The Effects And Mechanisms Of Asiatic Acid On Mouse Liver Fibrosis Induced By Bile Duct Ligation

Objective: Liver fibrosis is an important pathological process in the development of many liver diseases such as liver cirrhosis.However,there are no effective therapeutic drugs at present.This study aims to research the effects and relative mechanisms of Asiatic acid on alleviating liver fibrosis in bile duct ligated(BDL)mice,and provide experimental evidence for its application on the treatment of liver fibrosis.Methods: This study researched the therapeutical effects and relative mechanisms of Asiatic acid on liver fibrosis via in vivo and in vitro experiments.In vivo : Using BDL mice and dividing them into four groups: Sham operation control group,BDL model group,BDL mice with Asiatic acid(15 mg/kg)and BDL mice with Asiatic acid(30 mg/kg),then gaving them treatment for 5 days continually.The content of biochemical indicators were detected to analysis the liver function.The liver and spleen tissue were weighed and the liver index and spleen index were calculated.Liver tissue sections were stained with H&E and Masson staining for the pathological examination,the other sections were stained with TUNEL staining for the detection of hepatocyte apoptosis.?-SMA(?-smooth muscle actin)immunohistochemistry was used to detect the degree of liver fibrosis.RT-q PCR assay tested the m RNA levels of inflammatory factors,pro-fibrotic factors and the bile acid metabolism related factors.The detection of MDA(malondialdehyde),SOD(superoxide gasase)and Catalase(catalase)and GSH(reduced glutathione)were used to research oxidative stress in BDL mice.The expression levels of apoptosis-related proteins of Bax,Bcl2 and anti-oxidative-related proteins of nuclear factor erythroid 2related factor 2(Nrf2),heme oxygenase-1(HO-1)were determined by western blotting.At the cellular level,HL-7702 cells were treated with sodium glycochenodeoxycholate(GCDC)and divided into control group,model group and Asiatic acid-treated group with different doses.The cell viability ratio and apoptosis of HL-7702 cells were detected by MTT and flow cytometry assay respectively,and the expression level of Nrf2 was determined via western blotting.Results: The phenomenon of hepatomegaly,collagen deposition and inflammatory cells infiltration were showed in BDL group mice.Asiatic acid improved these liver tissue morphology and pathological features,and reduced the liver index and spleen index.We found that AA significantly prevented BDL-induced liver injury,which represented as decreased content of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(AKP),?-glutamyltransferase(?-GT),total bilirubin(TBIL),hydroxyproline(Hyp),total cholesterol(T-CHO)and total bile acid(TBA)in serum.Simultaneously,AA reduced hepatocytes apoptosis with altering protein expression level of Bcl2 and Bax.The immunohistochemistry analysis showed that AA inhibited ?-SMA expression in liver tissues of BDL mice.Real-time PCR results showed that AA decreased genes m RNA expression level of pro-fibrogenic factors including prostaglandin-endoperoxide synthase(Pgst2),IL-6,TNF-? and Chemokine ligand 3(CCL3);and also the pro-inflammation factors including Collagen type alpha 1 chain gene(Col3a1),Vimentin,aorta smooth muscle actin(Acta2)and TGF-?1.In addition,we found that AA increased the level of SOD,catalase and GSH,and decreased the content of MDA in BDL mice.Western blot showed that AA increased the protein expression levels of Nrf2 and HO-1.The date of bile acid metabolism related factors demonstrated that Asiatic acid regulated bile acid metabolism,including farnesoid X receptor(FXR),fibroblast growth factor15(FGF15),small heterodimerizing partner(SHP),Cholesterol-7-? hydroxylase(Cyp7?1).In vitro experiments showed that AA reduced GCDC-induced apoptosis and Reactive oxygen species(ROS)generation in a dose-dependent manner,while increased the protein expression of Nrf2 in HL-7702 cells.Conclusion: This study indicated that AA might be beneficial for inhibition of cholestasis liver fibrosis,which is associated with anti-apoptosis,as well as Nrf2-mediated anti-oxidation and regulation of bile acid metabolism.