从星形胶质细胞谷氨酸代谢通路探讨针刺治疗缺血性脑中风的作用机制

Excitatory toxicity of glutamate play main role in ischemic stroke, astrocyte can inhibit the release of glutamate through the glutamate cycle of glutamate-glutamine,in which GLT-1 and GS are key components: GLT-1 is responsible for transport and GS is responsible for degradation. It was shown that up-regulation of GLT-1 may reduce the glutamic acid concentration, and the phosphorylation of p38MAPK is closely related to it. Our pioneer study also found that acupuncture at Neiguan can facilitates proliferation of astrocytes as well as drastically reduced the excessive brain glutamate content in MCAO rats, thus we speculated that the mechanism of acupuncture treatment of cerebral ischemia may be related with astrocytic glutamate metabolism. We will construct MCAO modle rats and interfering the phosphorylation of p38MAPK and the key components of glutamate-glutamine cycle such as GLT-1 and GS by its’inhibitors, to verify the hypothesis that “Acupuncture can regulate astrocytic glutamate metabolism pathway in the process of cerebral ischemic, which may mediate its role against excitotoxicity caused by glutamate, and the effect is highly associated with the phosphorylation of p38MAPK.” This project will be not only a further study on our previous research, but also provide new experimental basis and research thought for clarifying protective effect of acupuncture on ischemia stroke.

谷氨酸的兴奋性毒性是缺血性脑中风的主要病理机制之一，星形胶质细胞可通过谷氨酸-谷氨酰胺循环降低谷氨酸浓度，谷氨酸转运体1（GLT-1）负责转运，谷氨酰胺合成酶（GS）负责降解。研究表明p38MAPK的磷酸化在脑缺血时与GLT-1的表达关系密切。项目组前期实验结果显示，针刺内关可以促进缺血脑组织星形胶质细胞的活化，降低谷氨酸含量，由此推测针刺治疗脑缺血的作用机制可能与星形胶质细胞的谷氨酸代谢通路有关。本项目拟建立MCAO大鼠模型，利用GLT-1抑制剂、GS抑制剂以及p38MAPK抑制剂分别对谷氨酸-谷氨酰胺循环的关键环节及p38MAPK的磷酸化进行干预，验证“针刺可能通过调控星形胶质细胞谷氨酸代谢通路而减轻脑缺血引起的谷氨酸兴奋性毒性，该调控作用与p38MAPK的磷酸化密切相关”的假说，本项目既是对以往研究的深入，也为阐明针刺治疗缺血性脑中风的作用机制提供新的实验依据和研究思路。

本项目采用线栓法建立大脑中动脉栓塞（MCAO）大鼠模型，通过检测大鼠神经体征评分、神经元的坏死及凋亡、脑梗塞体积，研究针刺内关穴对脑功能恢复的影响；通过检测星形胶质细胞谷氨酸代谢通路关键环节GLT-1、GS的表达及脑组织Glu、Gln的含量，研究针刺内关对脑缺血后谷氨酸-谷氨酰胺循环的作用；通过检测p38MAPK、p-p38MAPK的表达，研究针刺通过p38MAPK磷酸化对星形胶质细胞谷氨酸代谢的调控作用。并分析各因素之间的关系，探讨针刺内关穴影响脑缺血星形胶质细胞谷氨酸代谢通路的作用机制，为阐明针刺治疗缺血性脑中风的保护机制提供新的研究思路和实验依据。.结果显示：.1. 针刺内关穴能显著改善MCAO大鼠神经功能损伤，有效促进其精细运动功能的恢复；.2. MCAO大鼠脑组织中Glu、GS及Gln的含量异常升高，针刺内关穴早期（第3天）即能够显著降低MCAO大鼠脑组织中Glu、GS及Gln的含量，该作用可持续至针刺第7天，至针刺14天时效果不明显；.3. MCAO大鼠脑组织中GLT-1蛋白表达显著降低，针刺内关穴能够显著提高MCAO大鼠脑组织中GLT-1的蛋白表达，该作用在针刺第7天时最为明显；.4. MCAO大鼠早期（第3天）脑组织中P38MAPK、p-P38MAPK蛋白表达明显升高，至脑缺血恢复期（14天）p-P38MAPK反而明显降低，而针刺内关穴在恢复期可显著提高MCAO大鼠脑组织中P38MAPK、p-P38MAPK的蛋白表达。