Cx32通过调控NOX4的活化抗缺血性脑卒中的作用及机制研究

缺血性脑卒中严重危害人类生命，目前尚无有效的治疗方法和药物。在脑缺血阶段，NOX4是细胞内ROS的主要来源，且最新研究发现Cx32作为神经元上表达最强的缝隙连接蛋白能够促进细胞内ROS的生成。据此，我们推测Cx32通过调控NOX4的活化影响细胞内ROS的生成，进而参与缺血性脑卒中的发生发展。申请人通过预实验发现：OGD/R损伤后，神经元细胞膜上Cx32，细胞内Wnt5a和膜上Rac1的表达均升高，NOX4活化，ROS生成增加，细胞存活率下降；利用RNA干扰抑制Cx32水平后再进行OGD/R损伤可逆转上述过程。在预实验的基础上，本项目拟从离体细胞与整体动物水平验证科学假说：缺血性脑损伤后，Cx32通过Wnt5a激活非经典的Wnt信号通路，促进Rac1从胞浆向膜移位，激活NOX4，启动氧化应激机制，最终导致神经元细胞凋亡，为缺血性脑卒中的机制研究提供新的思路，具有重要的理论意义和应用价值。

Ischemic stroke causes a very extensive health problem to humankind. There are no effective treatment and ideal drugs for ischemic stroke. During the stage of ischemia, NOX4 is the major source of intracellular ROS. Latest studies found that Cx32, a connexin of the strongest expression on neurons, can promote the generation of intracellular ROS. Thus, we hypothesized that Cx32 affected the production of intracellular ROS through the regulation of NOX4, further participated in the occurrence and development of ischemic stroke. Our preliminary experiments found that after OGD/R injury, the expression of Cx32 on cell membrane, Wnt5a in neurons and Rac1 on cell membrane are all up-regulated, NOX4 is activated, the generation of intracellular ROS is increased, and the survival rate of neurons is decreased. However, inhibiting Cx32 level by RNA interference before OGD/R injury could reverse the above process. On the basis of the preliminary experiments, our project would establish animal and cell models to verify a scientific hypothesis: after cerebral ischemia, Cx32 activates non-canonical Wnt signaling pathway through Wnt5a, promotes the translocation of Rac1 from the cytosol to the membrane, induces the activation of NOX4, initiates the oxidative stress mechanism, and eventually leads to neuronal cell apoptosis. The validation of this hypothesis will suggest a new idea for researching the pathogenesis of cerebral ischemia, which is of important theoretical significance and application value.