脑缺血再灌注中nNOS介导的ASK1巯基亚硝基化促进其激活

nNOS mediated endogenous s-nitrosylation of apoptosis signal-regulating kinase promotes its activation during Cerebral Ischemia-Reperfusion

目的本文主要研究在脑缺血再灌注早期,神经性一氧化氮合酶(nNOS)来源的NO是否能够引起凋亡信号调节激酶蛋白1(ASK1)巯基亚硝基化,以及ASK1的巯基亚硝基化与其激活的关系。方法 SD大鼠分为正常对照组,缺血复灌6 h组,7NI给药组,AMT给药组,生理盐水对照组。采用四动脉结扎去结扎法构建大鼠全脑缺血再灌注模型。运用SDS-PAGE、免疫印迹、免疫沉淀和免疫组织化学方法对蛋白质的磷酸化以及蛋白质之间的相互作用进行研究;蛋白质的S-亚硝基化的检测主要是通过"生物素转化法"(Biotin-Swich method)。结果 nNOS的抑制剂7NI能够降低ASK1的亚硝基化水平,进而降低了ASK1的磷酸化水平(P<0.05)即ASK1的活性。结论 7NI通过降低nNOS介导的ASK1巯基亚硝基化,对SD大鼠海马区脑缺血再灌注损伤的神经元起到保护作用。

Objective The objective of this study is primarily to determine,during the early stages of ischemia-reperfusion,1）whether nNOS-derived NO could cause the s-nitrosylation of ASK1,2）whether ASK1 s-nitrosylation could promote the activation of it. Methods Transient cerebral ischemia was induced by a four-vessel occluded（4-VO） method.We perform the study mainly by SDS-PAGE,western bloting,immunoprecipitation and immunohistochemistry to exam phosphorylation and the interaction of proteins.S-nitrosylation was examined mainly by the biotin switch assay.Results 7NI,the inhibitor of nNOS could decrease ASK1 s-nitrosylation of ASK1,thereby,decrease the phosphorylation of ASK1（P0.05）.Conclusions 7NI could protect the neuron in hippocampus of brain from injury by reducing the nitrosylation of ASK1 which mediated by nNOS.