电针“委中穴”对多裂肌卫星细胞增殖过程中的自噬调控

Low back pain is always accompanied by the injury of multifidus muscles, which are located under erector apinae and vital to stabilize lumbar vertebra. Acupuncture is an effective treatment for low back pain, and a majority of researches have indicated that acupuncture could markedly accelerate repairation of damaged muscles through promoting activity and proliferation of muscle satellite cells, while the mechanism is still to be elucidated. Autophagy is a potent way to maintain cell stability and protect cells against nutrient stress while excessive autophagy could reduce cell vitality. Recently, we find increased expression of IGF-1 and decreased formation of autophagosome in multifidus muscle satellite cells after electroacupuncture at “Weizhong” acupoint, and we speculate that electroacupuncture at “Weizhong” acupoint can produce IGF-1 and positively regulate proliferation of muscle satellite cells and accelerate repairation of damaged multifidus muscles through inhibition of excessive autophagy induced by activating PI3K/Akt/mTOR signaling pathway. Consequently, we will verify the effect of electroacupuncture at “Weizhong” via rats experiment and cell culture. In detail, we apply methods such as transmission electron microscope, morphology and molecular biological techniques to study morphology of multifidus muscles during the process of repairation, formation of autophagosome and expression of autophagy marker proteins and autophagy-related signal molecule in proliferative satellite cells. We expect this work can attribute to clarify the classic theory “treating lumbar-back problems by Weizhong” from a new perspective.

位于腰部竖脊肌深面的多裂肌是稳定腰椎的“钢索”，多裂肌的损伤、萎缩等功能紊乱可以影响腰椎稳定性，引发腰痛。针刺治疗腰痛疗效显著，大量研究已证实针刺可显著促进肌肉干细胞——肌卫星细胞的增殖，加速肌肉修复，但其机制尚未明了。自噬是维持细胞稳态和抵抗逆境的重要机制，自噬过度可降低细胞活力。近来，我们发现电针委中穴可促进损伤多裂肌卫星细胞中IGF-1表达，同时自噬体形成明显减少。我们的科学设想是，电针委中穴可抑制自噬过度表达，促进肌卫星细胞增殖，可能与IGF-1介导PI3K/Akt/mTOR信号通路有关。因此，我们在大鼠多裂肌损伤模型和原代培养肌卫星细胞体系两个层面，以电针委中穴为干预手段，通过透射电镜、形态学、分子生物学等技术，观察多裂肌修复过程中形态学、肌卫星细胞自噬体的形成和自噬标志蛋白的表达，并应用PI3K抑制剂探讨自噬相关信号分子的变化，从新视角阐释“腰背委中求”这一针灸理论的作用机理。

中文摘要：.委中穴在治疗腰痛方面有独特的疗效，但其作用机制尚不清楚，本课题从PI3K/Akt/mTOR 信号通路抑制自噬环节这一角度出发，借助透射电镜、免疫组化等现代技术进行在体和离体的实验，从多方面阐释电针委中穴治疗腰多裂肌损伤的作用机制。研究结果如下：.1..评价局部肌肉注射布比卡因不同时间点大鼠多裂肌组织形态和超微结构变化。.2..建立大鼠腰多裂肌卫星细胞分离、纯化、培养及鉴定方法。.3..电针“委中”穴可促进IL-17和血清CK的表达下调，减轻炎症反应的程度，同时可增加 LIF 的表达含量，促进骨骼肌中葡萄糖的摄取，利于骨骼肌修复。.4..多裂肌损伤早期，电针“委中”穴干预可通过抑制 CaM、CaMKⅡ的过度激活，减少组织中 iNOS 的过多产生，减轻组织炎症损伤。.5..电针“委中”穴可促进自噬蛋白Beclin1的表达上调，提高自噬水平，促进 p62 蛋白的分解，有利于骨骼肌损伤后的修复。.6..电针“委中”穴通过调节 Desmin 的蛋白表达，加快损伤修复，促进再生肌纤维趋向成熟。.7..电针“委中“穴可在早期增加IGF1-R的表达，并下调IGFBP3的表达，促进多裂肌损伤后的修复。.8..电针“委中”穴通过良性调节ECM中Collagen I、MMP2的蛋白表达，减轻骨骼肌纤维化程度，促进多裂肌损伤后早期的再生修复。.9..电针“委中”穴对腰多裂肌损伤模型大鼠损伤初期的神经炎症干预有效，与其降低外周和中枢炎性因子IL-6、TNF-α、iNOS 含量，抑制M1型小胶质细胞的过度激活有关。.10..电针“委中”血清和电针“肾俞”血清均可通过磷脂酰肌醇３－激酶－蛋白激酶Ｂ通路促进肌卫星细胞的增殖，且有促进成肌分化的趋势。.11..电针“委中”血清可通过上调 MyoD、CDK4 的表达，并下调 P57 的表达，加速肌卫星细胞增殖的周期性进程，促进损伤多裂肌修复。.12..进行了“针灸治疗腰椎肌筋膜疼痛方案随机对照试验”的系统评价。.13..进行了“布比卡因诱导的形态学变化概述:一种新的骨骼肌损伤动物模型”的系统评价。.结论：由此可知，电针“委中”穴可以调节肌卫星细胞的增殖，促进腰部多裂肌的损伤修复，这可能和激活 PI3K/Akt/mTOR 信号通路有关。