基于急性脊髓损伤微环境调节机制的夹脊电针时效关系研究

Mitochondrial apoptosis pathway and GSK-3βprotein mediated Rho-ROCK II signal pathway is the key link of motor dysfunction after acute spinal cord injury leading to Caspase-3 protein in microenvironment. How to use the best time to adjust the relation between electro-acupuncture on acute SCI after micro environmental changes in neuronal cell apoptosis and promote axon regeneration is the key point of this research. This topic through the establishment of rat model of acute SCI, in order to methylprednisolone as control, using a functional classification microarray advanced techniques, according to the related index of molecular biology, the key target based on Caspase-3 pathway and Rho-ROCK pathway, through the intervention of electro-acupuncture at different time, key targets and the signal pathway, constructing interconnected network system, to elucidate the molecular mechanism of Jiaji electro-acupuncture time-effect relationship, in order to explore the effect of electro-acupuncture and drug interaction, and provide a scientific basis for clinical practical value optimization of Jiaji electro-acupuncture treatment of acute SCI scheme.

微环境中Caspase-3蛋白参与细胞凋亡线粒体通路及GSK-3β蛋白介导Rho-ROCKⅡ信号通路是导致急性脊髓损伤（spinal cord injury，SCI）后肢体运动功能障碍的关键环节，如何利用最佳时效关系调节夹脊电针在急性SCI后微环境变化中减少神经细胞凋亡及促进轴突再生是本课题研究关键点。本课题通过建立急性SCI大鼠模型，以甲基强的松龙为对照，运用功能分类芯片先进技术等方法，参照行为学和分子生物学相关指标，着眼于Caspase-3通路和Rho-ROCKⅡ通路的关键靶点，探讨夹脊电针通过干预不同时间、关键靶点及该信号通路，构建相互关联的网络体系，阐明夹脊电针时效关系的分子机制，为探索电针与药物协同作用、优化夹脊电针治疗急性SCI方案提供科学依据和临床实用价值。

急性脊髓损伤（acute spinal cord injury，ASCI）后微环境中细胞凋亡和Rho-ROCKⅡ信号通路介导的轴突再生障碍是导致急性脊髓损伤后肢体运动功能障碍的关键环节，如何利用最佳时效关系调节夹脊电针在ASCI后微环境变化中减少神经细胞凋亡及促进轴突再生是本课题研究关键点。本课题通过建立ASCI大鼠模型，以甲基强的松龙（MP）和Rho-ROCKⅡ信号通路抑制剂为对照，观察电针治疗对ASCI大鼠脊髓组织微环境相关因子表达的影响，结果表明：（1）急性脊髓损伤后6h以内是夹脊电针早期干预时间窗；（2）夹脊电针联合MP能改善ASCI大鼠运动功能评分，减少神经细胞凋亡，减轻脊髓继发性损伤；（3）大鼠急性脊髓损伤后兴奋性氨基酸毒性被激活，抑制ASCI大鼠微环境中calpain-2表达可以减少神经细胞凋亡，提示NMDAR/Calpain通路介导了ASCI大鼠神经细胞凋亡发生；（4）夹脊电针联合MP可以通过下调NMDA-NR1、calpain-2表达，抑制兴奋性氨基酸毒性，从而下调cleaved caspase-3的表达，抑制神经细胞凋亡，改善微环境，减轻脊髓继发性损伤，优于MP治疗组；（5）大鼠急性脊髓损伤后损伤区微环境中髓鞘相关抑制因子释放增加，抑制了轴突再生现象，导致了脊髓继发性损害；（6）ASCI后损伤区内，髓鞘相关抑制因子被有效激活，促进ASCI大鼠损伤区微环境中ROCKⅡ的表达，抑制轴突再生，提示Rho-ROCKⅡ信号通路介导ASCI大鼠轴突抑制因子的释放，从而抑制轴突再生；（7）ROCKⅡ抑制剂（Fasudil）通过抑制ROCKⅡ的活性，从而阻断ROCKⅡ激活作用底物，改善脊髓损伤后轴突再生障碍，促进脊髓损伤神经功能恢复；（8）夹脊电针通过抑制RhoA、Nogo-A、ROCKⅡ、MLC抑制因子在大鼠急性脊髓损伤后微环境中的表达，促进轴突再生，减轻脊髓继发性病理损害，促进受损神经功能恢复。上述研究结果对阐明夹脊电针时效关系的分子机制，为探索电针与药物协同作用、优化夹脊电针治疗ASCI方案提供了科学依据，具有重要的临床实用价值。