Analysis Of GSTA1 Expression Regulated By HNF-1 On Acetaminophen Induced Injury On Hepatocytes

With increasingly serious phenomenon of drug abuse,drug-induced liver diseases increased year by year,which is one of the most common clinical liver problem.Acetaminophen(APAP)is commonly used for antipyretic and analgesic,and the liver problems caused by it accounted for the largest proportion of drug-induced liver problems.APAP-induced liver injury model is the typical representative of drug-induced liver injury.Glutathione S transferase A1(GSTA1)is one of the enzyme systems in body,which can prevent the cells to oxidative stress and protect the body against foreign toxic substances and the production of reactive oxygen species.Hepatocyte nuclear factor-1(HNF-1)is identified to have a DNA-binding activity that interacts with several liver gene promoters and plays an important role in liver-specific transcription.Therefore,it plays an indispensable role in regulating the growth and metabolism of the liver.This study aimed to investigate the regulation of GSTA1 expression based on hepatocytes injury model with the inhibitory effect of C2-ceramide and the promoted effect of oltipraz on HNF-1.The regulatory effect of HNF-1 on expression of GSTA1 was determined.The key role of GSTA1 in drug-induced hepatocellular injury was clarified,which provide the vitro evidence for the study of drug-induced liver injury,a academic foundation for more study of its mechanism and defense of liver injury.In this study,hepatocytes were treated with a series of concentration of APAP.Based on the transaminase(ALT,AST)activity in culture supernatant and the level of indicators(MDA,SOD,GSH,GSH-Px)in hepatocytes,the degree of liver injury was determined in different concentration of APAP and the hepatocytes injury model was successfully replicated.On the basis of this model,the transaminase(ALT,AST)activity in supernatant was used as an index to screen the optimal concentration of C2-ceramide and oltipraz.Subsequently,the regulation of HNF-1 on GSTA1 was carried out.This study was divided into six groups: control group,APAP model group(model group),C2-ceramide group(C2 group),C2-ceramide + APAP group(C2 + APAP group),oltipraz group,oltipraz + APAP group(OL + APAP group).The levels of transaminase(ALT,AST)activity and hepatocytes injury indicators(MDA,SOD,GSH,GSH-Px)were measured by kit.Real-time RT-PCR was used to detect the relative expression of HNF-1 and GSTA1 mRNA in hepatocytes.The relative expression of HNF-1 and GSTA1 protein in hepatocytes was detected by Western blot.The content of GSTA1 in culture supernatant was detected by kit.The above study clarifies the regulation of HNF-1 by C2-ceramide and oltipraz in APAP-induced hepatocytes injury.The effects of different expression of HNF-1 on GSTA1 were analyzed and the regulatory effect of HNF-1 on GSTA1 expression was determined in drug-induced hepatocytes injury.Results:1.The different degrees of injury to hepatocytes can be produced through different concentrations of APAP for 10 h.the activities of two transaminases in supernatant,associated with control group,were increased and the significant difference was found in 15 m M APAP group.Hepatocytes index(MDA,SOD,GSH,GSH-Px)levels were obviously changed and there are poor cell status,cell deformation,shrinkage and so on.Finally,15 m M APAP was used as the model group for further study and the drug-induced hepatocytes injury model was successfully replicated.2.C2-ceramide and oltipraz were used to hepatocytes at concentrations of 6 μM and 8 μM,respectively,through the optimal concentration screening test.3.C2-ceramide and oltipraz were used into hepatocytes injury model.Compared with the control group,transaminase activity in culture supernatant and hepatocytes index in C2 and OL group,were no remarkerly change,indicating that 6 μM C2-ceramide and 8 μM oltipraz does not cause hepatocytes injury.Associated with the model group,the two transaminase activities of C2 + APAP group were significantly increased(p<0.01)and the hepatocytes index showed a significant change(p<0.01),indicating that C2-ceramide aggravated APAP-induced hepatocytes injury.The activity of transaminase in OL + APAP group was significantly decreased(p<0.01)and the hepatocytes indexes also showed a significant change(p<0.01),indicating that oltipraz could alleviate APAP-induced hepatocyte injury.4.The results of the expression of HNF-1 showed that,likened with control group,the mRNA and protein expression levels of HNF-1 in model group was significantly decreased(p<0.01),while there was no significant change in C2 and OL group.Associated with model group,the m RNA and protein expression levels of HNF-1 in C2 + APAP group were significantly decreased(p<0.01),while that in OL + APAP group was markedly increased(p<0.01).Above all,in the case of APAP-induced hepatocytes injury,C2-ceramide can lessen the expression of HNF-1,and oltipraz can raise its expression.5.The results of the expression of GSTA1 showed that,linked with control group,the relative expression of GSTA1 m RNA and protein in model group was obviously reduced(p<0.01),the content of GSTA1 in culture supernatant of model group was significantly increased(p<0.01),while there was no significant change in C2 and OL.Associated with model group,the relative expression of GSTA1 mRNA and protein in C2 + APAP group was obviously reduced(p<0.01),while that in OL + APAP group was significantly increased(p<0.01).The content of GSTA1 in culture supernatant of C2 + APAP group was considerably increased,while that of OL + APAP group was markedly decreased.The above results were consistent with the results of HNF-1 expression.In this study,APAP-induced hepatocytes injury model was successfully replicated.The optimum concentrations of C2-ceramide and oltipraz were determined.In the APAP-induced hepatocytes injury,C2-ceramide can aggravate the degree of hepatocytes injury,and oltipraz can reduce that.In the case of hepatocytes injury,C2-ceramide can down-regulate the relative expression of HNF-1,whereas oltipraz up-regulates that.It was confirmed that HNF-1 could regulate the expression of GSTA1 and the relative expression levels of mRNA were consistent with the relative expression of protein.HNF-1 can regulate the expression of GSTA1,which plays a vital role in protecting hepatocytes.However,the mechanism was still to be explored.