脑中风后神经元过度自噬恶化小胶质细胞炎症损伤的机制研究

Excessive neuronal autophagy and prominent activated microglial inflammation induced by cerebral stroke are the important causes resulting in nerve injury , whereas the crosstalk mechanism between them is unknown. Studies have revealed that neurons had an inhibitory effect on microglial activity, through ligation by its membrane-bound protein CX3CL1 with microglial membrane receptor CX3CR1, furthermore, the CX3CL1 expression was regulated by GSK-3β. Our previous study found that neuronal autophagy led to reduction of CX3CL1 expression, and in turn activated microglia and deteriorated inflammatory injury. We accordingly theorize that excessive autophagy in neurons may suppress GSK-3β activity and in turn decrease CX3CL1 expression, and the reduction of CX3CL1 then represses CX3CL1-CX3CR1 cross-communication, resulting in exacerbation of neural injury. For this hypothesis, first, gene expression profiles of neuronal autophagy and microglial inflammation after cerebral stroke are detected; Second, autophagy and microglial inflammatory activity are intervened at characteristic time points, to elucidate the interaction between them and its neurotraumatic mechanisms. Meanwhile, alter GSK-3β activity to explore its inflammatory injury by inhibited CX3CL1-CX3CR1 signaling. Finally, the regulatory mechanism of neuronal autophagy to modify microglial inflammation is investigated in an ischemia model of co-culture of neurons with microglia. This study is to provide new clue and theoretical guide for treatment of cerebral stroke.

脑中风诱导的神经元过度自噬及显著激活的小胶质细胞炎症是导致神经损伤的重要原因，但两者间的对话机制尚不清楚。研究表明，神经元通过其膜锚定蛋白CX3CL1与小胶质细胞膜受体CX3CR1结合，抑制小胶质细胞活性，而CX3CL1的表达受GSK-3β调控。我们前期研究发现脑中风后神经元自噬可致CX3CL1表达降低，激活小胶质细胞加剧炎症损伤。故推测脑中风后神经元过度自噬抑制GSK-3β活性进而降低CX3CL1表达，阻遏CX3CL1-CX3CR1信号对小胶质细胞炎症的抑制，恶化神经损伤。为此，首先检测脑中风后神经元自噬与小胶质细胞炎症表达时间谱；其次在特征时间点干预自噬和炎症，阐明两者间对话及其神经损伤机制；同时干预GSK-3β探讨抑制CX3CL1-CX3CR1通路引发的炎性损伤；最后利用神经元—小胶质细胞共培养缺血模型，探明神经元自噬对小胶质细胞炎症的调控机制，为脑中风治疗提供新的思路和理论依据。

脑卒中所致脑缺血可显著激活缺血半影区神经元的自噬活性，而被激活的自噬可对脑卒中病理进程可产生重要影响。研究发现，缺血性脑卒中急性期神经元往往发生过度自噬反而加重神经损伤。神经元与小胶质细胞间的对话机制主要由CX3CL1-CX3CR1介导。CX3CR1是仅表达于小胶质细胞而不表达于其它脑细胞的CX3CL1特异性受体。我们前期研究发现脑缺血可过度激活缺血性半影区神经元自噬，与此同时神经元上的CX3CL1表达显著降低，因而推测神经元上的CX3CL1表达降低可能由其发生自噬引发，由此削弱其通过CX3CL1-CX3CR1对话机制对小胶质细胞炎症抑制的效应。为验证该假说，本研究分别建立缺血性脑卒中大鼠模型及体外HT22（神经元）与BV2（小胶质细胞）共培养细胞缺血模型。结果表明，脑缺血诱导缺血半影区神经元自噬后可显著降低其CX3CL1的表达，而此时炎症信号Iba1及NF-κB p65增强伴随炎症因子IL-1β、TNF-α、IL-6和PGE2明显增加。而后通过侧脑室注射改变自噬活性，结果发现提高缺血半影区神经元自噬可进一步降低其CX3CL1的表达进而恶化小胶质细胞炎症，并最终加重脑梗死面积、神经元死亡、脑炎性水肿及神经功能缺失。而抑制自噬则通过提高神经元CX3CL1的表达对小胶质细胞炎症产生明显的抑制效应，并减轻脑缺血损伤。同时，外建立神经元（小鼠HT22细胞株）与小胶质细胞（小鼠BV2细胞系）共培养体系，并采用氧糖剥夺（OGD）构建细胞缺血模型。结果表明，提高HT22细胞自噬通过降低其CX3CL1的表达可加重小胶质细胞炎症，并恶化神经元死亡。相反，降低HT22自噬则显著抑制小胶质细胞炎症，并可提高神经元活力及存活。本项目初步阐明神经元自噬通过降低其CX3CL1表达加重小胶质细胞炎症，进而恶化脑缺血损伤的病理机制。本项目的研究结论提示，降低神经元自噬是减轻小胶质细胞炎性损伤的有效途径。