针刺通过Notch-1信号通路调控星形胶质细胞活化改善局灶性脑缺血神经功能机制研究

It is definite that acupuncture exert a enormous effects on the nervous functional deficiency of cerebral ischemia whereas the mechanisms remain unclear. Our previous studies clearly showed that the improvement of neural function is related to the astrocytes activation regulated by acupuncture. Recent studies also showed that the Notch-1 signaling pathway is closely related to astrocytes activation. We speculate that the mechanism of acupuncture regulating the activation of cerebral ischemic astrocytes to improve the nerve function is related to the role of this pathway. In order to verify the above hypothesis, this project aims to establish the MCAO model, using CATWALK gait analysis system to observe electroacupuncture effects on neural function damage, observe the damage of focal ischemia with small animal magnetic, the mRNA and protein expression of Jagged-1, Notch-1, GFAP, Hes-5 will be tested and detected. The effect of Notch-1 signaling pathway was discussed by lateral ventricle injection with agonist Jagged-1, inhibitor DAPT respectively. This project will contribute to interpret the mechanisms of electroacupuncture effects on astrocytes activation and on promotion of neural function recovery after cerebral ischemia, and provide new academic insights and theoretical basis for acupuncture treatment of stroke.

针刺可有效改善缺血性脑卒中神经功能缺损，但相关机制尚待阐明。通过前期研究发现，针刺改善神经功能的作用与其调控星形胶质细胞的活化有关。最新的研究表明，Notch-1信号通路与星形胶质细胞活化关系密切。我们推测，针刺调控脑缺血星形胶质细胞活化改善神经功能的机制与该通路的作用有关。为验证上述假说，本项目拟建立MCAO模型，利用CATWALK步态分析系统等观察针刺对神经功能的影响，利用小动物核磁观察缺血灶损伤，运用免疫荧光、qRT-PCR及Western blotting检测GFAP以及Notch-1信号通路分子mRNA及蛋白的表达情况；从正、反两方面探讨Notch-1信号通路作用，阐释针刺调控星形胶质细胞活化、促进脑缺血后神经功能缺损恢复的分子机制，为针刺治疗脑卒中提供新的学术见解和理论依据。

本项目按照项目计划，在针刺内关穴具有确切的神经功能保护效应基础上，以课题组的前期工作为基础，结合最新的关于缺氧时星形胶质细胞活化调控机制的研究成果，验证了电针通过 Notch-1通路调控星形胶质细胞的活化，从而减轻脑组织损伤，改善神经功能缺损的研究假说。. 研究结果显示，针刺是改善缺血性脑卒中神经功能缺损的有效手段，经针刺治疗后，MCAO模型大鼠的神经体征、走横木、步态、旷场等神经功能评分情况明显改善。在此基础上，利用免疫荧光双标开展的 Notch-1通路调控星形胶质细胞活化研究结果显示，针刺一方面可以调控星形胶质细胞细胞的活化，另一方面，可通过定位于星形胶质细胞内Notch-1信号通路实现对细胞活化的调控。项目利用RT-PCR、Western Blot、通路兴奋剂及通路抑制剂等方法进行的研究均证实了免疫荧光的研究结果。. 本项目通过对电针调控脑缺血星形胶质细胞细胞活化影响的相关机制展开研究与探讨，阐明了Notch-1信号通路在针刺调控星形胶质细胞活化的作用机制，上述研究工作的开展及成果将为针刺治疗缺血性脑中风的机制研究提供新的思路与靶点。