caspase-8介导小胶质细胞活化在脑梗死后炎症损伤及神经元死亡中的作用及机制研究

Microglia can be activated by the harmful factors released from dead neurons after cerebral infarction, then induced the over-expression of inflammatory factors and brain damage. Recently, several studies reported that the activation and neurotoxic effect of microglia depend on caspase-8 (Cas8). In our previous experiments, we found that Cas8 is expressed richly in microglia and up-regulates after global cerebral ischemia/reperfusion injury in the brain of rat model, suggested that Cas8 may be involved in the activation of microglia after cerebral ischemia. Based on these, we propose that, harmful factors released from dead neurons activate microglia through caspase-8 dependent manner; microglial activation leads to inflammatory cascade for the overexpression and release of inflammatory cytokines; inflammatory damage and neuronal death are enlarged by these cytokines. In this project, we tried to use kinds of technology and methods, such as signal pathway inhibitors, RNA interference to inhibit the expression of caspase-8, CreLysMCasp8fl/fl mice, to prove our hypothesis in cultured microglia and animal models of middle cerebral artery occlusion. The activation of microglia, the expression of inflammatory cytokines, neuronal death, brain infarct volume, behavior, the damage of blood brain barrier and brain edema will be detected. The implementation of the project is expected to reveal the role of caspase-8 in microglia activation and inflammatory cascade after brain infarction, thus providing a theoretical basis for the intervention of inflammatory injury after cerebral infarction.

脑梗死后神经元死亡可释放有害物质激活小胶质细胞导致炎症。已知caspase-8（Cas8）介导小胶质细胞活化引起炎症及神经元毒性。我们发现Cas8主要在大鼠脑的小胶质细胞表达，并在脑缺血后表达上调，提示Cas8参与缺血后小胶质细胞活化。我们推测：脑梗死后损伤神经元释放有害物质，通过Cas8通路，激活小胶质细胞产生炎症因子。炎症因子反作用于神经元加剧其死亡并诱发脑内炎症级联反应。本项目拟采用CreLysMCas8fl/fl小鼠在小胶质细胞敲除Cas8基因，结合凋亡、程序性坏死通路阻断剂、RNA干扰抑制Cas8活化或表达，通过分子生物学、组织病理学等手段，在脑梗死动物模型上检测小胶质细胞活化，炎症因子表达，神经元死亡，脑梗死面积，血脑屏障及脑水肿等变化，从细胞及动物水平验证上述假说。本课题有望揭示Cas8在小胶质细胞活化及脑梗死后炎症级联反应中的作用及机制，为脑梗死后炎症干预治疗提供理论基础。

脑组织中神经元对缺血缺氧最为敏感，脑缺血后梗死核心区的神经元由于离子泵和线粒体功能障碍在数分钟内即出现坏死，但半暗带区神经元死亡可以延迟到几天或几周。神经元死亡后将产生后续一系列反应，包括氧化应激、释放细胞毒性因子和促炎性物质、激活胶质细胞、破坏血脑屏障、血循环中的炎症细胞进入脑实质等。炎症级联反应在脑缺血损伤中起重要作用，抗炎治疗可能是脑保护治疗中有重要前途的研究方向。Pellino3是一种具有抗炎功能的E3泛素连接酶蛋白，其可减少caspase-8的裂解、TNF-α诱导的细胞死亡。Takeda G protein receptor 5（TGR5）是一种血浆膜结合的G蛋白偶联胆汁酸受体，存在于多种组织中（包括动物和人类大脑）。多项研究证实，TGR5通过抗炎症、抗凋亡和抑制氧化应激，被认为是治疗代谢性疾病和肾脏疾病的潜在靶点。. 因此，本研究采用MCAO模型，结合各种抑制剂、激动剂、siRNA等手段，观察小胶质细胞活化、炎症因子产生、神经元死亡、神经功能损伤等，我们发现：（1）TGR5-Pellino3通过抑制Caspase-8/NLRP3的表达，从而抑制MCAO的神经炎症反应，减轻脑损伤，改善神经功能；（2）激活TGR5可以通过BRCA1/Sirt1信号通路降低MCAO后的脑水肿和血脑屏障通透性；（3）在缺血半暗带，Pannexin1通道抑制剂(10panx) 通过抑制RIP3的表达、HMGB1的释放和减轻后续相关的炎症反应起到保护作用。.