GSSG通过抑制TNF-α介导的NF-κB活化诱导非酒精性肝损伤

摘要

Objective] To study the role of GSSG in hepatocyte death induced by inhibiting of TNF-α-mediated the NF-κB activation.[Methods]Human liver cell line HepG2 was used to establish the cell model. The effect of GSSG on TNF-α-induced cell death was determined by lactate dehydrogenase(LDH) release and Hoechst Staining. The intracellular levels of GSSG-protein adducts, and the protein expression of IKK-βand phospho-IKK-βwas determined by Western blotting. The intranuclear NF-κB(p65) and its DNA binding activity were detected by ELISA. The level of GSSG and GSH was detected by ELISA too.Use Real time PCR to detect the expression of NF-κB target genes. [Results](1)GSSG significantly increased the sensitivity of HepG2 cells for the killing effect of TNF-α. (2)H2O2 can significantly increase the level of GSSG in HepG2 cells.(3)GSSG significantly increased the level of glutathione-protein adducts by oxidizing sulfhydryl.(4)GSSG inhibited TNF-α-mediated NF-κB(p65) activation in HepG2 cells.(5)GSSG inhibited NF-κB activity by glutathione modification of IKK-β. [Conclusions]Oxidative stress plays a key role in the development of non-alcoholic fatty liver disease, and produces GSSG as a hepatocyte-sensitizing factor, which inhibits TNF-α-mediated NF-κB anti-apoptotic signaling pathway in hepatocytes, thus inducing alcoholic liver damage. This may be a new pathogenesis of nonalcoholic fatty liver disease.%[目的]通过体外细胞实验，研究氧化型谷胱甘肽[glutathione(Oxidized)，GSSG]通过抑制细胞内肿瘤坏死因子( tumor necrosis factor-α，TNF-α)介导的核转录因子(nuclear transcription factors kappa B，NF-κB)的活化诱导肝细胞死亡的作用机制。[方法]以人肝细胞株HepG2为细胞模型，利用乳酸脱氢酶(lactate dehydrogenase，LDH )测定法及Hoechst染色法检测GSSG、TNF-α作用后细胞培养液中LDH活性及细胞凋亡情况。通过蛋白免疫印迹法(western blot，WB)检测细胞中蛋白质谷胱甘肽化水平、IκB激酶β(inhibitor kappa B kinase-β，IKK-β)蛋白表达水平及其磷酸化水平。通过酶联免疫法(enzyme linked immunosorbent assay，ELISA)检测GSSG、还原型谷胱甘肽(glutathione，GSH)水平及NF-κB(P65)的结合活性。并利用实时定量荧光PCR(real time PCR，RT-PCR)检测NF-κB目标基因的表达情况。[结果](1)H2O2作用后可显著提高HepG2细胞内GSSG水平。(2)GSSG可以显著增加HepG2细胞对TNF-α杀伤作用的敏感性。(3)GSSG通过氧化巯基使蛋白质谷胱甘肽化水平显著升高。(4)GSSG抑制HepG2细胞内TNF-α介导的NF-κB活化。(5)GSSG通过谷胱甘肽化修饰IKK-β抑制NF-κB的活性。[结论]氧化应激产物GSSG通过抑制TNF-α介导的NF-κB活化诱导肝细胞死亡。这可能是一种新的非酒精性脂肪性肝病(nonalcoholic fatty liver disease，NAFLD)的发病机制。