METHODS: We collected EVs from LPS-stimulated BV2 cells expressing CCR5 (EVsM1) and from BV2 cells with CCR5 knockdown (EVsM1-CCR5). These were administered to POCD-induced mice. Protein interactions between CCR5, G-protein-coupled receptors (GPCRs), and Ras were analyzed using structure-based docking and co-immunoprecipitation (Co-IP). We assessed the phosphorylation of p38 and Erk, the expression of synaptic proteins PSD95 and MAP2, and conducted Morris Water Maze tests to evaluate cognitive function.
RESULTS: Structure-based docking and Co-IP confirmed interactions between CCR5, GPR, and Ras, suggesting a CCR5-GPCRs-Ras-MAPK pathway involvement in neuroinflammation. EVsM1 heightened neuroinflammation, reduced synaptic integrity, and impaired cognitive function in POCD mice. In contrast, EVsM1-CCR5 reduced neuroinflammatory markers, preserved synaptic proteins, enhanced dendritic spine structure, and improved cognitive outcomes.
CONCLUSIONS: EVsM1 induced neuroinflammation via the CCR5-GPCRs-Ras-MAPK pathway, with EVsM1-CCR5 showing protective effects on POCD progression, suggesting a new therapeutic strategy for POCD management via targeted modification of microglial EVs.
方法:我们从表达CCR5的LPS刺激的BV2细胞(EVsM1)和CCR5敲低的BV2细胞(EVsM1-CCR5)收集EV。将这些施用于POCD诱导的小鼠。CCR5,G蛋白偶联受体(GPCRs)之间的蛋白质相互作用,和Ras使用基于结构的对接和免疫共沉淀(Co-IP)进行分析。我们评估了p38和Erk的磷酸化,突触蛋白PSD95和MAP2的表达,并进行Morris水迷宫测试以评估认知功能。
结果:基于结构的对接和Co-IP确认了CCR5,GPR,还有Ras,提示CCR5-GPCRs-Ras-MAPK通路参与神经炎症。EVsM1加剧了神经炎症,突触完整性降低,和POCD小鼠的认知功能受损。相比之下,EVsM1-CCR5降低了神经炎症标志物,保存的突触蛋白,增强的树突脊柱结构,和改善认知结果。
结论:EVsM1通过CCR5-GPCRs-Ras-MAPK通路诱导神经炎症,EVsM1-CCR5对POCD进展具有保护作用,提出了一种通过靶向修饰小胶质细胞电动汽车来管理POCD的新治疗策略。