Wnt/β-catenin信号通路在大鼠糖尿病周围神经病变髓鞘再生中的作用及中药筋脉通对其影响的研究

The main pathological features of diabetic peripheral neuropathy (DPN) include axonal atrophy, nerve demyelination,and delayed regeneration of peripheral sensory nerve fiber.The myelination of nerve fibers is not only involved in nerve signal conduction,also has the protection, nutrition、insulation and other multiple roles.Although the conduction of myelinated nerve fiber is fast, the ability against damage is low and a slow recovery due to the regeneration of Myelin sheath.Mechanisms of remyelination still remain unclear,and how to reduce the damage of DPN by promoting remyelination has become a research hotspot in recent years. Wnt signaling pathway is a key regulator of cell proliferation and differentiation.Many scholars found that high glucose can activate Wnt signaling pathway,but the effects of Wnt/β-catenin Signaling Pathway on the myelin regeneration of peripheral nerve injury have been rarely reported. Jinmaitong capsule which has been used in clinic for many years，has been proved that it can improve algesia and thalposis of STZ induced DM rats and also the pathomorphology abnormality of sciatic nerve. It can up-regulate both NGF and CNTF mRNA and their expression in sciatic nerve. Otherwise, Jinmaitong medicinal serum could enhance the NGF and CNTF secretion and proliferation of Schwann cells (SCs). Based on our previous research achievement, our group plan to establish the rodent DPN model and Schwann cell(SCs) cultured in vitro to reach whether Jinmaitong capsule promotes remyelination of injured peripheral nerveafter high glucose caused by regulating Wnt/β-catenin signaling pathway，aimed to approach the mechanism of Jinmaitong capsule promoting nerve regeneration.

糖尿病周围神经病变（DPN）主要病理特征包括轴突萎缩、髓鞘脱失、外周感觉神经纤维再生缓慢等。神经纤维的髓鞘不仅参与神经电信号的传导，也具有保护、营养以及绝缘等多重作用。有髓神经纤维损伤后由于需要有髓鞘再生，恢复较慢。有关髓鞘再生的机制目前尚不清楚，如何促进髓鞘再生从而改善DPN是近年来研究的热点。Wnt信号通路是细胞增殖分化的关键调控环节，高糖能够激活Wnt信号通路。目前，Wnt/β-catenin信号通路在周围神经损伤后髓鞘再生方面的作用未见报道。临床应用中药复方筋脉通多年，证实其可减轻神经细胞氧化应激损伤，减少凋亡；筋脉通含药血清可促进高糖培养雪旺细胞的增殖活性及其分泌NGF和CNTF的水平。本课题在前期研究工作的基础上，拟利用分子生物学技术研究筋脉通是否通过调节Wnt/β-catenin信号通路来促进高糖所造成的周围神经损伤后的髓鞘再生，从而探讨筋脉通促进神经再生修复的部分作用机制。

糖尿病周围神经病变（diabetic peripheral neuropathy，DPN）是糖尿病最常见的慢性并发症之一，严重影响了患者的生活质量，但其发病机制尚未完全阐明。中药筋脉通是北京协和医院院内制剂，临床应用二十余年，取得了良好的临床疗效，但其能否通过调节Wnt/β-catenin信号通路促进坐骨神经髓鞘修复尚不明确。本实验以链脲佐菌素诱导的糖尿病大鼠模型及高糖培养雪旺细胞（Schwann cell, SCs）模拟DPN损伤，采用中药筋脉通进行干预，随后采用实时荧光定量多聚核苷酸链式反应法、免疫蛋白印迹法检测大鼠坐骨神经及SCs中Wnt家族配体3a(Wnt family member 3a,Wnt3a)、Wnt抑制因子1(Wnt inhibitory factor 1, Wif-1)、糖原合成激酶3β(glycogensynthase kinase 3β, GSK3β)、β-连环蛋白和髓鞘蛋白零（myelin protein zero, P0）的mRNA及蛋白表达，以及GSK3β、β-catenin磷酸化蛋白的表达；酶联免疫吸附法检测大鼠血清及细胞上清液中Wnt3a、Wif-1蛋白的表达。整体及细胞实验研究结果显示，随着造模时间的延长，糖尿病大鼠出现坐骨神经损伤，并逐渐加重；DPN大鼠坐骨神经及高糖培养的SCs中，Wnt/β-catenin信号通路表达受到抑制，P0表达降低，髓鞘修复障碍。筋脉通能够促进大鼠坐骨神经中P0的表达，提高SCs增殖率，缓解DPN大鼠坐骨神经病理损伤，促进其髓鞘再生，以10倍人体剂量筋脉通效果最佳。本课题从整体、细胞及分子水平进行研究，结果表明，筋脉通可通过缓解DPN大鼠坐骨神经及高糖培养SCs中Wnt/β-catenin信号通路受到的抑制，起到改善髓鞘修复障碍的作用。阐明了筋脉通防治DPN的可能分子机制，为推广应用筋脉通防治DPN 提供理论基础和实验依据。