NADPH氧化酶2在脑缺血再灌注损伤不同时期的不同作用及机制研究

Ischemic stroke is the first cause of mortality in our country. Early and rapid restoration of cerebral blood flow is critical to minimize persistent brain damage following stroke but results in cerebral ischemia/reperfusion injury (CIRI). With the reperfusion of cerebral blood flow, oxidizing enzymes are activated and generate a huge burst of reactive oxygen species (ROS). NADPH oxidase (NOX) 2 is principle enzyme of the production of ROS. Physiologically, ROS act as signaling molecules, but excessive production of ROS during cerebral ischemia/reperfusion tremendously contributes to secondary brain tissue injury. Our pilot experiments showed that infarct volume was smaller, neurological deficiency and behaviors were improved, infiltration of microglia/macrophages was inhibited and expression of inducible nitric oxide synthase (iNOS), a pro-inflammatory mediator, was decreased in NOX2-/- mice during early phase (shorter than 3 days) of middle cerebral artery occlusion (MCAO) as compared with wild type (WT) mice. However, during reparative phase (longer than 7 days), deterioration of neurological deficient, worsening of behavioral manifestations and increased infiltration of macrophages were observed in MCAO NOX2-/- mice, suggesting that NOX2 maybe exert protective effect and promote restoration of injured brain tissue. In present project, we try to investigate the dynamic expression and production of NOX2-ROS during different periods after CIRI, determine the distinctive effects of NOX2 at different phases of stroke and clarify the corresponding mechanisms by using immunological, molecular biological technologies and Bio-marker system in in vivo NOX2-/- MCAO model and in vitro oxygen-glucose deprivation/reoxygenation (OGD/RX) models of primary cultured microglia and RBMVEC (rat brain microvascular endothelial cells).

脑缺血后缺血区血管再通发生脑缺血再灌注损伤(CIRI)。NADPH氧化酶2（NOX2）激活致过量活性氧（ROS)生成，是再灌注后脑损伤的重要原因。我们预实验在NOX2-/-小鼠大脑中动脉阻塞（MCAO）模型中发现：CIRI3天内，脑梗塞体积减小、行为学和缺血区炎症反应改善，验证NOX2参与CIRI急性期脑损伤；然而，我们首次发现MCAO7天后，NOX2-/-小鼠神经功能和行为学恶化、缺血区炎症细胞增多，提示CIRI修复期，NOX2可能具有保护效应。我们推测，CIRI不同时期，NOX2-ROS产量不同，发挥的效应不同，具体机制不清。本项目拟利用在体NOX2-/-小鼠MCAO及离体原代细胞氧糖剥夺/复氧复糖模型，运用免疫学、分子生物学等方法，探讨CIRI不同时期NOX2-ROS生成的动态变化，发挥的不同效应及相应机制，拟进一步完善CIRI的发生机制，为以时程不同个性化精准治疗脑缺血提供新靶点。

缺血性脑卒中（IS）后，阻塞血管随着自然再通或医源性再通恢复血流灌注的同时，糖、氧供给需求急速增加，诱发细胞“氧化爆发”，导致过量活性氧（ROS)生成，诱导脑缺血再灌注损伤(CIRI)。NADPH氧化酶2（NOX2）是中枢神经系统主要的NADPH氧化酶之一，也是CIRI时ROS 的主要来源，其在CIRI急性期表达升高，参与脑组织损伤已经得到证实。然而，本项目中，我们发现：1、NOX2基因敲除或其抑制剂APO抑制NOX2，脑梗塞体积减小的同时，改善急性期但加重恢复期大脑中动脉阻塞（MCAO）模型小鼠的行为学缺陷；2、NOX2基因敲除抑制急性期脑缺血区中枢小胶质细胞及外周来源的巨噬细胞浸润，且减少促炎性巨噬细胞M1亚型的极化，这可能通过活化AKT/mTOR信号途径实现，但在恢复期促进血液来源单核细胞迁移浸润至梗塞灶；3、MCAO不同时期，NOX2-ROS动态变化，急性期大量生成而恢复期小剂量生成；4、MCAO不同时期，自噬动态激活并具有NOX2-ROS依赖性，其在急性期过度激活，而恢复期低水平激活；5、MCAO恢复期NOX2-ROS通过自噬依赖性方式抑制NLRP3炎症小体激活，改善脑组织炎症；6、MCAO恢复期NOX2-ROS通过自噬依赖性方式促进血管生成；7、MCAO 7天后小鼠缺血脑组织转录组学测序，KEGG通路富集分析发现PI3K/Akt信号通路可能参与NOX2在缺血性脑卒中恢复期的作用；8、免疫印迹验证和证实MCAO恢复期NOX2-ROS-自噬可能通过PI3K/Akt/NF-κB信号通路抑制NLRP3炎症小体表达，改善炎症反应，诱导血管生成。基于以上结果，本项目首次证明了NOX2在缺血性脑卒中不同阶段具有不同作用，其在急性期对缺血脑组织发挥损伤效应，而恢复期发挥保护效应。据此，可以NOX2为靶标，以其在不同时间的不同水平和功能为依据，从而精准调控不同时程的自噬活化程度，进而改善脑组织炎症反应，并促进血管新生，减轻脑损伤而促进脑组织修复。本项目的发现进阐明了活性氧在缺血性脑卒中中的双向效应，为个性化精准治疗缺血性脑卒中提供了新理论和新方向。