| In recent years,with the increasing incidence of drug-induced liver injury in the world,acetaminophen?APAP?is one of the most commonly used analgesic and antipyretic drugs.As the dose problem,it has become the main cause of acute drug-induced liver injury,it is of great significance to study the mechanism of APAP-induced drug-induced liver injury.Glutathione-S-transferase A1?GSTA1?is a phase II drug metabolic detoxification enzyme,which can promote the combination of glutathione with toxic metabolites and excrete them out of the body,and improve the body's antioxidant capacity.Hepatocyte nuclear factor-1?HNF-1?is one of the members of liver-specific transcriptional proteins,which plays an important role in regulating liver growth and development and metabolic processes.C-Jun N-terminal kinase?JNK?is an important component of the mitogen-activated protein kinase family,which is mainly involved in the signal transduction of apoptosis and plays an important role in regulating cell inflammation,growth and differentiation.This experimental study is based on the model of APAP-induced liver injury to further determine the role of the JNK signaling pathway and HNF-1 and GSTA1 in drug-induced liver injury,which provides a solid theoretical basis for the study of liver injury mechanisms and the development of new drugs.In this experiment,mice were administered with APAP of 175 mg·kg-1,and the model of liver injury was successfully replicated by detecting the activity of serum transaminase?ALT,AST?.40mice were randomly divided into 8 groups:control group,model group,C2-ceramide+APAP group(274 mg·kg-1,342 mg·kg-1,410 mg·kg-1C2-ceramide),oltipraz+APAP group(318 mg·kg-1,340mg·kg-1,362 mg·kg-1oltipraz).The serum transaminase index was determined to screen the optimal dose of C2-ceramide and oltipraz.50 mice were randomly divided into 10 groups:control group,model group,C2-ceramide+APAP group,oltipraz+APAP,SP600125+C2-ceramide+APAP group?group 3?,SP600125+oltipraz+APAP group?group 3?,SP600125 dose gradient 2mg·kg-1,3 mg·kg-1,4 mg·kg-1,the optimal dose of SP600125 was selected by measuring activity of serum transaminase.On this basis,40 mice were randomly divided into 8 groups:control group,SP600125 group,model group,APAP+SP600125 group,APAP+C2-ceramide group,APAP+oltipraz group,APAP+SP600125+C2-ceramide group,APAP+SP600125+oltipraz group.The activity of serum transaminase,SOD,GSH,GSH-Px and MDA were detected,and histopathological analysis was performed by H&E staining,and cell apoptosis was detected by Hoechst 33258 staining.The relative expression of HNF-1 and GSTA1 m RNA in liver tissues was detected by RT-PCR.The expression levels of HNF-1 and GSTA1 as well as the expression levels of JNK signaling pathway related proteins and apoptosis-related proteins in liver tissues were detected by Western blot.Results:?1?410 mg·kg-1,340 mg·kg-1,and 3 mg·kg-1 were determined to be the optimal doses of C2-ceramide,oltipraz and SP600125.?2?Compared with the control group,the activity of transaminase in the model group was significantly higher?p<0.01?;compared with the model group,the activity of transaminase in the APAP+C2-ceramide group was significantly higher?p<0.01?,the activity of transaminase in the APAP+oltipraz group was significantly lower?p<0.01?;the activity of transaminase was significantly lower after adding sp600125 on the basis of liver injury?p<0.01?;other indexes of liver injury were significantly changed?p<0.01?.Under the light microscope,the hepatocytes in the model group appeared structural rupture,inflammatory cell infiltration,hepatic cord blur and irregularity,the cell damage in the APAP+C2-ceramide group was more serious,the cell damage in the APAP+C2-ceramide+SP600125 group was improved,and the situation in the other groups was similar to that in the control group.Under the fluorescence microscope,apoptosis was observed in the model group,especially in the APAP+C2-ceramide group,which was improved in the APAP+C2-ceramide+SP600125 group,and similar to the control group in other groups.In conclusion,on the basis of APAP induced liver injury model,C2-ceramide can aggravate liver injury,while oltipraz or SP600125 can reduce liver injury.?3?The m RNA and protein expression of GSTA1 and HNF-1 were reduced significantly with control group?p<0.01?when APAP was used to induce liver injury;the m RNA and protein expression of GSTA1 and HNF-1 decreased were significantly after C2-ceramide treatment?p<0.01?,and the m RNA and protein expression of GSTA1 and HNF-1 were significantly increased after oltipraz or SP600125 treatment?p<0.01?;the m RNA and protein expression of GSTA1 and HNF-1 were significantly increased in the application of C2-ceramide and SP600125?p<0.01?.The above results showed that C2 ceramide and oltipraz can regulate the expression of HNF-1,and the change trend of GSTA1 expression was the same as that of HNF-1.After SP600125,the expression of HNF-1 and GSTA1 increased,and the degree of liver injury was alleviated.?4?In this experiment,the relative expression of JNK and phosphorylated JNK?p-JNK?,c-Jun and phosphorylated c-Jun?p-c-Jun?,Bax and Caspase-3 were detected.The expression of p-JNK/JNK,p-c-Jun/c-Jun was increased significantly with control group?p<0.01?;p-JNK/JNK,p-c-Jun/c-Jun were significantly increased after C2-ceramide?p<0.01?,p-JNK/JNK,p-c-Jun/c-Jun were significantly decreased after adding oltipraz or SP600125?p<0.01?;at the same time,the application of C2-ceramide and SP600125,the expression of p-JNK/JNK,p-c-Jun/c-Jun was significantly decreased?p<0.01?.The above studies showed that JNK signaling pathway was activated,downstream substrate phosphorylation,and apoptosis cells increased during liver injury;when activation of JNK signaling pathway was inhibited,downstream substrate phosphorylation decreased,apoptosis cells decreased,and liver injury degree decreased.In this study,the model of drug-induced liver injury induced by APAP was successfully replicated,and the optimal dosage of SP600125,C2-ceramide and oltipraz was determined.In APAP induced liver injury,the application of C2-ceramide and oltipraz showed that HNF-1 had a positive regulatory effect on GSTA1 expression.JNK signaling pathway was activated in liver injury,and JNK signaling pathway was inhibited after the application of inhibitors.Meanwhile,the expression of HNF-1 and GSTA1 increased,and the degree of liver injury decreased.Therefore,JNK signaling pathway,HNF-1 and GSTA1 are involved in APAP-induced drug-induced liver injury,the expression of HNF-1 is positively correlated with the expression of GSTA1,while the activation of JNK signaling pathway is negatively correlated with the expression of HNF-1 and GSTA1. |
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