糖痹康调节AMPK/PGC-1α/SirT3信号通路干预线粒体应激防治糖尿病周围神经病变的机制研究

Diabetic peripheral neuropathy (DPN) has a high morbidity and disability rate. Traditional Chinese Medicine (TCM) has a significant effect for on TangBiKang (PATENT NO: ZL200810167551.1), which is valid in clinical medicine, could protect nerve of DPN by decreasing Schwann cells apoptosis which is due to oxidative stress. The updated pathology research has indicated that mitochondrial stress is at the core in the course of the occurrence and development of DPN. Regulating the AMPK/PGC-1α/Sirt3 signaling pathway to remitting mitochondrial stress injury is the key molecular mechanism of the treatment for DPN. Then “TangBiKang could anti-oxidative stress to induce the apoptosis of Schwann cells, is that related to the mechanism that TangBiKang can regulate the AMPK/PGC-1α/Sirt3 signaling pathway to release mitochondrial stress?” The pre-experimental had found that TangBiKang may protect the rats’ nerve which had get DPN by regulating fatty acid degradation and metabolism. As a result, we regard mitochondrial stress of Schwann cell as a starting point to discuss the mechanism of the effect of protection for DPN in vivo and cell level by lipidomics technology and mitochondrial defect in animal model. So that we can provide a basis for theoretical and experimental evidence of the TangBiKang, offer new ideas for the study on curing DPN by TCM.

糖尿病周围神经病变（DPN）发病率和致残率高，中医药对改善DPN临床症状疗效显著，科学内涵亟待阐明。前期研究临床有效中药方糖痹康（专利号：ZL200810167551.1）可通过减少氧化应激引起的雪旺细胞凋亡实现DPN神经保护作用。最新研究表明，线粒体应激在DPN发生发展过程中处于核心地位，调节AMPK/PGC-1α/SirT3信号通路改善线粒体应激损伤是治疗DPN的关键分子机制，那么“糖痹康抗氧化应激诱导雪旺细胞凋亡的作用是否与调节AMPK/PGC-1α/SirT3信号通路减轻线粒体应激的分子机制相关”？预实验发现糖痹康可通过调节脂肪酸降解及代谢对DPN大鼠起到神经保护作用。由此我们以“雪旺细胞线粒体应激”为切入点，利用脂质组学等技术和线粒体缺陷动物模型，拟从整体和细胞水平探讨糖痹康对DPN神经保护作用机理，以期为糖痹康治疗DPN提供理论和实验依据，为中药抗DPN研究提供新思路。

糖尿病周围神经病变（DPN）是世界范围内最常见的神经病变形式，影响约50%的糖尿病患者。目前临床针对DPN的主要治疗方式是对症治疗，仅能缓解疼痛，无法延缓神经病变的进展。有研究表明线粒体应激在DPN发生发展过程中处于核心地位，调节AMPK/PGC1α/SirT3信号通路改善线粒体应激损伤是治疗DPN的关键分子机制。中医药在DPN的防治方面具有丰富经验，团队前期多项临床和基础实验研究已证明糖痹康可以有效地减轻DPN中的氧化应激损伤，修复坐骨神经，减少雪旺细胞凋亡。但糖痹康减轻糖尿病大鼠坐骨神经炎症的具体机制尚不明确。本课题分别通过链尿佐菌素联合高脂饲料诱导和自发性2型糖尿病ZDF大鼠建立两种DPN大鼠模型，分别观察糖痹康对大鼠的一般状态、行为学检测、坐骨神经病理结构及相应蛋白表达水平的干预作用，探讨糖痹康调节AMPK/ PGC-1α/ SirT3通路干预线粒体应激对DPN的神经保护作用。体内实验均表明糖痹康能够抑制线粒体应激、提高坐骨神经传导速度和血流量、改善肢体远端神经感觉、改善神经病理损伤，并通过激活AMPK/ PGC-1α/ SirT3通路对神经起保护作用。通过PGC-1αsi RNA、SirT3 siRNA 和 TFAM siRNA 三个细胞转染实验分别探讨中药复方糖痹康治疗DPN 的关键作用靶点。通过制备糖痹康含药血清、培养雪旺细胞，分别施加PGC-1α si RNA、SirT3 siRNA 和 TFAM siRNA因素分组实验。结果表明施加PGC-1αsi RNA、SirT3 siRNA 和 TFAM siRNA后细胞增殖降低，活性氧、凋亡率、氧化应激增加，线粒体膜电位、细胞能量代谢明显降低，相应通路蛋白的基因表达、雪旺细胞胞质及线粒体的蛋白表达受抑制。糖痹康含药血清给药干预后，细胞增殖增加，活性氧、凋亡率、氧化应激降低，线粒体膜电位、细胞能量代谢升高，相应通路蛋白的基因表达、雪旺细胞胞质及线粒体的蛋白表达明显增多。综上，体内实验表明糖痹康通过激活AMPK/ PGC-1α/ SirT3通路对神经起保护作用；细胞实验表明PGC-1α、SirT3、TFAM分别可作为糖痹康治疗DPN 的关键作用靶点。本研究通过体内、体外实验阐明了糖痹康对DPN 神经保护作用机理，为中医药治疗DPN提供更多依据。