Study On The Roles Of JNK Signaling Pathway With HNF-1 And GSTA1 In Acetaminophen-induced Injury On Hepatocytes

Acetaminophen(APAP)is commonly used as an analgesic in clinical,but can cause drug-induced liver injury when it is taken in excess because of an accumulation of N-acetyl-p-benzoquinonimine which is a toxic metabolite of APAP.At present,APAP-induced hepatocyte injury model is widely used to study liver diseases.Hepatocyte n uclear factor-1(HNF-1)is a transcription factor which can regulate expressions of specific gen es in liver.Glutathione S-transferase-A1(GSTA1)is a phase II drug metabolic enzyme which is important in detoxification and cell protection.c-Jun N-terminal kinase(JNK)has been proved to regulate cell metabolism,differentiation and apoptosis.In this research,based on the model of hepatocyte injury which was induced by APAP,the regulation of HNF-1 on GSTA1 and the role of JNK signaling pathway in HNF-1 and GSTA1 were studied to provide a new target for DILI.Based on the model of APAP-induced hepatocyte injury,the regulation of HNF-1 on GSTA1 was investigated firstly.Hep G2 were divided into 8 groups and then treated with vehicle(control group),10 μM APAP(model group),10 μM APAP with 6,8 or 10 μM C2-ceramide(C2 groups),10 μM APAP with 6,8 or 10 μM oltipraz(OL groups).Transaminase(ALT and AST)activities in culture supernatant were determined;m RNA expression of GSTA1 in cells was detected by RT-PCR;HNF-1 and GSTA1 expressions were detected by Western blot;recombinant plasmid p GSTA1-1298-LUC and plasmid p GSTA1-HNF1-LUC in which GSTA1 promoter HRE was mutant were constructed and transfected into Hep G2 then the activity of luciferase was measured.Furthermore,the role of JNK signaling pathway in HNF-1 and GSTA1 were then detected.Hep G2 were divided into 7 groups: control group,APAP(10 μM)model group,C2 group(APAP 10 μM+C2 8 μM),OL group(APAP 10μM+OL 8 μM),AP+SP+OL group(APAP 10 m M,SP 2 μM and OL 8 μM)and AP+SP+C2 group(APAP 10 m M,SP 2 μM and C2 8 μM).Transaminase(ALT and AST)activities in culture supernatant,SOD activity,MDA content,GSH content and GSH-Px activity in hepatocytes were determined;m RNA expressions of HNF-1 and GSTA1 in cells were detected by RT-PCR;JNK,p-JNK,c-Jun,p-c-Jun,HNF-1,GSTA1,Bax and Caspase-3 expression were detected by Western blot.Results:(1)The model of hepatocyte injury can be replicated successfully when treated Hep G2 with 10 m M APAP.(2)The effects of C2 and OL on hepatocyte injury showed that C2 could aggravate APAP-induced hepatocyte injury and OL was contrary.m RNA expression of GSTA1 can be reduced extremely by C2(p<0.01)and the function of OL was contrary(p<0.01).Except that,C2 could downregulate the expressions of HNF-1 and GSTA1 while OL upregulate their expressions.(3)The results of regulation of HNF-1 on GSTA1 showed that after transfecting plasmid of p GSTA1-1298-LUC,the activity of luciferase was reduced significantly by the decreasing expression of HNF-1(p<0.01).While the activity of luciferase was increased significantly by the increasing expression of HNF-1(p<0.01).The results showed that HNF-1 could regulate the expression of GSTA1.After transfecting mutant plasmid p GSTA1-ΔHNF1-LUC,the changed expression of HNF-1 had no effect on GSTA1 expression.All the above results demonstrated that HNF-1 can regulate the expression of GSTA1 by binding with HRE.(4)The results of JNK inhibitor,C2 and OL on hepatocyte injury showed that JNK inhibitor and OL could significantly reduce the transaminase activities,but increase m RNA expression of HNF-1 and GSTA1 significantly(p<0.01).The transaminase activities were increased while m RNA expression of HNF-1 and GSTA1 were decreased significantly(p<0.01)after C2 treatment.Compared with C2 group,AP+SP+C2 downregulated the transaminase activities but upregulated m RNA expression of HNF-1 and GSTA1(p<0.01).Compared with OL group,the expression of HNF-1 and GSTA1 in AP+SP+OL group had no significant change.Other hepatocyte indexes had significant change(p<0.01).These results suggested that APAP-induced hepatocyte injury was related to JNK activation.Inhibition of JNK could alleviate hepatocyte injury and significantly increase the expression of HNF-1 and GSTA1.(5)The results of detection of JNK and apoptosis signaling pathways showed that inhibitor of JNK and OL could significantly reduce the ratio of p-JNK/JNK、p-c-Jun/c-Jun,and reduce the expression of Bax and Caspase-3(p<0.01),while these were increased significantly in C2 group(p<0.01).Compare with C2 group,p-JNK/JNK、p-c-Jun/c-Jun,Bax and Caspase-3 in AP+SP+C2 group were decreased significantly(p<0.01).Compare with OL group,p-JNK/JNK in AP+SP+OL group had no significant change.These results indicated that JNK signaling pathway was activated,phosphorylation of JNK and its downstream protein and apoptotic p rotein expression were increased in APAP-induced hepatocyte injury.In the present research,APAP-induced hepatocyte injury was replicated.It can be concluded that expression of GSTA1 can be influenced by C2 and OL,and HNF-1 can regulate the expression of GSTA1 through binding with the binding-site HRE of GSTA1 promoter.Also,JNK signaling pathway is involved in hepatocyte injury,and activation of JNK can downregulate expression of HNF-1 and GSTA1.Inhibiting JNK can reduce the expressions HNF-1 and GSTA1 and alleviate hepatocyte injury.Therefore,JNK signaling pathway with HNF-1 and GSTA1 are involved in the process of drug-induced liver injury and play an important role in hepatocyte injury.