mechanism Of Hugan Qingzhi Tablets And Its Active Ingredients In Preventing And Treating Non-alcoholic Fatty Liver Disease

Background and objective:Non-alcoholic fatty liver disease(NAFLD)is caused by the exclusion of alcohol,hepatitis virus,drugs and other definite liver damage factors.It is characterized by diffuse liver cell bullous fat,with or without Genetic-environment-metabolic stress-related clinicopathological syndrome with inflammation as the main pathological feature.With the change of people’s lifestyle in recent years,the incidence of NAFLD has been increasing year by year,and there is a trend of aging.Hugan Qingzhi Tablet(HQT)is derived from famous old Chinese medicine prescriptions.It consists of traditional Chinese medicines such as Zexie,Hawthorn,Sanqi,and Puhuang.It is mainly used in the prevention and treatment of non-alcoholic fatty liver disease Significant effect.In vivo and in vitro tests have confirmed that HQT at 1.08g/kg can effectively improve liver steatosis,reduce blood lipids and inflammatory responses in NAFLD rats.The team used UHPLC-QqQ-MS detection in the early stage and found that HQT contained a large number of flavonoids such as isorhamnetin,quercetin,rutin,cattailin,and hypericin.In group studies,we found that HQT can up-regulate the expression of microbial metabolic pathways in liver of NAFLD rats.It can regulate the intestinal flora structure of NAFLD rats,inhibit harmful bacteria,promote the growth of beneficial bacteria,and enhance the intestinal flora of NAFLD rats Flavonoid biosynthesis.Therefore,we speculated that the flavonoid monomer component in HQT may be the active component for its prevention and treatment of NAFLD.This experiment combined with network pharmacological methods to explore the effective active ingredients of Huganqingzhi tablets in the prevention and treatment of NAFLD and its mechanism of action.Methods1.Use the TCM-SP and BATMAN-TCM databases to collect the active monomer components of Huganqingzhi Recipe and their target targets;collect the non-alcoholic fatty liver disease(NAFLD)treatment targets from the OMIM database.Cytoscape(Version 3.3.0)builds a drug-active ingredient network and an active ingredient-acting target network.The DAVID database was used for the enrichment analysis of potential target genes for the effect of Hugan Qingzhi Recipe on non-alcoholic fatty liver disease.2.A high-fat diet was used to establish a NAFLD rat model,and the rats were randomly divided into 3 groups:Normal fat diet(NFD)group,High fat diet(HFD)group,1.08g/kg HQT+In the fat feed group,the liver lipid content,inflammation index level and related pathway protein of each group of rats were measured after the end of 12 weeks.3.A high-fat diet was used to establish the NAFLD model of C57 mice,and 30 C57 mice were randomly divided into three groups:normal group(NC),high fat diet(HFD)group,and 100 mg/kg hyperin(HYP))+ High-fat diet group.After the end of 19 weeks,the liver TG,TC content and inflammation index levels of C57 mice in each group were measured,and the pathology of liver tissue was observed with oil red O staining and HE staining sections.4.Using 16SrRNA sequencing technology to defecate the intestinal bacteria of the feces of three groups of mice.5.Gas chromatography was used to detect the short-chain fatty acids in the feces of the three groups of mice;intestinal mucosa HE staining,WB and qRT-PCR were used to detect the expression of tight junction protein in intestinal mucosal epithelial cells.Results1.Use the TCM-SP and BATMAN-TCM databases to collect the active monomer components of Huganqingzhi Recipe and their target targets;collect the non-alcoholic fatty liver disease(NAFLD)treatment targets from the OMIM database.Cytoscape(Version 3.3.0)builds a drug-active ingredient network and an active ingredient-acting target network.The DAVID database was used for the enrichment analysis of potential target genes for the effect of Hugan Qingzhi Recipe on non-alcoholic fatty liver disease.2.A high-fat diet was used to establish a NAFLD rat model,and the rats were randomly divided into 3 groups:Normal fat diet(NFD)group,High fat diet(HFD)group,1.08g/kg HQT+In the fat feed group,the liver lipid content,inflammation index level and related pathway protein of each group of rats were measured after the end of 12 weeks.3.Animal experiments show that hypericin(HYP)can reduce triglyceride(TG),cholesterol(cholesterol,CHOL),alanine aminotransferase(ALT),and aspartate aminotransferase(AST)levels in serum of NAFLD mice Mouse NAFLD model liver tissue homogenate triglyceride(TG),cholesterol(cholesterol,CHOL)and other indicators.4.After hypericin treatment in high-fat diet mice,the intestinal flora was significantly changed back to normal mice.The increase in F/B ratio caused by high-fat diet was also significantly reversed.The abundance of A.muciniphila in the intestine It significantly increased the degree of intestinal flora in NAFLD mice caused by high-fat diet.5.The content of short-chain fatty acids in the feces of the three groups of mice was detected by gas chromatography.Compared with the NC group,the levels of acetic acid,propionic acid and butyric acid in the intestines of the HFD group were reduced(p<0.05).The levels of acetic acid and propionic acid in the intestine of mice in the HYP group were significantly higher than those in the HFD group,which may be related to hypericin promoted the growth of beneficial bacteria producing acetic acid and propionic acid in the intestines of the mice.Compared with HFD group,the expression of tight junction protein in ileal mucosal epithelial cells was increased(p<0.05),and the damage of intestinal mucosal barrier was relieved.ConclusionsIn this experiment,a network of "HuganQingzhi Tablets Active Ingredients-Disease-Targets" was successfully constructed through the method of network pharmacology.The results of in vivo experiments were consistent with the results of network pharmacology screening,combined with network pharmacology and the previous application of UHPLC-QqQ-MS obtained monomer components,screened out possible monomer components for preliminary verification.Pre-experimental studies have found that earlier studies have found that hypericin can significantly slow down the weight gain of high-fat diet mice,effectively improve the liver tissue morphology and lipid droplet deposition in NAFLD model mice,and reduce TG and TC in liver tissues.Levels,while reducing the levels of TG,TC,AST,and ALT in serum.In addition,after hypericin treatment in high-fat diet mice,the intestinal flora was significantly changed back to normal mice,and the increase in F/B ratio caused by the high-fat diet was also significantly reversed.The abundance of A.muciniphila in the intestine Significantly increased,effectively regulated the intestinal flora disturbance of NAFLD mice caused by high-fat diet,and significantly restored the phenomenon of low SCFAs induced by NAFLD.We speculate that hypericin may participate in the protection of NAFLD by regulating the intestinal flora and increasing the production of SCFAs and SCFAs by repairing the intestinal barrier.