NADPH氧化酶在NOD2介导的脑缺血再灌注损伤中的作用及机制

The innate immune response is involved in the pathophysiology of cerebral ischemia-reperfusion (I/R) injury. NOD2 (nucleotide-binding oligomerization domain protein 2) is one of the pattern recognition receptors involved in innate immune response and is genetically associated with several inflammatory diseases. Our recent studies demonstrated that the expression of NOD2 in both mRNA and protein levels was significantly increased in the ischemic penumbra of mice subjected to 2 h middle cerebral artery occlusion (MCAO) and 24 h reperfusion; moreover, compared with wild type mice, NOD2-deficient mice had lower infarct volumes. However, the NOD2-dependent signalling pathway after cerebral I/R is far from being fully characterized. NOD2 cause inflammation via activation of nuclear factor-kappa B (NF-κB) which is a central regulator of inflammatory response. NADPH oxidase type 2 (NOX2) is well-known as a major source for superoxide radical generation, which play a significant role in brain I/R injury and is associated with inflammation by activated NF-κB. Our preliminary data also showed that NOD2 and NF-κB P65 subunit were up regulated in the microglia cells with oxygen-glucose deprivation (OGD), Apocynin could effectively down regulate NOD2 expression and NF-κB activation by inhibiting NOX. Taken together, our data suggested the role of NOD2 in cerebral I/R injury was regulated by NOX2-mediated oxidative stress. However the detailed mechanisms are needed to be further investigated. In this study, we will explore the role and machenism of NOD2 in cerebral I/R injury by using microglia OGD model in vitro and mice MCAO model in vivo. We will also investigate the role of NOX2 signalling in NOD2-mediated brain damage in I/R. Our study will firstly provide evidence that NOD2-mediated innate immunity participates in brain damage after stroke. With the consideration that the immune response occurs early after injury, it would be useful to develop new therapies to inhibit NOD2 signaling to produce a neuroprotective effect.

固有免疫介导的炎症反应在缺血性脑血管疾病病理生理机制中的作用日益引起人们的重视，NOD2是新近发现的胞内模式识别受体，其介导的免疫炎症反应在机体防御和炎症性疾病中发挥着关键作用，迄今尚无NOD2在脑缺血再灌注损伤中的明确作用及机制的研究。我们前期研究证实脑缺血再灌注后缺血区NOD2表达显著升高，而且NOD2敲基因鼠在缺血再灌注损伤后梗死体积明显小于野生型小鼠，提示其在脑缺血再灌注损伤中可能发挥了重要作用。此外，我们还发现抑制NADPH氧化酶（NOX2）可以明显抑制小胶质细胞糖氧剥夺诱导的NOD2表达及NF-κB活化。本课题拟采用小鼠的大脑和小胶质细胞的缺血再灌注损伤模型，体内外分别应用基因敲除和沉默技术，深入探讨NOD2引起脑缺血再灌注损伤的机制及其和NOX2/NF-κB信号通路的关系。本课题将首次揭示NOD2信号通路在缺血再灌注损伤的作用及机制，并为疾病的防治提供新的策略。

脑卒中是全世界发病率和死亡率极高的脑血管疾病，其中缺血性脑卒中约占80%。传统的溶栓治疗使缺血脑组织得到及时再灌注的同时，也能进一步加重其病理损害，造成缺血再灌注脑损伤。近年来，越来越多研究证明固有免疫介导的炎症反应与脑缺血之间存在密切联系。位于胞浆内的模式识别受体核苷酸结合寡聚化结构域(nucleotide-binding oligomerization domain 2，NOD2)是固有免疫领域的重要分子和研究热点，其在缺血再灌注脑损伤中的作用及机制尚未阐明。本项目利用小鼠大脑中动脉线栓法建立的脑缺血模型再灌注损伤模型，发现脑缺血再灌注损伤后NOD2的表达明显上调并且在小胶质细胞及星形胶质细胞中有大量表达。脑室内注射NOD2激动剂MDP加重了缺血再灌注脑损伤。而NOD2敲基因鼠较野生型小鼠在缺血再灌注损伤后梗死体积明显缩小，神经功能学评分明显改善；同时明显抑制了缺血再灌注损伤诱导的NF-κB, p38MAPK和JNK的活化； IL-1β、IL-8、IL-6、TNFа等炎症因子的产生以及NADPH氧化酶2型（NOX2）表达和氧自由基的产生。此外体外抑制NOX2活性可以明显降低小胶质细胞NOD2活化诱导的IL-6表达。以上结果提示脑缺血再灌注后上调的NOD2活化后激活NOX2，产生的ROS，从而启动转录因子NF-κB的活化，进一步诱导免疫炎症反应，参与缺血再灌注脑损伤。本课题首次揭示NOD2 /NOX2信号通路在脑缺血再灌注损伤的作用及机制，为疾病的防治提供了新的策略。