Salvianolic Acid A Alleviates Chronic Ethanol-induced Liver Injury By Activating SIRT1/?-catenin Pathway

Background:Alcoholic liver disease(ALD)is caused by long-term heavy drinking,including fatty liver,hepatitis,liver fibrosis and cirrhosis of the liver.In recent years ALD has emerged as a growing public health problem worldwide.Studies have shown that ?catenin plays an important role in the growth,development,regeneration and metabolism of the liver.Salvianolic acid A(SalA)is a water-soluble component extract of the root of Salvia miltiorrhiza Bunge,and its therapeutic effect of ALD is not reported.Objective:To explore whether SalA can protect against chronic ethanol-induced liver injury.To investigate whether SIRT1-mediated ?-catenin deacetylation can modulate the ?-catenin nuclear accumulation in the process of ALD and to explore whether SalA can protect against ALD via the SIRT1/?-catenin pathway.Methods:1.60 male Sprague-Dawley rats were fed a Lieber-DeCarli ethanol diet.They were randomly divided into 5 groups of 12 animals each:1)control group,2)control+SalA(16 mg/kg/d)group,3)Lieber-DeCarli ethanol diet(ED)group,4)ED+SalA(8 mg/kg/d)group,and 5)ED+SalA(16 mg/kg/d)group.Rats underwent the intragastric administration of SalA(8 and 16 mg/kg/d)or the same volume of normal saline for 8 weeks.After exposure to the Lieber-DeCarli ethanol diet for 8 weeks,all of the animals were euthanized.The liver tissues were harvested for histopathologic assessment and blood sample were collected for measuring Alanine Aminotransferase(ALT),Aspartate Aminotransferase(AST),Triglyceride(TG),Cholesterol(TC),ethanol,ammonia,Malondialdehyde(MDA),glutathione(GSH),citrate synthase and citrate synthase.The liver proteins expression of SIRT1,?-catenin,acetyl-?-catenin,SOD-2,CYP2E1 were determined by Western Blotting.2.(1)The Aml-12 and L02 cells were divided into five groups:the control group,the ethanol(EtOH)group and EtOH+SalA(0.5?M,5?M,50?M)groups.The EtOH group and the EtOH+SalA groups were treated with EtOH(100mM)for 24 hours.CCK-8 method was used to detect cell survival rate.(2)The Aml-12 cells were divided into three groups:the control group,the ethanol(EtOH)group and EtOH+SalA(50?M)groups.Nile Red staining method was used to detect lipid droplets in Aml-12 cells.(3)The Aml-12 cells were divided into four groups:the control group,the SalA(50?M)pretreatment groups,the ethanol(EtOH)group and EtOH+SalA(50?M)groups.The p-catenin mediated transcription activity was determined by luciferase reporter.The ?-catenin nuclear localization and the changes of ADH1 were determined by Immunofluorescent staining.(4)The Aml-12 cells were divided into four groups:negative control group,negative control+SalA(50?M)group,siRNA SIRT1 group and siRNA SIRT1+SalA(50?M)group.The protein expression of SIRT1,?-catenin,acetyl-?-catenin were determined by Western Blotting.Results:SalA significantly inhibited ethanol-induced increase of ALT,AST,TG,TC,ethanol,ammonia,MDA and decrease of GSH,the activity of citrate synthase and citrate synthase.SalA treatment significantly alleviated the accumulation of lipid droplets caused by alcohol,increased the expression of CYP2E1 and decreased the expression of SOD-2.Moreover,SalA decreased the acetylation of ?-catenin and significantly increased ?-catenin nuclear distribution by up-regulated SIRT1.Conclusion:The results demonstrated that the SIRT1/?-catenin pathway is a key therapeutic target in controlling liver injury caused by chronic alcohol and SalA provides protection against chronic ethanol-induced liver injury through the SIRT1-mediated deacetylation of p-catenin.