GSK-3β在脑缺血再灌注损伤时对Nrf2活性的调控及机制研究

Modulation of Nrf2 has a unique role in ischemic reperfusion injury.Glycogen synthase kinase-3β(GSK-3β) is one of the kinases implicated in the regulation of Nrf2. However, the mechanistic connection between GSK-3β and Nrf2 remains largely unexplored. In the presnt study, we will verify the hypothesis that GSK-3β as a direct upstream regulatory kinase of Scr subfamily kinases that contributes to nucleocytoplasmic trafficking of Nrf2, and modulates the Nrf2 protein stability through β-TrCP. We will further study the effects of GSK-3 βon Nrf2 against ishcheia reperfusion injury in vitro, following oxygen-glucose deprivation (OGD), and in vivo, after middle cerebral artery occlusion (MCAO) in rats. By using RNA interfering(RNAi).PCR. westen immnoblot analysis. immunofluorescende staining. electrophoretic mobility shift assay (EMSA) . immunoprecipitation (IP) and site-directed mutagenesis, the effects of GSK-3β on activation of Nrf2 . Scr subfamily kinases .β-TrCP and the related target genes will be measured.The aim of this study is to dicuss the regulation mechanism of GSK-3β in Nrf2 post-transcription activity oncerebral ischemia/reperfusion, which will provide a academic basis for a more complete understanding of the inner protective mechamisms in the brain,and which may also provide a promising opportunities to prevent stroke.

Nrf2活性的维持是神经细胞抵抗脑缺血再灌注损伤的关键。糖原合成酶激酶-3β（GSK-3β）是调控Nrf2活性的重要激酶，但具体调控机制尚不清楚。基于前期研究，我们提出："在脑缺血再灌注应激损伤时，GSK-3β可以通过磷酸化Scr酪氨酸激酶家族成员，参与 Nrf2的出核转运；同时调控β-TrCP，参与Nrf2泛素化降解，从而成为Nrf2抗氧化系统调控的关键分子"。本课题在体内外脑缺血再灌注模型上，采用RNA干扰、药理阻滞、Western blot、荧光定量PCR、免疫荧光、IP、EMSA、定点突变等技术研究GSK-3β对Nrf2功能及活性的影响，对Scr酪氨酸激酶家族、β-TrCP-泛素化蛋白酶体降解各信号分子及靶基因的影响,阐明在缺血再灌注氧化应激损伤时，GSK-3β对Nrf2活性的影响及调控机制，将进一步完整阐明机体内源性保护机制的具体环节，从而为脑卒中的防治提供新策略。

Nrf2活性的维持是神经细胞抵抗脑缺血再灌注损伤的关键。糖原合成酶激酶-3β（GSK-3β）是调控Nrf2活性的重要激酶，但具体调控机制尚不清楚。本课题在体内外脑缺血再灌注模型上，采用RNA干扰、药理阻滞、Western blot、荧光定量PCR、免疫荧光、EMSA等技术研究GSK-3β对Nrf2功能及活性的影响，阐明在缺血再灌注氧化应激损伤时，GSK-3β对Nrf2活性的影响及调控机制。通过本项目的研究，我们发现：在脑缺血再灌注应激损伤时，GSK-3β在脑缺血再灌注中对中枢神经系统神经元有损伤作用，同时，GSK-3β可以通过参与Nrf2的出核转运从而成为Nrf2抗氧化系统调控的关键分子。进一步阐明了机体内源性保护机制的具体环节，从而为脑卒中的防治提供新策略。