基于miRNAs介导突触可塑性探讨电针改善缺血性脑卒中后学习记忆障碍的作用机制

The learning and memory impairment is a core symptom of cognitive impairment after ischemic stroke, and closely related to the synaptic plasticity. The effect of electroacupuncture(EA) on learning and memory impairment by stimulatiing in Baihui and Shenting acupoints is clear in clinical, but its mechanism is uncertainty. Previous researches have shown that between of miRNAs and NMDA receptors crossing talk were critical molecule of the synaptic plasticity. Therefore EA stimulate which brain regions to produce effect of miRNA mediated synaptic plasticity? Therefore, to build MCAO model in rats in this project, stimulate in Baihui and Shenting acupoints by EA, observe of learning and memory behavior, identify of EA stimulating active brain regions by using small animal MRI imaging system. Using of electron microscopy methods to distinguish of synaptic plasticity in the structure, and the synaptic plasticity in the function by observing of LTP or LTD phenomenon by electrophysiological techniques and calcium activity by detecting of laser confocal scanning microscopy (LCSM). Biological chip screening different miRNAs and verify its function of interacting with the NMDA receptor in vitro by using of biologic methods and to identify miRNA and NMDA receptor signaling pathways in the function of synaptic plasticity in acupuncturing related to brain regions in vivo. To illuminate that improve learning and memory impairment by EA stimulation in miRNAs-NMDA receptor signaling pathway regulating of LTP or LTD and synaptic plasticity.

学习记忆障碍是缺血性脑卒中后认知障碍的核心症状，与突触可塑性的变化紧密相关。临床上电针百会、神庭穴对其疗效明确，但具体机制尚未清楚。研究已表明miRNAs与突触可塑性的关键分子NMDA 受体cross talk，那么电针刺激哪些脑区产生效应miRNAs介导突触可塑性呢?因此，本课题拟通过构建MCAO大鼠模型，进行电针百会、神庭穴治疗，观察大鼠学习记忆行为学改变；利用小动物MRI成像分析系统探明电针刺激的脑区；采用电镜观察突触结构的可塑性；应用电生理技术观察LTP/LTD现象以及LSCM技术检测它们中钙变化揭示突触功能的可塑性；针对脑激活和抑制脑区利用生物芯片筛选效应miRNAs分子，利用生物学技术体外鉴定miRNAs与NMDA受体相互作用并体内验证它们在突触可塑性中发挥的功能。阐明电针刺激产生的特异性miRNAs介导NMDA受体信号通路调控LTP/LTD和突触可塑性，从而改善学习记忆障碍。

电针“百会”、“神庭”穴治疗缺血性脑卒中模型大鼠14天，跳台实验和Morris水迷宫行为学实验证实电针提高学习记忆能力，T2WI显示海马、内嗅皮层等脑区梗死体积减少，fMRI分析发现电针干预后双侧杏仁核、双侧海马、右侧岛叶皮层、双侧内嗅皮层等脑区BOLD信号局部一致性升高。进一步高尔基染色显示电针治疗缓解海马CA1、内嗅皮层树突棘脱落、萎缩，电生理检测发现其fEPSP 斜率的百分比增加，突触后膜相关学习记忆蛋白AMPA受体、NMDA受体的磷酸化水平升高，提示电针“百会”、“神庭”穴促进缺血性脑卒中模型大鼠缺血侧海马、内嗅皮层树突棘形态的修复和易化海马CA3-CA1和内嗅皮层-海马CA1的LTP反应，促进突触结构和功能的可塑性。miRNA芯片显示：缺血侧海马CA1差异表达≥1.5 miRNAs共48个miRNAs，其中36个miRNAs表达下调，12个miRNAs表达上调；缺血侧内嗅皮层差异表达≥1.5 miRNAs 共90个，其中70个miRNAs 表达下调，20个miRNAs表达上调；其中rno-miR-668和rno-miR-3084b-5p在缺血侧海马CA1和内嗅皮层均表达上调；而rno-miR-28，rno-miR-10a，rno-miR-let-7b，rno-miR-425，rno-miR-7b，rno-miR-154，rno-miR-540，rno-miR-let-7f-2，rno-miR-186，rno-miR-219a，rno-miR-3072，rno-miR-344a，rno-miR-190b，rno-miR-100，rno-miR-92a的表达水平在两个脑区均下调；两个脑区均上调和下调的miRNAs进行生物信息学分析，cGMP-PKG信号通路参与学习记忆功能，体外证实miR-219a负调控其信号通路关键节点PRKG2靶基因；在体miR-219a mimics注射可阻断电针下调miR-219a的效应，并影响PRKG2下游CREB磷酸化，进而减少学习记忆即刻早期基因c-fos和c-jun的表达，提示电针“百会”、“神庭”穴抑制模型大鼠miR-219a的表达，上调PRKG2，促进CREB的磷酸化，从而促使学习记忆相关蛋白的合成。此外，电针百会、神庭穴可能通过调控海马CA1区miR-134，促进LIMK1磷酸化，改善树突棘形态，进而改善空间学习记忆功能。