JNK/MCP1与AIF/RIP3正反馈环路调控程序性坏死介导全脑缺血再灌注损伤的机制研究

我们前期研究证实程序性坏死是全脑缺血再灌注损伤的重要机制之一。全脑缺血损伤后受体相互作用蛋白3（RIP3）作为程序性坏死的标志蛋白，以AIF-RIP3蛋白复合体形式向细胞核内位移诱导大鼠神经元DNA断裂，同时激活胶质细胞JNK介导的炎症通路，导致神经细胞死亡。如何调控AIF-RIP3成为探讨程序性坏死的关键。研究发现炎症因子与上游信号通路可能存在相互作用，但全脑缺血损伤后JNK及其炎症趋化因子MCP1是否对AIF-RIP3产生影响尚不明确。本项目拟建立大鼠全脑缺血再灌注损伤模型和体外细胞缺血模型，以特异性药物阻断剂、siRNA、重组腺相关病毒等作为干预手段，应用免疫荧光、共沉淀和激光共聚焦等方法，探索缺血损伤后AIF-RIP3合成及细胞核转移是否受到JNK/MCP1信号通路的调控，从而验证以RIP3为中心的程序性坏死与炎症通路的正反馈环路，可能是促进全脑缺血损伤的重要机制。

Necroptosis is one of the important pathological mechanisms following cerebral ischemic injury. Our preliminary study confirmed that nuclear translocation of receptor-interacting protein 3 (RIP3) is involved in necroptosis after global cerebral ischemia/reperfusion (I/R) injury. The nuclear translocation and co-localization of RIP3 and apoptosis induced factor (AIF) indicates an interaction in which the AIF-RIP3 protein complex causes rat neuronal DNA fragmentation, a process which is associated with the JNK phosphorylation mediated inflammation pathway. Exploring the mechanism of AIF-RIP3 is the key to studying necroptosis. However, the effect of JNK and MCP1 on the regulation of AIF-RIP3 synthesis in global cerebral ischemic injury is not clear. This study is intended to demonstrate that the AIF/RIP3 pathway is a central mechanism of necroptosis and is mediated by a positive feedback loop involving activation of the JNK/MCP1 inflammatory pathway which promotes cerebral ischemic injury. We used both the rat global cerebral ischemia model and oxygen-glucose deprivation model on mixed cultures of neurons and glial cells to study this mechanism both in vitro and in vivo. And we utilized specific inhibitor, siRNA, recombinant adeno-associated virus, co-immunoprecipitation, immune-probe and laser confocal observation to explore whether the synthesis of the AIF-RIP3 protein complex and its nuclear translocation activates the JNK/MCP1 signal pathway to produce a positive feedback of inflammation after ischemic injury.