基于JNK通路的益气活血通络法对糖尿病周围神经保护作用机制研究

On the base of summarizing and analysing pathogenesis and treatment of diabetic peripheralneuropathy, raising:deficiency of both qi and yin , static blood blocking collaterals is the pathological basis of diabetic peripheralneuropathy.Considering:inflammatory injury and decreasing of nerve growth factor both plan an important role in the pathogenesis of diabetic peripheralneuropathy and regulate JNK signal channel,destroying the structure and function of peripheral nerve,rusulting in peripheral nerve injury.The evolution process of qi deficiency-yin deficiency-blood stasis consistents with inflammatory injury and neural function and structure injury. On the basis of achievement of prophase clinical research,based on JNK signal channel, using modern molecular biology experimental techniques, investigating the effects of Yiqihuoxuetongluo recipe on neural structure and function and changes of relative index of inflammatory cytokines and nerve growth factor of db/db mice; Meanwhile,vitro modelling high sugar state, observing the expression of key moleculars of JNK signal channel in endothelial cells and dorsal root ganglia. To investigate the regulating effects of interaction relationship between Yiqihuoxuetongluo recipe inhibits inflammatory injury and promotes NGF to protecte peripheral nerve and JNK signal channel. Explaining the mechanism of Yiqihuoxuetongluo recipe protecting diabetic peripheral nerve，defining the target, and providing new accurate point and breakthrough point for optimizing Chinese medicine treating diabetic peripheralneuropathy.

项目组在总结分析糖尿病周围神经病变（DPN）发病机制和治疗现状基础上，提出：气阴两虚，瘀血阻络是DPN 发生的病理基础。认为：炎性损伤和神经生长因子（NGF）减少不仅在DPN的发病机制中占重要作用，而且可调控JNK信号通路，破坏周围神经结构和功能，导致周围神经损伤。气虚-阴亏-血瘀演进过程与炎性损伤、神经功能结构受损相一致。鉴此，本课题在我们前期临床研究成果基础上，基于JNK信号通路，运用现代分子生物学技术，观察益气活血通络方干预对先天性糖尿病模型小鼠周围神经结构功能的影响和炎性因子、神经生长因子相关指标的变化。体外模拟高糖状态，观察内皮细胞和背根神经节内JNK信号通路关键分子的表达。探讨益气活血通络法抑制炎性损伤、促进NGF对周围神经保护和JNK之间互动关系的调节作用，阐明益气活血通络法对糖尿病周围神经的保护作用机制，明确其作用靶点，为优化中医药治疗DPN提供新的切入点和突破口。

本研究探讨了益气活血通络方保护糖尿病周围神经病变（DPN）的作用及机制。主要研究内容与结果如下：1）通过观察糖尿病周围神经病变模型大鼠及自发性2型糖尿病db/db模型小鼠周围神经超微结构和功能的变化，发现益气活血通络方能明显改善周围神经的结构及功能。2）利用酶联免疫吸附法及Western blotting技术检测炎性因子、神经生长因子及其受体、JNK信号通路相关蛋白在先天性糖尿病模型小鼠体内的表达，证实了益气活血通络方能抑制JNK信号转导通路的激活，减轻db/db小鼠的炎症反应，激活 NGF 及其受体的表达，促进神经元生长，从而明确了益气活血通络方治疗糖尿病周围神经病变的作用靶点。3）益气活血通络方含药血清作用于人脐静脉内皮细胞，可降低炎性因子的表达，减轻细胞凋亡的发生，证实与JNK信号通路激活有关。4）益气活血通络方含药血清作用于背根神经节神经元细胞，可上调NGF及其高亲和力受体的表达，并下调Bax和P53的基因表达，且与JNK通路的调节有关。以实验动物为研究对象，以细胞实验验证其机制，环环相扣，验证了预定设想，完成了预期计划。