初探中医“虚损劳衰”学说及“肾络微型癥瘕”理论的分子基础

Diabetic nephropathy (Diabetic kidney diseases, DKD), which is the leading causes of terminal renal failure, is one of the diabetic chronic microvascular complications. It is suggested that renin angiotensin system (RAS) inhibitors is standard care for preventing the progress developing into end-stage renal disease.This project is based on the theory of diabetic nephropathy pathogenesis which is named as "Xu Sun Lao Shuai" and "renal collaterals miniature remove lump". In our studies , we will test the indexes of the RAS system (ACE-AngⅡ-AT1R and ACE2- Ang(1-7)-Mas), endothelial system (vWF, PAI-1, t-PA), and neuroendocrine system(pituitary - target gland axis) and will summarize of their pathological physiology characteristics and changing trends at different periods. Then we will investigate the correlation between the indexes and TCM hypothesizes of Kidney disease pathogenesis. We hope that we can looking for some DKD medium-term risk markers and provide the molecular pathology of TCM hypothesizes of Kidney disease pathogenesis. In addition, we will analyze the molecular mechanisms, targets and timing of "Zhi Xiao Wen Shen Ning” particles which is a formula according to nourished qi, warmed kidney and dissipation pathological products. We also will investigate the correlation between the foregoing indexes and TCM hypothesizes of Kidney disease pathogenesis. We hope our research can lay a solid experiment foundation for treatment of DKD Using traditional Chinese medicine therapy.

糖尿病肾病（Diabetic kidney disease, DKD）为糖尿病慢性微血管并发症，也是终末肾衰竭的主要病因。研究认为肾素血管紧张素系统（RAS）抑制剂是减少DKD患者发展为终末期肾脏病风险的标准治疗。本项目根据糖尿病肾病“虚损劳衰”学说及“肾络微型癥瘕”的中医假说，通过STZ诱导的糖尿病肾病大鼠动物模型，观察体内RAS系统（ACE-AngⅡ-AT1R和ACE2-Ang(1-7)-Mas）、内皮系统（vWF、PAI-1、t-PA）、神经内分泌系统（垂体―靶腺轴）不同时间点的病理生理特点和变化趋势，探讨其与“肾虚证”、“微型癥瘕证”的关系，寻找DKD 早中期风险标志物，为中医肾病“虚损劳衰”学说及“肾络微型癥瘕”中医病理假说提供分子研究基础；并以方测证证实上述两个中医病理假说理论，验证总结益气温肾散结法及其代表方止消温肾宁颗粒治疗DKD的作用机理，分子机制、作用靶点及作用时机。

目的：本研究旨在通过STZ诱导的DKD大鼠阳虚血瘀证动物模型，以RAS系统关键因子、内皮系统关键因子、血液流变学及肾脏病理等指标为切入点，探讨“肾虚证”与RAS系统,“肾络微型癥瘕”与内皮系统损伤标志物及血流流变学的关系，为中医肾病“虚损劳衰”、“肾络微型癥瘕”病理假说及散结消聚治法提供分子研究基础。方法：1.实验一进行糖尿病肾病阳虚证大鼠动物模型的证型评价。采用STZ单次腹腔内注射的方法，构建糖尿病肾病大鼠动物模型，分别于成膜后6w、10w、12w、16w进行实验，检测大鼠空腹血糖、24h尿蛋白定量、肾功能等指标，收集腹主动脉血，对模型大鼠阳虚证相关指标进行评价。2.实验二根据实验一中证候评价结果，选取阳虚证模型大鼠进行实验，分为正常组，阳虚证模型组，缬沙坦组，益气温肾散结中药组（低、中、高剂量）。灌胃8周后检测大鼠空腹血糖、24h尿蛋白定量、肾功能、血脂等指标；测定阳虚证相关指标及血浆纤维连接蛋白（fibronectin，FN）、血管性假性血友病因子（vWF）、肾脏组织型纤溶酶原激活物（t-PA）、纤溶酶原激活物抑制剂-1（PAI-1）mRNA 的表达水平。结果：1.实验一：STZ诱导的DKD大鼠模型，其阳虚证相关指标(cAMP、cAMP、T3、T4、ACTH等)在第10周至12周阶段会出现较为明显的降低。2.实验二：模型组会出现阳虚证相关指标（cAMP、cGMP、T3、T4、ACTH）降低，益气温阳散结组方能在一定程度上改善阳虚证指标，显著降低AngII、Mas蛋白表达，提高AT1、ACE2蛋白表达，通过上调肾脏ACE2-Ang(1-7)-Mas轴、下调ACE-AngⅡ-AT1轴相关蛋白表达，从而明显改善糖尿病肾病阳虚证，以高剂量中药组疗效尤为显著。同时增强ACE2-Ang(1-7)-Mas轴对ACE-AngⅡ-AT1轴的拮抗能力，抑制AT1等因素对足细胞的损伤，并改善血液流动学。结论：实验一初步确定糖尿病肾病大鼠动物模型的阳虚证型时间点为成膜后12周左右。实验二证明益气温阳散结组方能在一定程度上改善cAMP、cGMP、T3、T4、ACTH阳虚证指标，减轻阳虚证，以高剂量中药组尤为显著。同时增强ACE2-Ang(1-7)-Mas轴对ACE-AngⅡ-AT1轴的拮抗能力，抑制AT1等因素对足细胞的损伤，并改善血液流动学。