Vitexin Protects Against Ethanol-induced Liver Injury Through Sirt1/p53-mediated Apoptotic Pathway

Alcoholic liver disease(ALD)involves hepatocellular injury induced by ethanol.Nowadys,it has been paid more and more attention by scholars because of the increasing morbidity and mortality.Besides,there are no especially effective drug therapies to this diseases.Apoptosis is a significant mechanism in alcohol-induced liver injury.Studies revealed Sirtl is an important target of the treatment of ALD.p53 is a downstream effector of Sirtl,which play important roles in mitochondria mediating cell apoptosis.Literature indicates that vitexin has good anti—apoptotic activities.Therefore,based on this,this subject put forward"vitexin inhibit the alcohol-induced hepatocyte apoptosis,and improve alcoholic liver injury via stimulating Sirtl,then restraining p53 acetyl-p53 expression" as the scientific hypothesis.We study vitexin protect against ethanol-induced liver injury in mice and L02 cell.ObjectiveTo preliminary evaluate whether vitexin has a efficacy in protecting against ethanol-induced liver injury in acute and subacute animal model,and assess vitexin impact on Sirt1/p53-mediating cell apoptosis pathway in animal model,including Sirtl,p53,Bcl-2,Bax and caspase3.Finally,using L02 cell line as model,we further investigate the efficacy and mechanism of vitexin in vitro.Methods1 The animal experiments(1)Acute alcohol-induced liver injury modelMale KM mice randomly divided into normal,model,silyymarin(80mg/kg),vitexin low,middle and high dose(20,40,80mg/kg)groups.According to the above dose,drug treatment groups are given to a single administration.1h later,we use 4.8g/kg bw ethanol to inducing acute alcoholic liver injury in mice.Test indicators:liver index,AST,ALT,TC,TG in serum.(2)Subacute alcohol-induced liver injury modelMale KM mice randomly divided into normal,model,silymarin(80mg/kg),vitexin low,middle and high dose(20,40,80mg/kg)groups.During the first week,except normal group,other groups are fed with 30%ethanol(10ml/kg)via administering intragastrically every ante meridiem.Treatment groups are given corresponding drugs treatment every post meridiem.Since the second week,the ethanol concentration will be increased gradually,the ethanol concentration of the next three weeks are 40%,50%,55%respectively.Test indicators:liver index,AST,ALT,TC,TG,TP,TBIL,UA in serum and liver pathology.2 L02 cell experimentsL02 cells are treated with different concentration of vitexin and silymarin with a certain concentration of ethanol.Assessing the vitexin efficacy via cell viability assay and measuring the level of AST in supernatants.3 Investigate the mechanism of vitexinThe apoptosis degree of each group of cells were measured with Fluorescent Cell Imager and Flow Cytometerseparately in vitro.Then assessing related gene and protein expression in Sirtl/p53-mediating cell apoptosis pathway in vivo and vitro.Results(1)Vitexin protects against acute alcohol-induced liver injury in miceThe liver index,AST,ALT,TC in serum in model group increased significantly compared with normal group(P<0.05 or P<0.01),after vitexin interventing,all this indictors decreased significantly(P<0.05 or P<0.01).(2)Vitexin protects against subacute alcohol-induced liver injury in miceIn subacute model,model group had a higher mortality.And the liver index,AST,AST/ALT,TC,TG,UA in serum significantly increased(P<0.05 or P<0.01).Vitexin could inhibit them significantly(P<0.05 or P<0.01)and improve the liver pathologic changes to a certain extent.(3)Vitexin protects L02 cell against ethanol-induced injuryIn model group,the cell viability and the level of AST were reduced significantly(P<0.01),various concentration of vitexin could reverse this indictors significantly(P<0.05 or P<0.01).(4)The mechanism of vitexin in vivoAfter vitexin interventing,Sirtl and Bcl-2 expression were elevatecd significantly(P<0.05 or P<0.01),and ac-p53,p53,the ratio of ac-p53/p53,,caspase3 were significantly declined(P<0.05 or P<0.01)in vivo and in vitro.And L02 cells were treated with vitexin,the apoptotic rate declined significantly(P<0.05 or P<0.01).ConclusionVitexin can protect against alcohol-induced liver injury effectively.The mechanism of vitexin to protect liver could be via stimulating Sirtl,regularing the p53 and ac-p53 expression,then affecting the downstream mitochondria mediating cell apoptosis pathway and inhibiting apoptosis.