基于NMDA受体/KCC2通路的栝楼桂枝颗粒调控脊髓运动神经元过度兴奋抗脑卒中后肢体痉挛的机制研究

The over-excitation of motor neurons in the spinal cord causes the hyperfunction of myotatic reflex, which is the main reason of spasticity after cerebral ischemia. An important mechanism underlying the over-excitation of motor neurons is the activated NMDA receptor mediated down-regulation of the cation-chloride cotransporters KCC2 caused the turnover of the GABA function from inhibition to excitation. Gualou Guizhi Granule (GLGZG) is the effective remedy to treat post ischemia spasticity. Out preliminary work revealed that the prescription alleviated the hyperfunction of the myotatic reflex, but the mechanism underlying the effect remain unclear. The present project is designed to verify the effects of GLGZG on the hyperfunction of the myotatic reflex in a rat model of post-ischemic spasticity, and explore the effects of the prescription on the over-excitation of the spinal motor neuron using immunofluorescence and laser confocal microscope. Then the spinal glutamic acid and its receptor NMDA receptor, KCC2 and pKCC2 are determined in their mRNA level and protein expression to explore the effects of the prescription on NMDAR/KCC2 pathway. Then the chloride ion homeostasis is detected to clarify the modulation of the prescription on the KCC2 function by confocal living cell analysis. At last, the antagonist to KCC2 is utilized to further analyze the effect of the NMDAR/KCC2 pathway in the effects of the prescription. The project is designed to elucidate the molecular mechanism of the anti-spasticity effects of GLGZG on the post-ischemic spasm, to provide theoretical foundation for its clinical application.

缺血性脑卒中后脊髓运动神经元（MN）过度兴奋导致牵张反射亢进是发生肢体痉挛的主要原因，而NMDA受体过度活化使K+-Cl-联合转运体KCC2下调，导致GABA能神经兴奋性翻转是MN过度兴奋的重要机制。栝楼桂枝颗粒是治疗脑卒中后肢体痉挛的有效制剂，前期研究表明其可减轻脑缺血后牵张反射，但对脊髓MN的调控作用及机制不明。本项目拟在大鼠脑缺血后肢体痉挛模型上明确栝楼桂枝颗粒对脊髓牵张反射作用的量效和时效规律；继而采用免疫荧光结合激光共聚焦表征该方对MN过度兴奋的作用；结合谷氨酸及NMDA受体、KCC2/pKCC2mRNA和蛋白测定，探讨该方对NMDA受体/KCC2通路的作用；采用激光共聚焦活细胞分析检测MN氯离子稳态，明确该方对KCC2功能的调控；最后进行KCC2阻断，进一步分析NMDA受体/KCC2通路在该方药效中的作用。阐明该方调控MN过度兴奋缓解肢体痉挛的分子机制，为其临床应用提供理论依据

脑缺血再灌注后脊髓运动中枢过度兴奋导致牵张反射亢进是缺血性脑卒中后肢体痉挛发生的直接原因。栝楼桂枝汤是中医临床缓解肢体痉挛症状的一种补充替代疗法。栝楼桂枝颗粒是治疗脑卒中后肢体痉挛的有效制剂。本项目证实了栝楼桂枝颗粒显著降低脑缺血损伤后脊髓前角运动神经细胞的兴奋性，并从脊髓NMDA受体/KCC2通路的变化角度探讨了其机制。研究发现，栝楼桂枝颗粒显著降低脑缺血损伤后脑脊液兴奋性氨基酸，提高抑制性氨基酸含量。脑缺血损伤后，患侧脊髓组织KCC2显著下调，细胞内氯离子浓度显著升高，栝楼桂枝颗粒可显著逆转KCC2的下调，降低细胞内氯离子浓度，降低脊髓神经细胞兴奋性。同时，NMDA受体亚型NMDAR1、NMDAR2A/B以及Glutamate2在脑缺血损伤的脊髓组织中显著上调，栝楼桂枝颗粒可降低NMDA受体亚型的升高。通过本课题的研究，我们证实了脊髓组织可能是栝楼桂枝颗粒发挥解痉作用的重要靶器官。缺血性脑卒中发生后，兴奋性氨基酸大量释放，脊髓运动神经元 NMDA受体过度激活，导致 KCC2 功能下降，细胞内氯离子外排受阻，GABA 能神经信号由抑制性翻转为兴奋性，使运动神经元过度兴奋，加重牵张反射的亢进；而栝楼桂枝颗粒可能通过抑制 NMDA 受体的激活恢复 KCC2 功能，降低运动神经元过度兴奋，减轻牵张反射亢进，从而缓解肢体痉挛。.本项目的研究结果不仅可为该方治疗缺血性脑卒中后肢体痉挛的临床应用提供理论依据，还可以为该方治疗其他疾病所致的肢体痉挛症状提供理论基础。进一步研究将继续深入探讨该方何种成分对NMDA受体/KCC2通路起到主要作用，为其开发为治疗肢体痉挛的中药新药奠定理论基础。