一氧化氮参与缺血性脑中风早期血脑屏障损伤的作用靶点及干预研究

Blood-brain barrier (BBB) damage is a key event of ischemic stroke. However, the molecular and cellular events related to early BBB damage remain poorly understood. Our previous study had demonstrated that cerebral ischemia caused matrix metalloproteinase 2(MMP2)-mediated occludin degradation and caveolin-1 (Cav-1)-mediated redistribution of claudin-5 by initiating MMP2 release and Cav-1 translocation in brain vascular endothelial cells. These events result in early BBB damage in ischemic stroke. And nitric oxide (NO) induced by early ischemia is associated with occludin degradation and claudin-5 rearrangement. It suggests that NO may be a key factor that involves in early BBB damage in ischemic stroke via mediating MMP2 release and Cav-1 translocation. This study aims to reveal the mechanism of NO-mediated early BBB damage in ischemic stroke using in vitro [oxygen-glucose deprivation (OGD)] and in vivo [middle cerebral artery occlusion (MCAO)] stroke models. The protein and mRNA levels of MMP2, Cav-1, occludin, claudin-5,and so on, endothelial cell monolayer permeability, and BBB leakage into brain tissue were detected after NO was scavenged or NO production and the possible downstream signaling pathways related to NO were blocked in these models. By means of this research, we could uncover and identify the potential targets inducing early ischemic BBB damage, which might provide clues for developing new treatment strategies to protect the BBB against ischemic damage.

血脑屏障（BBB）损伤是缺血性脑中风的关键事件，但对缺血早期BBB发生的分子和细胞事件知之甚少。我们前期的工作显示：紧密连接蛋白occludin的降解和claudin-5的重排是早期BBB损伤的重要标志，分别由血管内皮细胞释放的基质金属蛋白酶2（MMP2）和caveolin-1（Cav-1）的胞内转位介导它们的发生，中风早期诱导产生的一氧化氮（NO）与这些紧密连接蛋白的变化相关。提示NO可能通过诱导MMP2释放和Cav-1转位参与早期BBB损伤。本研究拟采用缺血性脑中风细胞和动物模型，通过清除NO、抑制NO的产生以及阻断NO可能介导的下游通路后，检测MMP2、Cav-1、occludin、claudin-5等蛋白质水平和细胞内分布以及单层血管内皮细胞通透性和BBB的漏出，旨在揭示NO介导中风早期BBB损伤的精细途径并筛选潜在的作用靶点，为预防缺血性脑中风早期BBB损伤的药物开发提供依据。

血脑屏障（BBB）损伤是缺血性脑中风的关键事件，但目前对缺血早期BBB发生的分子和细胞事件知之甚少，如果能弄清楚中风早期 BBB 损伤的机制，对阻止或延缓 BBB 的损伤，具有重要意义。本项目主要开展了以下研究：1）详细研究了一氧化氮（NO） 介导缺血性脑中风早期 BBB 损伤的信号通路，发现NO介导BBB呈二阶段损伤，第一阶段早期缺氧会迅速诱导细胞NO产生，NO导致caveolin-1从细胞骨架解离，caveolin-1解离后携带claudin-5从细胞骨架上解离，并将claudin-5运送至溶酶体附近，第二阶段随着继续缺氧， claudin-5最终在溶酶体内被降解。2）研究了老年人血脑屏障损伤的机制，褪黑素下降导致老年人容易发生细菌感染和败血症，导致血脑屏障损伤，发现褪黑素通过阻止脂多糖抑制Ampk激活以及降低脂多糖引起的产生自由基的NADPH的催化亚基gp91的表达减轻脂多糖引起的血脑屏障损伤。这一机制的发现，为细菌感染导致BBB损伤以及感染与大脑疾病的关系提供了科学依据。3）研究了干细胞对脑中风的治疗作用，发现干细胞和中药冰片结合治疗可以有效降低小鼠梗死面积和细胞凋亡，提高神经再生和功能恢复。4）发现了脑卒中炎性微环境产生的新机制，从单细胞水平发现脑卒中后神经元是产生炎性物质的主要细胞，C/EBP是启动炎性物质产生的基础。本研究的完成为预防缺血性脑中风早期 BBB 损伤以及控制损伤后炎性微环境的药物开发提供了科学依据。. 本研究以第一作者或者通讯作者已经发表了4篇SCI研究论文，总的影响因子超过18分，另外一篇挂基金号的第一作者的文章准备投Cell。以第一发明人获得实用新型专利2项，第一发明人申请国家发明专利3项。培养了3名研究生。达到了本项目申报时的预期研究结果。