【摘要】 目的：探讨福他替尼（Fostamatinib）对血管性痴呆（Vascular Dementia,VaD）中小胶质细胞NLRP3的作用机制。方法：(1)构建双侧颈总动脉狭窄（bilateral common carotid artery stenosis,BCAS）小鼠模型，通过八臂迷宫行为学验证小鼠认知功能受损情况，设置假手术组（Sham）及BCAS术后3 d、1个月、3个月多个时间点，采用免疫荧光染色，劳克坚牢蓝染色（LFB）等方法评估小胶质细胞活化与白质损伤程度，选取损伤最显著的时间点作为造模组；(2)设置对照组、BCAS造模组及（BCAS+福他替尼治疗）组，采用免疫荧光染色和LFB染色方法探究福他替尼对BCAS小鼠的小胶质细胞激活，白质损伤，认知功能障碍及相关炎症因子表达的影响；(3)体外建立原代小胶质细胞外源性髓鞘刺激模型，给予福他替尼或siRNA处理，通过Western blot和实时荧光定量PCR技术，检测炎症通路关键蛋白及基因的表达变化。结果：(1)与对照组相比，造模组BCAS术后1个月时小鼠记忆功能受损，LFB染色白质损伤评分最高，同时髓鞘碱性蛋白（MBP）荧光强度减弱最为显著，Sholl分析结果发现小胶质细胞活化程度也最为明显；(2)与造模组相比，福他替尼治疗组小鼠参考记忆有所改善，白质损伤评分降低，Sholl分析结果显示小胶质细胞活化减少，免疫荧光染色促炎因子NLRP3和SYK的表达均显著下降（P<0.05）；(3)体外福他替尼处理可显著降低髓鞘处理后的小胶质细胞中NLRP3、SYK、ASC、Caspase-1及IL-18等炎症相关因子的表达（P<0.05）；使用siRNA敲低SYK后，炎症因子表达下调结果与前一致（P<0.05）。结论：福他替尼可能通过抑制SYK改善小胶质细胞NLRP3炎症小体炎症通路的活化，减轻神经炎症反应，缓解髓鞘损伤，促进脑白质缺血后结构重建和认知功能的恢复。更多还原

【Abstract】 Objective: To investigate the mechanism underlying the effects of Fostamatinib on NLRP3 in microglia within vascular dementia(VaD). Methods:(1) A bilateral common carotid artery stenosis(BCAS)mouse model was established. The cognitive impairment in mice was validated through the eight-arm maze behavioral test. Sham-operated(Sham) group, as well as multiple time points at 3 days, 1 month, and 3 months after BCAS surgery, were set up. Immunofluorescence staining and Luxol fast blue(LFB) staining were employed to evaluate the activation of microglia and the extent of white matter damage. The time point with the most pronounced damage was selected as the model establishment group.(2) Control group, BCAS model group,and(BCAS + fostamatinib treatment) group were established. Immunofluorescence staining and LFB staining were utilized to investigate the effects of fostamatinib on microglia activation, white matter damage, cognitive dysfunction, and the expression of related inflammatory factors in BCAS mice.(3) An ex vivo primary microglia model with exogenous myelin stimulation was established. The cells were treated with fostamatinib or siRNA.Western blot and real-time fluorescence quantitative PCR techniques were used to detect changes in the expression of key proteins and genes in the inflammatory pathway. Results:(1) Compared with the control group, the mice in the model group exhibited impaired memory function at 1 month after BCAS surgery, with the highest white matter damage score on LFB staining and the most significant reduction in the fluorescence intensity of myelin basic protein(MBP). Sholl analysis revealed the most pronounced activation of microglia.(2)Compared with the model group, the fostamatinib treatment group showed improved reference memory in mice,a reduced white matter damage score, and decreased microglia activation according to Sholl analysis.Immunofluorescence staining demonstrated a significant decrease in the expression of the pro-inflammatory factors NLRP3 and SYK(P<0.05).(3) In vitro, fostamatinib treatment significantly reduced the expression of inflammatory-related factors such as NLRP3, SYK, ASC, Caspase-1, and IL-18 in microglia after myelin treatment(P<0.05). Consistent with these findings, knocking down SYK using siRNA also resulted in downregulated expression of inflammatory factors(P<0.05).Conclusion: Fostamatinib may improve the activation of the NLRP3 inflammasome inflammatory pathway in microglia by inhibiting SYK, thereby reducing neuroinflammatory responses, alleviating myelin damage, promoting structural reconstruction after cerebral white matter ischemia, and facilitating the recovery of cognitive function.更多还原