基于miR-181b/PKC信号轴介导NVU稳态探讨巨刺法改善MCAO大鼠运动功能的作用机制

Motor dysfunction is a major symptom of ischemic cerebral apoplexy, which is closely related to the homeostasis of neural vascular unit (NVU). The phosphorylation of Cx43ser368 sites is the key to the maintain the homeostasis of NVU, miR-181b/PKC signal axis can promote phosphorylated modification of Cx43ser368 site. Opposing needling have curative effect to improve motor dysfunction after ischemic stroke, previous study found Opposing needling can improve PKC protein concentration in white matter of the MCAO rat, increase the expression of astrocytes marker which were the NVU important components, but the mechanism was unclear. This study on the basis of the homeostasis of NVU modulated by miR-181b/PKC axis targeted Cx43ser368 phosphorylation, opposing needling will be adopted to treat MCAO rats, to observe the exercise behavior changes, DTI tracked white matter changes in nerve fiber bundles, then take the bilateral white matter, laser confocal observation the express changes with neurons and microglia, radial glial cells and endothelial cells, Western blot will be used to detect the changes of proteins(APQ4, ZO-1, occludin), Real-time fluorescence quantitative to test miR-181b expression level, ELISA to detect concentration changes of PKC, so as to clarify the mechanism of opposing needling in improving motor dysfunction after ischemic stroke.

运动功能障碍是缺血性脑卒中的主要症状，与神经血管单元（NVU）稳态紧密相关。Cx43 ser368位点磷酸化是维持NVU稳态的关键，miR-181b/PKC信号轴可促进Cx43ser368位点磷酸化修饰。巨刺法改善缺血性脑卒中后运动障碍疗效确切，我们前期研究发现巨刺法可提高MCAO模型鼠白质PKC蛋白浓度，上调NVU重要组份星形胶质细胞表达，但机制不清。故本项目以miR-181b/PKC轴调控Cx43ser368磷酸化参与维持NVU稳态为切入点，对MCAO模型鼠进行巨刺干预，观察运动行为改变，DTI追踪白质神经纤维束变化，取双侧脑白质，激光共聚焦观察神经元、小胶质细胞、星型胶质细胞、内皮细胞表达改变，Western blot检测APQ4、ZO-1、occludin蛋白变化，实时荧光定量检测miR-181b表达水平，ELISA检测PKC浓度变化，阐明巨刺改善缺血性脑卒中后运动障碍的作用机制。

临床证实巨刺法改善缺血性脑卒中后运动功能障碍疗效确切，有文献资料通过动物实验肯定了巨刺的脑保护效应，但其中枢作用机制尚无统一定论。本课题制备改良的MCAO模型鼠模拟临床急性脑缺血再灌注损伤病理，对MCAO模型大鼠进行巨刺法干预，观察大鼠行为学、功能核磁信号（DTI）、脑白质神经血管单元各组份（βIII-Tublin、Iba1、GFAP、CD31）及其缝隙连接功能（ZO-1、occludin、AQP4）、Cx43 ser368位点磷酸化程度、miR-181b/PKC信号通路标志蛋白表达之间的关系，结果发现巨刺法可明显改善大鼠的神经行为学评分，提高大鼠的运动能力，促进脑组织纤维束完整性，通过调控miR-181b/PKC信号通路，促进Cx43 ser368位点磷酸化进而调整缝隙连接功能，恢复MCAO大鼠脑白质神经血管单元稳态。