Preventive Effect And Mechanism Study Of Aqueous Extract Of Qing Brick Tea On Non-alcoholic Fatty Liver Disease

Background With the continuous change of dietary habits and living conditions,non-alcoholic fatty liver disease(NAFLD)has gradually become a serious global health problem.The latest statistics show that the global prevalence of NAFLD has exceeded 25%,with the prevalence in Asia reaching 27.37%.NAFLD is a complex liver disease that includes many different disease manifestations: simple fatty liver disease,nonalcoholic steatohepatitis,liver fibrosis,and liver cancer.NAFLD is also associated with the development of several extrahepatic complications such as type 2 diabetes,cardiovascular disease,chronic kidney disease,and certain types of extrahepatic malignancies,and the risk of these extrahepatic complications is positively correlated with the severity of NAFLD.At present,the treatment of NAFLD mainly includes lifestyle change,drug therapy,intestinal microecological regulation and surgical intervention,but there is no clear and effective treatment method.Therefore,it is particularly important to study drugs or natural products that can prevent the development of NAFLD.Objective A mouse model of NAFLD was established to observe the preventive effect of tea extract on NAFLD,and to explore its possible mechanism from the lipid metabolism pathway,"entero-liver axis" pathway and toll-like receptor 4(TLR4)signaling pathway.Methods(1)In vivo experiment: The mice model of NAFLD was established by high-fat diet combined with 2.5% DSS,and randomly divided into normal control group,model control group,low dose and high dose groups of Qing brick tea extract.After the experiment,the mice were euthanized,liver weight was weighed to calculate liver index,serum samples were collected,serum ALT,AST,LDL-C,HDL-C,TG and T-CHO levels were detected,and the steatosis of the mice liver was observed by HE staining and oil red O staining.Western blot analysis was performed to detect the expression levels of the key proteins of lipid metabolism AMPK,p-AMPK,FAS,ACC,p-ACC,SREBP1 and the key proteins of TLR4 signaling pathway TLR4 and p65 in mouse liver tissue,as well as the expression levels of the intestinal mucosal barrier related proteins ZO-1 and Claudin-1.The m RNA expression levels of pro-inflammatory factor IL-6,IL-1β,TNF-α and anti-inflammatory factor IL-10 were detected by Real-time PCR.(2)In vitro experiment:palmic acid(PA)combined with lipopolysaccharide(LPS)is used to construct the high lipid and inflammatory environment of Hep G2 cells.They were divided into blank group,model group,low,medium and high dose of Qing brick tea extract group,AMPK inhibitor Compound C group + Qing brick tea high dose group.Western blot analysis was performed to detect the protein expression levels of AMPK,p-AMPK,FAS,ACC,p-ACC,SREBP1,TLR4 and p65.The m RNA expression levels of pro-inflammatory factor IL-6,IL-1β,TNF-α and anti-inflammatory factor IL-10 were detected by Real-time PCR.Results(1)The mouse model of NAFLD was successfully established.Compared with the normal control group,the liver index,serum liver function and lipid protein level of the mice in the model control group were significantly increased.The results of HE staining and oil red O staining showed that a large number of lipid vacuoles appeared in the liver of mice in the model control group.Western blot results showed that compared with the normal control group,the protein expression levels of p-AMPKα,p-ACC,ZO-1 and Claudin-1 in the liver of mice in the model group decreased.The protein expressions of FAS,SREBP1,TLR4 and p65 were increased.Real-time PCR results showed that the m RNA levels of pro-inflammatory factors increased and the m RNA levels of anti-inflammatory factors decreased in the liver and colon of the model control group.(2)Compared with the model control group,the serum liver function and lipid protein levels of mice in the intervention group were decreased,the protein expression levels of p-AMPKα,p-ACC,ZO-1 and Claudin-1 were increased,and the protein expressions of FAS,SREBP1,TLR4 and p65 were decreased.The m RNA levels of pro-inflammatory factors IL-6,IL-1β and TNF-α decreased,while the m RNA levels of anti-inflammatory factor IL-10 increased.(3)In vitro experiment results showed that compared with normal cells,the protein expression levels of p-AMPKαand p-ACC in model group were decreased,the protein expressions of FAS,SREBP1,TLR4 and p65 were increased,the m RNA levels of pro-inflammatory factors were increased,and the m RNA levels of anti-inflammatory factors were decreased.The expression of the above indexes could be reversed in the cells of the Qing brick tea extract group.AMPK inhibitor Compound C group could reduce the expression of key protein of lipid metabolism in Hep G2 cells in Qing brick tea high-dose group.Conclusion Qing brick tea extract can effectively prevent the occurrence of NAFLD,and its mechanism may be related to enhancing AMPK related pathway,promoting the repair of intestinal mucosal barrier and inhibiting TLR4 inflammatory signaling pathway.