Effect Mechanism Of Yinchenhao Decoction In Regulating Bile Acid And Fat Metabolism In Mice With NAFLD Combined With Cholestasis Based On FXR Expression

Objective:Based on the activation of nuclear receptor FXR,the mechanism of Yinchenhao Decoction regulating NAFLD and NAFLD combined with cholestasis of bile acid and fatty acid metabolism was discussed.Method:The pathological model of NAFLD was induced by high fat diet（HFD）.The pathological model of NAFLD combined with cholestasis was induced by HFD+ANIT.In the first experiment,48 male C57BL/6J mice were randomly divided into normal control mice and model mice.Model mice used HFD.Model mice were randomly divided into two groups starting from the 8th week,one group was fed with HFD,and the other group was administered with ANIT on the basis of HFD,once a week until 12 weeks.On the 8th week and the 12th weekend of the modeling,samples of mouse serum and liver tissue were collected twice.Each collection specimen was set to normal control group,HFD group and HFD+ANIT group.In the second experiment,58 male C57BL/6J mice were randomly divided into normal control mice and model mice.The model mice were fed with HFD.On the first day of the 9th week of modeling,the model mice were randomly divided into HFD group,HFD+ANIT group,HFD+Yinchenhaotang group,HFD+ANIT+Yinchenhaotang group.In the third experiment,20 male C57BL/6J Fxr^-/-mice were randomly divided into standard feed group and HFD group,and fed with standard feed and HFD,respectively.On the 9th weekend of modeling,standard feed-fed mice were randomly divided into Fxr^-/-standard feed group（FN）and Fxr^-/-standard feed plus Yinchenhaotang intervention group（FNY）;HFD-fed mice They were randomly divided into Fxr^-/-HFD group（FH）and Fxr^-/-HFD plus Yinchenhaotang intervention group（FHY）.Histopathological changes of mice liver were observed by H&E,oil red O staining and immunohistochemistry.The routine biochemical methods were used to detect serum liver function and blood lipid changes in mices.Real-time quantitative PCR and Western blotting were used to detect liver tissue bile acid transporters BSEP,NTCP,OAPT4a1,MRP2,MRP3,MRP4,liver tissue nuclear hormone receptor FXR and fat metabolism related proteins FAS,ACC1,SREBP-1c,Ch REBP,Changes in CD36,LXR-alpha,and SCD1 expression.Result:1.The histopathology of NAFLD combined with cholestasis mice is characterized by vesicular hepatic steatosis,prominent bile duct hyperplasia in the portal area,and inflammatory cell infiltmiceion;its serology is ALT,AST,ALP activity and TBIL,DBIL,TC,TG content is significantly increased as a feature.2.The expression of bile acid nuclear hormone receptor FXR,bile acid transporter BSEP,MRP2,OATP4a1 was decreased in NAFLD combined with cholestasis mice,and the expression of NTCP,MRP3 and MRP4 was increased,and bile acid was reduced to hepatic bile duct.Increased bile acid in sinusoids leads to hepatic cell cholestasis;NAFLD combined with cholestasis mice liver tissue fatty acid synthesis key transcription factor SREBP-1c,Ch REBP and its target genes ACC1,FAS increased,catalytic conversion of satumiceed fatty acids into single The expression of SCD1,a micee-limiting enzyme of satumiceed fatty acids,is abnormal,and the expression of CD36,an important factor in fatty acid transport and oxidation,is abnormal.The increase in fatty acid synthesis in hepatocytes is greater than that in oxidative metabolism,leading to fat accumulation in hepatocytes.3.Yinchenhao Decoction can significantly reduce liver fat accumulation in NAFLD combined with cholestasis mice,reduce inflammatory cell infiltmiceion and biliary epithelial cell prolifemiceion in the portal area,improve serum liver function and blood lipid levels,and reduce liver tissue bile acid transporters BSEP,FAS,CD36,LXR-alpha protein expression;elevated liver tissue nuclear hormone receptor FXR protein expression.4.Yinchenhao Decoction has no significant regulatory effect on serum liver function,blood lipids and liver histopathology in normal diet Fxr^-/-mice;serum ALB,TBIL,DBIL,TBA and blood lipid levels in high-fat diet Fxr^-/-mice No significant regulation,but can significantly reduce serum ALT,AST,ALP activity in high-fat diet Fxr^-/-mice,reduce small bile duct hyperplasia in the liver portal area,reduce liver tissue fat accumulation.5.Yinchenhao Decoction has no significant regulatory effect on the expression of bile acid transporter MRP3,BSEP,OATP4a1,NTCP,FAS,SREBP-1c,CD36 and LXR-alpha in normal diet Fxr^-/-mice,while can reduce the expression of ACC1 protein in liver tissue of Fxr^-/-mice with normal diet,increased SCD1 protein expression.Yinchenhao Decoction has no significant effect on the expression of MRP3,BSEP,NTCP,FAS,ACC1,SREBP-1c and LXR-alpha protein in liver tissues of high-fat diet Fxr^-/-mice,while can reduce the high-fat diet Fxr^-/-CD36 protein expression in mouse liver tissue increased OATP4a1 and SCD1 protein expression.Conclusion:1.The use of high-fat diet combined with ANIT can induce NAFLD and cholestasis lesions in mice.NAFLD combined with cholestasis mice have bile acid metabolism,abnormal fat metabolism and significant down-regulation of nuclear hormone receptor FXR.2.Yinchenhao Decoction increases FXR m RNA and protein expression,regulates bile acid transport and reduces fat synthesis,and is the main mechanism of its treatment of NAFLD combined with cholestasis.