基于P2Y1受体介导的海马区胶质细胞神经炎性反应调控探讨电针对MCAO大鼠认知功能改善的实验研究

Previous studies have found that electroacupuncture(EA) at Baihui and Shenting can effectively improve cognitive impairment after cerebral ischemia and the underlying mechanisms have strong relationship with the anti- neuroinflammation effect of EA. The recent research showed that ischemia increases the extracellular ATP released by damaged cells, which stimulates astrocytic P2Y1, resulting in high GFAP expression. The inflammatory responses generated by the activated astrocytes disturb synaptic plasticity and may cause a decline in cognitive function . Using a middle cerebral artery occlusion model,we are going to explore the mechanism of electroacupuncture on regulating the cognitive impairment. Hippocampus-dependent cognitive function is evaluated by a contextual fear conditioning test. The glial neuroinflammatory responses are investigated by immunohistochemical evaluation and diffusion tensor MRI (DTI). We used a P2Y1-specific antagonist, a glial ADP/ATP receptor that induces the release of proinflammatory cytokines, to examine the links among P2Y1-mediated JAK/STAT3 signaling, the neuroinflammatory response, and cognitive function and the modulation effect of EA.

课题组前期研究发现电针“百会”“神庭”能有效改善脑缺血后认知功能障碍，但其作用机制尚不明确。近来研究表明脑缺血后细胞外ATP释放增加，刺激海马区胶质细胞P2Y1受体产生，促使胶质细胞活化，前炎性细胞因子大量释放，干扰突触可塑性，进而影响LTP和认知功能。本研究拟通过电针干预局灶性脑缺血MCAO大鼠“百会”“神庭”穴，采用fear conditioning、Barnes迷宫检测大鼠认知功能，弥散张量成像及免疫荧光法检测海马区神经炎性反应相关的结构及细胞改变，ELISA法检测海马区前炎性细胞因子（TNF-α、IL-6、IL-1β）的分泌，Real-time qPCR 和WB检测海马区胶质细胞神经炎性反应重要受体P2Y1及其激活的JAK/STAT3信号通路相关因子的动态表达。以此，基于P2Y1受体介导的海马区胶质细胞神经炎性反应调控探讨电针“百会”、“神庭”对MCAO大鼠认知功能改善的作用机制。

本项目研究发现电针百会、神庭可以改善脑缺血损伤后神经功能缺损，减少脑梗死体积，改善脑缺血损伤后认知功能，学习记忆能力。本研究除了揭示电针调控嘌呤受体P2Y1介导的神经炎性反应，还发现电针可调控另一嘌呤受体P2X7及其介导的神经炎性反应。研究结果表明，脑缺血模型组大鼠左侧缺血周围海马CA1区和内侧前额叶皮质胶质细胞样P2X7R和P2Y1R、活化的小胶质细胞ED1和星形胶质细胞GFAP阳性表达高于假手术组。而电针组大鼠胶质细胞样P2X7R和P2Y1R，以及ED1和GFAP阳性表达较模型组、非穴组减少。脑缺血模型组大鼠左侧缺血周围海马CA1区和内侧前额叶皮质P2X7R+/ED1+、P2X7R+/GFAP+、P2Y1R+/ED1+及P2Y1R+/GFAP+双阳性共标表达高于假手术组。而电针组大鼠P2X7R+/ED1+、P2X7R+/GFAP+、P2Y1R+/ED1+及P2Y1R+/GFAP+双阳性共标较模型组、非穴组降低。模型组大鼠左侧缺血周围海马和内侧前额叶皮质炎症因子1L-1β、TNF-α水平高于假手术组。而电针组大鼠炎症因子1L-1β、TNF-α水平较模型组、非穴组降低。且这些标志性蛋白的表达受到JAK2/STAT3信号通路的调控。与模型组和非穴组大鼠比较，电针组p-JAK2及p-STAT3的表达降低，差异具有统计学意义。因此，电针百会、神庭穴可能通过抑制嘌呤受体P2X7R 、P2Y1R表达，改善缺血再灌注损伤大鼠左侧缺血周围海马和内侧前额叶皮质神经炎症反应，包括减少小胶质细胞、星形胶质细胞活化及炎症因子1L-1β、TNF-α表达，改善MCAO/R大鼠认知功能障碍，这种作用可能与调控JAK2/STAT3信号通路有关。