基于PARP通路探讨益气活血中药治疗糖尿病周围神经病变的分子机制

Diabetic peripheral neuropathy (DPN) is ischemic neuronal injury caused by hyperglycemia. Intracellular oxidative stress induced by high glucose involved in the injury process. Activation of ROS, PKC and NF-κB is three key factors for oxidative stress pathway, and the activation of PARP is one of the key factor of DPN. To aim at DPN pathogenesis of blood stasis due to Qi deficiency, we have over 20 years of clinical research in application of replenishing Qi and activating blood traditional Chinese medicine in treating DPN. Through the previous research it was found that the prescription may reduce the expression of inflammatory factors of DPN nerve tissue, and this effect is closely correlated with PARP, so it is speculated that the possible mechanism in treatment of DPN is to play a role through effecting activation of PARP, ROS, PKC, and NF-κB, inhibiting PARP activation may be the key of this prescription. By immunohistochemistry, electron microscopy, Laser Confocal, RT-PCR, electrophysiological techniques, exploring the treatment of DPN clinical features and molecular mechanisms from an overall, cellular and molecular levels at different aspects done in this research to verify the objectivity of the hypothesis. A new theoretical and experimental basis for DPN prevention and cure through replenishing Qi and activating blood by traditional Chinese medicine provided in this research, and new ideas for inheritance of experience of famous veteran TCM doctors offered.

糖尿病周围神经病变(DPN)是高血糖导致的缺血性神经细胞损伤。高糖引起的细胞内氧化应激参与了损伤过程。ROS、PKC、NF-κB的活化是氧化应激通路的三个关键因素,而PARP活化是DPN发病的关键因素之一。针对DPN气虚血瘀病机，我们应用益气活血中药治疗DPN已有20多年临床研究积累。前期研究发现该方可降低DPN神经组织炎性因子表达，此效应与PARP关系密切，故推测该方治疗DPN的可能机制，是通过影响PARP、ROS、PKC、NF-κB的活化而发挥作用，抑制PARP活化可能是该方作用的关键。本研究采用免疫组化、透射电镜、激光共聚焦、RT-PCR、神经电生理等技术，从整体、细胞及分子水平等不同层面探讨该方治疗DPN的疗效特点及分子机制，验证该假说的客观性。结果可为中医益气活血防治DPN提供新的理论和实验依据，为传承名老中医经验提供新思路。

糖尿病周围神经病变（DPN）是糖尿病常见的慢性并发症之一，是糖尿病患者致死致残的重要原因之一。DPN是以高血糖为发病中心环节、神经损伤和血管内皮损伤同时存在所致的糖尿病微血管并发症。线粒体电子传递链产生过氧化物导致的血管损伤加重了DPN。ROS过量产生可以激活聚腺苷二磷酸核糖聚合酶（PARP）和蛋白激酶C，继而激活NF-kB，介导炎症信号转导途径，产生更多氧化应激，导致血管内皮功能障碍。近期研究表明，PARP的活化可以影响细胞能量代谢、信号转导和神经轴突转运，导致细胞代谢障碍及死亡，参与DPN的发病、损伤过程，是引发DPN必需的启动因子和关键步骤。.益气活血中药“糖痛方”是名老中医林兰教授治疗DPN的有效验方。前期实验表明，糖痛方可通过减少雪旺细胞凋亡率与Caspase-3、Caspase-9蛋白及mRNA表达，提高Bcl-2蛋白及mRNA表达，显示出其具有抑制细胞凋亡的作用。研究发现这些结果都与影响氧化应激和炎性因子活性与表达有关，提示对DPN发病的上游通路的关键因子PARP、ROS、PKC和NF-kB的研究，可能对进一步揭示该药治疗DPN疗效分子免疫机制有重要的理论和实践意义。. 本次实验采用免疫组化、透射电镜、激光共聚焦、RT-PCR、神经电生理等技术，从在体和体外两个方面，从调控DPN发病的氧化应激上游通路的关键因子PARP、ROS、PKC、NF-kB活性及自噬相关指标的表达，探究中药糖痛方治疗糖尿病周围神经病变的分子机制。. 结果显示，中药糖痛方能够抑制DPN模型大鼠坐骨神经ROS的产生，降低PARP、PKC、及NF-kB的蛋白表达，同时糖痛方能够改善DPN大鼠坐骨神经髓鞘的变性，肿胀，改善神经纤维的形态结构，降低自噬相关蛋白LC3、Beclin-1的表达，抑制雪旺细胞的过度自噬。糖痛方能够抑制高糖损伤的雪旺细胞自噬相关蛋白LC3、Beclin-1、Atg5、Atg7、FoxO3a的过度表达，减轻自噬的消极作用，进而减少细胞凋亡。 . 此次实验从整体、细胞及分子水平等不同层面探讨糖痛方治疗糖尿病周围神经病变的疗效特点和分子机制，为中医益气活血防治DPN提供新的理论和实验依据；为传承名老中医经验提供新的思路。