GSSG Sensitizes Hepatocytes To TNFa Cytotoxicity Via Inducing NF-κB Inhibition Contributes To Liver Injury In Nonalcoholic Fatty Liver Disease

Objective To investigate the mechanism of oxidized glutathione(GSSG)synergistic tumor necrosis factor α(TNFα)induced hepatocyte death from the level of signal pathway regulation.To investigate the effect of GSSG on NF-κB pathway.The mechanism of GSSG inducing non-alcoholic fatty liver disease(NAFLD)by inhibiting the activation of TNFa mediated nuclear transcription factors kappa B(NF-κB)was elucidated from both in vitro and in vivo.Methods Male C57BL/6 mice were fed with either control or high fat diet(HFD)for 8 weeks.The levels of ALT and TNFa in serum,and the levels of TG,TNFα,GSSG and GSH in the liver were measured by HE staining and enzymatic methods.The expression of NF-κB regulatory gene and the glutathione modification of protein IKK-β in NF-κB regulatory pathway were analyzed by Real-time PCR,Western Blot and immunoprecipitation.The LDH activity and apoptosis of cells that induced by GSSG and TNFα were detected by LDH kit.The level of GSSG,GSH and the binding activity of NF-κB(p65)were detected by ELISA kit.The expression of NF-κB regulatory factor IKK-β was detected by Western Blot and immunoprecipitation technique,and the glutathione of IKK-β was detected by Real-time PCR.Restules(1)Long-term high-fat diet will reduce the level of GSH and increased the level of GSSG and TNFa in the liver.(2)GSSG accumulation can significantly increased the sensitivity of HepG2 cells to TNFα killing.(3)GSSG prevented TNFα induced activation of IKK-β,an upstream kinase in NF-κB signaling pathway,via inducing IKK-βglutathionylation.(4)Long-term HFD feeding increased hepatic IKK-β-SSG formation,leading to suppressed IKK-β activation and resultant NF-κB suppression.Conclusion Our data suggest that GSSG represents a potent and clinically relevant sensitizer to TNFα-induced hepatotoxicity in NAFLD and represents a potential therapeutic target for NAFLD.