Study On The Role Of JNK Signaling Pathway And GSTA1 In Acetaminophen Induced Acute Hepatic Injury In Mice

Acetaminophen?APAP?is an analgesic-antipyretic,which is the main ingredient of many compound coldrex.It could cause serious acute liver toxicity including hepatic necrosis,hepatic failure,and even death when used in overdose.APAP-induced hepatic injury model is a classic one,which is particularly important for studies on mechanism of hepatic injury.The c-Jun N-terminal kinase?JNK?is a member of the mitogen-activated protein kinase family,which could be activated by stimulation of environment and cytokines,participating in regulatory processes of inflammation,apoptosis,growth and differentiation.Oxidative stress in the APAP-induced liver injury may be related to the activation of JNK.Glutathione S-transferase A1?GSTA1?is a phase II metabolic detoxification enzyme that catalyzes the binding of glutathione with N-acetyl benzoquinone,toxic metabolite of APAP,increasing the ability to resist oxidative stress and detoxification for heterotoxin.This study aimed to ascertain the role of JNK signaling pathway and GSTA1 in APAP-induced liver injury by giving JNK inhibitor SP600125 based on the modle of APAP-induced acute hepatic injury,providing a foundation for further research on mechanism of drug-induced hepatic injury.In this experiment,mice were administrated with different doses of APAP.The activities of ALT and AST in serum were determined to find different dosages that could cause different degrees of injury.Then the optimal dose of SP600125 was selected by the same way.After the pre-test,the mice were divided into five groups:control group,low-dose APAP group(150 mg·kg^-1APAP),high-dose APAP group(175 mg·kg^-1 APAP),low-dose APAP with SP group(150 mg·kg^-1APAP+2 mg·kg^-1 SP600125),high-dose APAP with SP group(175 mg·kg^-1 APAP+2 mg·kg^-1SP600125).Serum ALT,AST and hepatic indices?MDA,SOD,GSH,GSH-Px?were measured using commercial kits.The content of GSTA1 in serum and liver was detected by ELISA.Histopathological examination of liver was performed by H&E.Hoechst 33258 staining was used to test cells apoptosis.The relative expression levels of p-JNK,JNK,p-c-Jun,c-Jun,p-c-Fos,c-Fos,p-ASK1,ASK1,p-MKK4,MKK4 and GSTA1 were measured by Western blot.Based on all the above experiments,I tried to confirm the relervance of JNK activation and GSTA1 with acute hepatic injury induced by different dosages of APAP in mice.Results:?1?Different dosages of APAP can cause different degrees of hepatic injury in mice.The serum transaminase activities were markedly increased?p<0.05?at the dose of 150 mg·kg^-1;the serum transaminase activities were significantly increased?p<0.01?at the dose of 175 mg·kg^-1.Hence,the dosages at 150 mg·kg^-1 and 175 mg·kg^-1 were ascertained which could cause different degrees of hepatic injury.?2?The optimal dose of JNK inhibitor SP600125 to mice was deermined at 2 mg·kg^-1 based on the modle of acute hepatic injury.?3?Serum aminotransferases were significantly decreased?p<0.01?,and malondialdehyde content was markedly decreased?p<0.05?and superoxide dismutase activity,glutathione content,and glutathione peroxidase activity were significantly increased?p<0.01?in liver,and GSTA1contents in serum and liver in high-dose APAP with SP group were significantly changed?p<0.01?compared with high-dose APAP group.Malondialdehyde content was markedly decreased?p<0.05?but superoxide dismutase activity was markedly increased?p<0.05?,and glutathione content,and glutathione peroxidase activity were significantly increased?p<0.01?in liver in low-dose APAP with SP group compared to low-dose APAP group.GSTA1 contents in serum and liver in low-dose APAP with SP group were significantly changed?p<0.01?compared with low-dose APAP group.Slight hepatic cord disorders and a few bleeding points were observed in low-dose APAP group in histopathological examination,and disapperance of hepatic cell cords and inflammatory cells,congestion and cell fragmentation were observed in the high-dose APAP group;but bleeding points were largely disappeared in low-dose APAP with SP group;while regular hepatic cell cords,less inflammatory cells and less bleeding points were observed in high-dose APAP with SP group.Apoptosis assay by Hoechst 33258 showed that the apoptosis,which was charactorized of karyopyknosis,induced by APAP was dose-dependent,but the apoptosis was significantly reduced with administration of SP600125.The above results indicated that JNK inhibitor reduced APAP-induced injury and apoptosis in mice.?4?The expressions of upstream and downstream proteins of JNK signaling pathway showed that p-JNK/JNK,p-c-Jun/c-Jun,p-c-Fos/c-Fos,p-MKK4/MKK4 and p-ASK1/ASK1 ratios in high-dose APAP group were significantly higher than those in control group?p<0.01?.The p-JNK/JNK,p-c-Jun/c-Jun,p-MKK4/MKK4 and p-ASK1/ASK1 ratios were lower in the high-dose APAP with SP group than those in high-dose APAP group.Interestingly,administration of SP600125 didn't change the ratio of p-c-Fos/c-Fos,which probably proved that phosphorylation of c-Fos wasn't regulated by JNK signaling pathway.It was demonstrated that the JNK signaling pathway was activated in different degrees liver injury in mice and phosphorylations of upstream and downstream proteins of JNK were increased.After administration of SP600125,the phosphorylation levels of JNK,c-Jun,ASK1,and MKK4 decreased,but the phosphorylation level of c-Fos did not change.The above results indicated that activation of JNK signaling pathway and phosphorylations of upstream and downstream proteins of JNK were related to the APAP-induecd hepatic injury,and they were positively correlated with the degrees of liver injury.?5?Results of GSTA1 expression by Western blot showed that the relative expression of GSTA1 was significantly lower in the APAP group than that in control group?p<0.01?.The relative expression of GSTA1 in the low-dose APAP with SP group was markedly higher than that in the low-dose APAP group?p<0.05?;however the relative expression of GSTA1 in high-dose APAP with SP group was significantly higher than that in high-dose APAP group?p<0.01?.The results showed that the expression of GSTA1 decreased by APAP,but the expression level increased with the administration of SP600125.This indicated that with the decreasing expression of GSTA1 in liver,hepatic ability to resist injury decreased when liver injury occurred.JNK inhibitor protected the liver by inhibiting the activation of JNK with increasing expression of GSTA1 and ability of the liver to resist injury.This study replicated APAP-induced acute hepatic injury model in mice successfully and ascertained the optimal dose of SP600125 to mice.JNK signaling pathway was activated in the acute hepatic injury induced by APAP.Administration of JNK inhibitor SP600125 can reduce the phosphorylation of proteins in JNK signaling pathway,increase the expression of GSTA1 in liver,and decrease APAP-induced hepatic injury and apoptosis.Finally this study clarified the role of JNK signaling pathway in APAP-induced hepatic injury.JNK signaling pathway may regulate the expression of GSTA1 to protect against peroxidative injury,but the mechanism still needs further study.