The Ameliorative Mechanism Of Cyanidin Chloride On Ethanol Induced Triglyceride Accumulation In Hepatocytes

Objective:To investigate the ameliorative mechanism of cyanidin chloride on ethanol-induced triglyceride accumulation in AML-12 hepatocytes.Method:AML-12 hepatocytes of ethanol-induced lipid accumulation were used in this study.The cells were divided into control group(1 μL/ml DMSO,no ethanol,no cyanidin chloride),ethanol group(50 mM ethanol),cyanidin chloride group(different doses of 1 μM,10 μM,100 μM,respectively),and ethanol+ cyanidin chloride group(50 mM ethanol+different doses of cyanidin chloride).After 24 hours of drug intervention,the content of triglyceride(TG)in AML-12 hepatocytes was determined by enzymatic method.The accumulation of lipid droplets in AML-12 hepatocytes was observed by oil red O staining.Regulatory element binding protein 1(SREBP-1),acetyl-Co A carboxylase(ACC),stearoyl-CoA desaturase 1(SCD-1),fatty acid synthase(FASN),peroxisome proliferator Activation of receptor alpha(PPAR alpha)and splicing factor Slu7(Slu7)mRNA expression in AML-12 hepatocytes.were detected by real-time quantitative PCR(qRT-PCR).Western blotting(Westen Blot)was used to detect the protein expression of Slu7 in AML-12 hepatocytes.In addition,ethanol-induced lipid accumulation model in AML-12 hepatocytes was used.The cells were divided into siRNA control group(50 nM siRNA control,no ethanol),ethanol group(50 mM ethanol),and different doses of siSlu7 interference group(10 nM,50 nM,100 nM siSlu7,respectively),Ethanol+different doses siSlu7 interference group(50 mM ethanol+different doses of 10 nM,50 nM,100 nM siSlu7,respectively).The expression of Slu7 mRNA in AML 12 hepatocytes was detected by qRT-PCR,and the content of TG in AML-12 hepatocytes was determined by enzymatic method.Result:(1)Compared with the control group,the intracellular triglyceride content in the ethanol group was significantly increased(P<0.01).Compared with the ethanol group,1 μM of cyanidin chloride had significant effect on ethanol-induced triglyceride content in AML-12 hepatocytes(P<0.05),but when the concentration was increased to 10 μM and 100 μM,the cyanidin chloride significantly reduced ethanol-induced triglyceride content in AML-12 hepatocytes(P<0.01).Moreover,with the increase of drug concentration,the intracellular triglyceride content decreased significantly(ethanol+100 μM cyanidin chloride group compared with ethanol+10 μM cyanidin chloride group,P<0.01).Different concentrations(1 μM,10 μM and 100 μM)of cyanidin chloride had no significant effect on the triglyceride content of AML-12 hepatocytes(P>0.05,compared with the control group).From the drug dose point of view,the 10 drug dose is significant effective to protect ethanol-induce fat accumulation.Therefore,we used 10 μM cyanidin chloride for subsequent experiments.(2)Oil red O staining showed that the lipid droplets in the ethanol group were increased compared with the control group.The effect of cyanidin chloride on the accumulation of lipid droplets in AML-12 hepatocytes was not obvious.However,10 μM of cyanidin chloride significantly reduced the amount of lipid droplets in the cells compared to the ethanol group.(3)qRT-PCR results showed that compared with the control,sterol regulatory element binding protein 1(SREBP-1),acetyl-CoA carboxylase(ACC),stearoyl-CoA desaturase 1(SCD-1)and fatty acid synthase(FASN)in ethanol group were significantly increased,while cyanidin chloride significantly down-regulated ethanol-induced SREBP1,ACC,SCD1 and FASN mRNA expression levels(P<0.05,compared with the ethanol group).In addition,compared with the control group,the expression of peroxisome proliferator-activated receptor alpha(PPARa)mRNA in AML-12 hepatocytes was significantly decreased(P<0.01),while cyanidin chloride was significantly up-regulated.PPARa mRNA expression in AML-12 hepatocytes(P<0.01),and cyanidin chloride was significantly up-regulated.ethanol-down regulated PPARa mRNA expression in AML-12 hepatocytes(P<0.01,compared with the ethanol group).(4)Compared with the control group,the mRNA and protein level of Slu7 in AML-12 hepatocytes in ethanol group was increased(P<0.05),while cyanidin chloride significantly down-regulated ethanol-induced Slu7 expression in AML-12 hepatocytes(P<0.01,compared to the ethanol group).(5)Compared with the control group,10 nM siSlu7 reduced the expression level of Slu7 mRNA in AML-12 hepatocytes(P<0.01),but when the concentration reaches to 50 nM and 100 nM,siSlu7significantly reduced(about 60%)the mRNA expression level of Slu7 in AML-12 hepatocyte(P<0.01).From the dose effect,the 50 nM dose was best to reduce the expression of SIu7 mRNA,so we used 50 nM siSlu7 for subsequent experiments.(6)Compared with the control group,the intracellular triglyceride content in the ethanol group was significantly increased(P<0.05).siRNA Slu7 had no significant effect on the triglyceride content of AML-12 hepatocytes(P>0.05).Interestingly,compared with the ethanol group,siSlu7 attenuated the ethanol-induced triglyceride content in AML-12 hepatocytes(P<0.01).Conclusion:Cyanidin chloride can inhibit ethanol-induced triglyceride accumulation in AML-12 hepatocytes,possibly through inhibiting the mRNA and protein expression of Slu7,and regulating the mRNA expression of SREBP1,ACC,SCD1,FASN and PPARa.