细胞焦亡介导脑缺血再灌注后血脑屏障破坏及通心络的干预机制研究

Previous studies of the applicant indicated that Tongxinluo has a remarkable protection on blood brain barrier after cerebral ischemia-reperfusion injury. It is closely related to the both function of Tongxinluo, which can alleviate brain injury and inflammatory response. Numbers of studies have shown that pyroptosis can induce cell damage and promote its inflammation. The main target molecules regulated by the signal of pyroptosis mediated the blood-brain barrier damage after cerebral ischemia-reperfusion. Our previous found that inhibit pyroptosis can alleviate cerebral ischemia-reperfusion injury. So we think that the protective mechanism of Blood Brain Barrier damage in Tongxinluo may be related to inhibit pyroptosis. Therefore, our research project intends to use male C57BL/6 mice, preparation of cerebral ischemia-reperfusion injury model, with the help of the techniques of RNA interference and plasmid expression, combined with a variety of means of molecular and cellular biology, we explore pathogenesis of pyroptosis in Blood Brain Barrier damage after focal cerebral ischemia-reperfusion from the perspective of Caspase-11/GSDMD signaling pathway and its main target signal molecular, and further excavate the protective mechanism of Tongxinluo in Blood Brain Barrier damage . These explorations can lay the foundation to find new targets for pharmacotherapy, provide a new breakthrough to clarify the mechanism of cerebral ischemia-reperfusion damage and provide experimental basis for modern scientific mechanism of Tongxinluo in the treatment of ischemic stroke.

申请人前期研究显示通心络在脑缺血再灌注损伤中具有显著的血脑屏障保护作用，这与通心络可同时缓解脑细胞损伤和抗炎症反应密切相关，既往研究表明细胞焦亡兼具诱发细胞损伤和促发炎症反应的特性，焦亡信号调控的主要靶分子参与脑缺血再灌注血脑屏障损伤的调控。结合我们前期发现抑制细胞焦亡可减轻脑缺血再灌注损伤，故我们认为通心络保护血脑屏障机制与细胞焦亡存在密切联系。因此，本项目拟利用雄性C57BL/6小鼠制备脑缺血再灌注损伤模型，采用慢病毒（RNA干扰、质粒过表达）及侧脑室注射感染等方法，结合多种分子细胞生物学手段，明确细胞焦亡介导脑缺血再灌注后血脑屏障破坏，并从Caspase 11/GSDMD信号通路及其主要靶分子角度，深入探讨脑缺血再灌注后细胞焦亡机制，挖掘通心络保护血脑屏障新机制，以期为寻找药物调控新靶点，阐明缺血再灌注脑损伤机理提供新的突破口，同时为通心络治疗缺血性中风的现代科学机制提供实验依据。

申请人前期研究显示通心络在脑缺血再灌注损伤中具有显著的血脑屏障保护作用，这与通心络可同时缓解脑细胞损伤和抗炎症反应密切相关，既往研究表明细胞焦亡兼具诱发细胞损伤和促发炎症反应的特性，焦亡信号调控的主要靶分子参与脑缺血再灌注血脑屏障损伤的调控。结合我们前期发现抑制细胞焦亡可减轻脑缺血再灌注损伤，故我们认为通心络保护血脑屏障机制与细胞焦亡存在密切联系，因此，本项目利用雄性SD大鼠制备脑缺血再灌注损伤模型，采用慢病毒（RNA干扰、质粒过表达）及侧脑室注射感染等方法，结合多种分子细胞生物学手段，我们完成研究证实：1）细胞焦亡介导脑缺血再灌注血脑屏障破坏，且通心络可通过减少细胞焦亡，改善脑缺血再灌注损伤；2）Caspase-11/GSDMD信号通路对脑缺血再灌注损伤后的细胞焦亡具有重要调控作用，其中通路核心蛋白GSDMD在脑缺血再灌注损伤后细胞焦亡过程中扮演“分子开关”的重要角色。3）通心络可通过抑制Caspase-11/GSDMD信号通路的激活，阻断关键蛋白GSDMD蛋白剪切活化，阻滞细胞焦亡发生，减轻炎症级联反应，达到保护脑缺血再灌注后血脑屏障的功效。Caspase-11/GSDMD信号通路极有可能成为缺血再灌注脑损伤发生的新机制，其中信号通路核心蛋白GSDMD在通心络调控脑缺血再灌注损伤后细胞焦亡过程中扮演重要角色，具有成为脑缺血再灌注血脑屏障损伤药物调控新靶点的潜力，本研究同时为通心络治疗缺血性中风的现代科学机制提供坚实的实验依据。