PSD-93调控CX3CL1/CX3CR1信号介导急性缺血性卒中后炎症反应的机制研究

The glutamate mediated excitotoxicity in neuron and microglia mediated neuroinflammation are main pathological mechanisms that lead to ischemic cerebral injury. And CX3CL1/CX3CR1 is the primary and specific adhesion molecule and receptor mediating the crosstalk between neurons and microglia. Our previous study has found that PSD-93 would promote the activation of NMDA receptor NR2B and then mediate the release of early inflammatory cytokines induced by ischemic brain injury, but the specific mechanism is still unclear. In this study, we will construct the middle cerebral artery occlusion (MCAO) model and detect the assembly of PSD-93•NR2B•CX3CL1 signaling module and clarify the relationship between the signaling module and activation of microglia. To further ascertain the binding sites between PSD-93 and NR2B，CX3CL1, mammalian two-hybrid, genetic mutations, plasmid construction, co-immunoprecipitation were applied. And peptide interfering the assembly of PSD-93•NR2B•CX3CL1 signaling module will be used to verify its function. Furthermore, gene knockout and pull-down technologies will also be used to clarify the effect of CX3CL1/CX3CR1 signaling on neuronal apoptosis and microglia activation.

神经元内谷氨酸兴奋毒和小胶质细胞介导的炎症反应是缺血后脑损伤的主要病理机制，而CX3CL1/CX3CR1是介导神经元与小胶质细胞对话的主要粘附分子及其受体。我们前期的研究发现，PSD-93通过促进NR2B的激活介导了脑缺血早期炎症因子的释放，但具体作用机制尚不清楚。本项目通过构建小鼠MCAO模型，了解PSD-93•NR2B•CX3CL1信号模块的组装及与小胶质细胞活性的关系；应用哺乳动物双杂交、基因敲除、质粒构建、免疫共沉淀等方法确定PSD-93和NR2B、CX3CL1结合的位点，并验证其功能；进一步应用基因敲除、pulldown等技术阐明CX3CL1/CX3CR1信号对神经元凋亡和小胶质细胞活化的作用。本课题重点探讨了PSD-93•NR2B•CX3CL1信号模块通过CX3CL1/CX3CR1信号介导神经元与小胶质细胞的对话，引发炎症反应的发生，为卒中的脑保护治疗提供新的思路。

前期的研究发现，PSD-93介导缺血性脑损伤引起的谷氨酸兴奋毒，进而诱导小胶质细胞炎症反应。然而，PSD-93如何介导突触后致密部神经元和小胶质细胞激活的机制尚不清楚。CX3趋化因子配体1 (CX3CL1)是一种特异性表达于神经元的趋化因子，其受体CX3CR1在小胶质细胞中高度表达。在本项目中，我们研究了PSD-93和CX3CL1在急性缺血性脑卒中神经元与小胶质细胞间的相互作用。首先，我们应用雄性C57BL/6小鼠建立大脑中动脉闭塞模型(MCAO)模型，发现PSD-93和CX3CL1的结合高峰在I/R 6 h，而这一时间点可溶性CX3CL1的表达亦明显增加，M1型（CCL2, COX-2, TNF-α）小胶质细胞开始极化，并在R24h时达到高峰，提示PSD-93与CX3CL1的结合可能促进其被剪切成可溶性的形式，进而激活小胶质细胞，启动炎症反应的发生。其次，应用酵母双杂交、基因敲除、质粒构建、免疫共沉淀等方法确定二者的结合位点分别位于PSD-93的420-535氨基酸序列和CX3CL1的357-395氨基酸序列。最后，前期研究的基础上设计融合小肽Tat-CX3CL1 (357-395aa)抑制PSD-93和CX3CL1的相互作用，发现Tat-CX3CL1 (357-395aa)可抑制PSD-93和CX3CL1的相互作用，促进小胶质细胞由M1型（IL-1β，TNF-α, iNOS，CCL2, Cox-2）向M2型（IL-4, IL-10, VEGF, CD163）的极化，保护缺血再灌注后神经元，改善卒中后的记忆功能。因此，特异性阻断PSD-93-CX3CL1的相互作用可减少I/R诱导的早期神经炎症状态的转化及后期神经功能的损伤，为缺血性卒中提供新的治疗靶点。