从RhoA/ROCK通路探讨益气生肌重构ECs细胞骨架治疗糖尿病血管神经病变的作用机制

Making concepts of “Qi-Blood Relationship”, such as “Promoting Qi and Generating Muscle”, “Activating Blood and Dredging Collaterals” in Chinese medicine as the theoretical basis, starting from the “Channel-Blood Vessel Identity”, HUVECs after injury of high glucose induces are planned to be the models. Promoting Qi and Activating Blood TCM Diabetic granules’ drug serum interferes. Tracer HRP detects HUVEC endothelial monolayer permeability, flow cytometry instrument detection of HUVEC apoptosis, Western blot method to detect ZO - 1, occludin, RhoA, ROCK, and P - ROCK protein expression, as well as Immunofluorescence testing F - actin dynamics; observing in vivo its influences on high sugar and ischemia injury model rats skeleton femoral artery endothelial cells and related channels, trying to discuss Promoting Qi and Activating Blood Compound TCD’s mechanism of stabilizing cytoskeleton, improving endothelial cells monolayers permeability, and protecting endothelial function, from aspects of cell, molecular, signal pathways, etc.. It aims at effective TCD for treating diabetic peripheral vascular neuropathy and its target. Therefore, the research provides methodology and scientific basis for “Promoting Qi and Generating Muscle”, “Promoting Qi to Contain Essence”, and “Theory of Collateral Diseases”, etc.

本课题以中医的“益气-生肌”，“活血-通络”等有关“气血关系”为理论基础，从“脉络-血管系统”的同一性为出发点，拟采用高糖诱导损伤后人脐静脉血管内皮细胞(HUVECs)为模型，益气活血中药消渴通痹颗粒含药血清进行干预，HRP作为示踪剂检测HUVEC内皮细胞单层通透性、流式细胞仪检测HUVEC细胞凋亡、Western blot法检测ZO-1、occludin、RhoA、ROCK及p- ROCK蛋白表达量，免疫荧光法检测F-actin肌动蛋白的动态变化；并在体观察其对高糖及缺血损伤模型大鼠股动脉血管内皮组织细胞骨架及相关通路的影响，试图从细胞、分子、信号传导途径等多方面，探讨益气活血复方中药稳定细胞骨架结构，改善内皮细胞通透性，保护内皮功能的机制，寻找治疗糖尿病周围血管神经病变的有效中药及分子靶点，为丰富和发扬中医“益气-生肌”、“益气-固摄”和“络病学说”等理论，提供方法学借鉴和科学依据。

本课题以中医的“益气-生肌”，“活血-通络”等有关“气血关系”为理论基础，从“脉络-血管系统”的同一性为出发点，探讨消渴通痹颗粒治疗糖尿病周围神经病变的分子机制。采用高糖诱导的HUVEC模型，用消渴通痹颗粒含药血清和ROCK特异性抑制剂Y27632干预，实验分为9组：正常对照组，高糖损伤组，高糖＋抑制剂组，中药（低、中、高剂量）组，中药（低、中、高剂量）+抑制剂组。各组细胞传代8～15代时，进行以下研究：HRP检测内皮细胞单层通透性，流式细胞术检测内皮细胞凋亡率，免疫荧光法检测细胞骨架F-actin的动态变化，Western blot检测F-actin及RhoA、ROCK蛋白的表达。研究结果表明：消渴通痹颗粒能降低高糖诱导的内皮细胞的通透性、改善内皮细胞的凋亡、低剂量消渴通痹颗粒可能通过抑制高糖诱导的RhoA/ROCK信号通路的激活、降低RhoA、ROCK等相关信号分子的表达来稳定内皮细胞骨架（F-actin），保护血管内皮细胞，治疗糖尿病周围神经病变。