从ROS介导的PINKl/Parkin线粒体自噬途径探讨糖络宁治疗糖尿病周围神经病变的作用机制

Diabetic peripheral neuropathy (DPN) has high incidence and high disability rate, currently there is lacking of generally accepted therapeutic treatments. This research group has been using Tang Luo Ning with the functions of replenishing qi and nourishing yin, activating blood and resolving stasis for DPN treatment for many years, the curative effect of which is definite. Preliminary study has shown that Tang Luo Ning can significantly reduce peroxide injury of sciatic nerve, and after the drug intervention the expression quantity LC3 is closely related to ROS within the schwann cells. The latest research suggests that mitophagy is closely related to the improvement of oxidative stress. PINK1/Parkin signaling pathways is the key regulating pathway of mitophagy. The problem to solve here is whether Tang Luo Ning treats DPN by regulating mitophagy and maintaining metabolic balance of mitochondrial through the intervention of PINK/Parkin pathway induced by ROS, so as to improve oxidative stress, and to restrain and mitigate the death of nerve cells. This study proposes to use the combination of in vivo and in vitro experiemnts and the experimental techniques such as immunofluorescence, transmission electron microscope, western blot, etc, to observe the impact of Tang Luo Ning on sciatic nerve and the functional activity of the mitochondria of schwann cells, ROS expression, mitophagy, and PINK/Parkin pathway, in order to explore the mode of action of Tang Luo Ning’s treatment on DPN, and to provide experimental basis for DPN treatment of replenishing qi and nourishing yin, activating blood and resolving stasis.

糖尿病周围神经病变（DPN）发病率高、致残率高，目前缺乏公认有效的治疗措施。本课题组多年来采用益气养阴、化瘀通络的中药糖络宁治疗DPN疗效确切。前期研究表明，糖络宁能明显减轻坐骨神经的过氧化损伤，该方干预后的施万细胞中ROS与LC3表达量密切相关。最新研究提示线粒体自噬与改善氧化应激密切相关，PINK1/Parkin是调节线粒体自噬途径的关键信号通路。糖络宁是否通过干预ROS介导的PINKl/Parkin信号通路来调控线粒体自噬，维持线粒体自身代谢平衡，从而改善氧化应激，抑制或减缓神经细胞的死亡来治疗DPN是本课题要解决的科学问题。本实验拟采用体内、体外实验相结合，应用免疫荧光、透射电镜、蛋白印迹等技术观察糖络宁对坐骨神经及施万细胞线粒体功能活性、ROS表达、线粒体自噬、PINKl/Parkin信号通路的影响，探究糖络宁治疗DPN的作用机制，为益气养阴、化瘀通络法治疗DPN提供实验依据。

【项目背景】糖尿病周围神经病变（DPN）发病率高、致残率高，目前缺乏公认有效的治疗措施。本课题组多年来采用益气养阴、化瘀通络的中药糖络宁治疗DPN疗效确切。前期研究表明，糖络宁能明显减轻坐骨神经的过氧化损伤，该方干预后的施万细胞中ROS与LC3表达量密切相关。最新研究提示线粒体自噬与改善氧化应激密切相关，PINK1/Parkin是调节线粒体自噬途径的关键信号通路。糖络宁是否通过干预ROS介导的PINKl/Parkin信号通路来调控线粒体自噬，维持线粒体自身代谢平衡，从而改善氧化应激，抑制或减缓神经细胞的死亡来治疗DPN是本课题要解决的科学问题。【主要研究内容】本实验拟采用体内、体外实验相结合，应用免疫荧光、透射电镜、蛋白印迹等技术观察糖络宁对坐骨神经及施万细胞线粒体功能活性、ROS表达、线粒体自噬、PINKl/Parkin信号通路的影响，探究糖络宁治疗DPN的作用机制，为益气养阴、化瘀通络法治疗DPN提供实验依据。【重要结果】血糖代谢指标和坐骨神经鼠尾热痛阈值检测、坐骨神经神经传导速度测定、神经纤维轴索染色结果提示糖络宁能降低血糖、改善DPN模型大鼠周围神经损伤状态；糖络宁可以降低DPN模型大鼠血清及坐骨神经中ROS含量、改善线粒体膜电位；糖络宁可以降低高糖培养下的施万细胞LC3-I、LC3-II、PINKl、Parkin蛋白表达、减少细胞自噬。【关键数据及科学意义】本实验研究结果证实，糖络宁可有效改善氧化应激对坐骨神经及施万细胞线粒体功能的影响，糖络宁通过PINKl/Parkin信号通路影响施万细胞线粒体自噬。