Programmed Necrosis And Apoptosis In Inflammatory Injury Induced By HBx In The Liver Of Mice

ObjectiveTo investigate the mechanism of inflammatory injury induced by HBx in liver of mice,the models expressing HBx were constructed to evaluate inflammatory injury of liver and analyze the role of programmed necrosis and apoptosis;to investigate the effect of NAC on inflammatory injury induced by HBx in liver of mice,NAC was used as intervention and the effect of NAC on apoptosis and programmed necrosis induced by HBx was analyzed.Methods(1)Twenty-five adult male ICR mice were randomly divided into five groups.The plasmid pcDNA3.1-flag-HBx,pcDNA3.1,pGEM-HBx,pGEM-HBV-?HBx and transfection reagent were injected into the caudal vein of mice rapidly.24 hours after injection,RT-PCR and Western blot were performed to detect the expression of HBx in liver;the expression of HBsAg in serum was detected by ELISA.ELISA and qPCR were respectively used to detect the expression of TNF-α in serum and liver.The histological changes of liver were observed by HE staining.qPCR was used to detect the expression of RIP3,JNK,Bcl-2/Bax,Caspase8 and Caspase3,and the expression of RIP3,JNK and phosphorylated JNK were detected by Western blot.(2)16 adult male ICR mice were randomly divided into four groups.The plasmid pcDNA3.1-flag-HBx,pcDNA3.1 and transfection reagent were injected into the caudal vein of mice rapidly.Intraperitoneal injection of NAC was performed half an hour before transfection in the intervention groups.24 hours after transfection,RT-PCR was performed to detect the expression of HBx in liver;the expression of TNF-α,RIP3,JNK,Bcl-2/Bax,Caspase8 and Caspase3 were detected by qPCR.Results(1)The results of RT-PCR,Western blot and ELISA confirmed that the plasmid was successfully expressed in mice and HBx could up-regulate the level of HBV replication in liver of payw1.2 group(P<0.05).The results of ELISA showed that the expression of TNF-α in serum of HBx group was significantly higher than that of Control group(P<0.0001)and there was no significant difference between payw1.2 group and payw*7 group.The results of qPCR showed that the expression of TNF-α and RIP3 in liver of HBx group was obviously higher than that of Control group(P<0.01).The expression of JNK and Bcl-2 in liver of HBx group was higher than that of Control group(P<0.05),while the expression of Caspase8 and Caspase3 was down-regulated(P<0.05)and no significant difference was found in the expression of Bax.The results of qPCR also showed that the expression of TNF-α and Bcl-2 in liver of payw1.2 group was higher than that of payw*7 group(P<0.05).There was no no significant difference between payw1.2 group and payw*7 group in the expression of RIP3,JNK,Bax,Caspase8 and Caspase3.The results of HE staining showed that there were different degrees of pathological changes in liver of all groups except Control group.The results of Western blot showed that the expression of RIP3 in liver of HBx group was higher than that of Control group(P<0.01),as well as the phosphorylation level of JNK,but there was no significant difference in the expression of RIP3,JNK and phosphorylated JNK between payw1.2 group and payw*7 group.(2)The results of RT-PCR confirmed the expression of HBx in mice of HBx+NAC group and HBx group.The results of qPCR showed that the expression of Bcl-2 in liver of pc+NAC group was obviously higher than that of pc group(P<0.01),while the expression of Caspase3 was significantly down-regulated(P<0.001)and no significant difference was found in the expression of TNF-α,RIP3,JNK,Bax and Caspase8.The expression of RIP3 in liver of HBx+NAC group was lower than that of HBx group(P<0.01),as well as the expression of TNF-α,Bcl-2 and Caspase3(P<0.05).The expression of Bax in liver of HBx+NAC group was higher than that of HBx group(P<0.001),as well as the expression of Caspase8(P<0.05),but there was no significant difference in the expression of JNK.The expression of JNK and Bax in liver of HBx+NAC group was higher than that of pc+NAC group(P<0.01),as well as the expression of TNF-α and RIP3(P<0.05),but there was no significant difference in the expression of Bcl-2,Caspase8 and Caspase3.ConclusionsHBx can promote the replication of HBV and mediate inflammatory injury of liver by inducing programmed necrosis and inhibiting apoptosis of hepatocytes in mice.NAC can inhibit programmed necrosis induced by HBx and alleviate inflammation of liver in mice effectively.