基于AGEs/RAGE/NF-κB/MCP-1炎症路径多靶点探究早期糖尿病肾病"气阴两虚、络脉瘀阻"病机

The main pathogenesis of early diabetic nephropathy in TCM is "qi and yin deficiency, collateral stasis", which is an instruction for clinical diagnosis and treatment and a good efficacy has been achieved. It is believed in modern medical research that "inflammation" is a key factor in the incidence of diabetic nephropathy, and AGEs / RAGE/NF- kappa B / MCP -1 inflammation path is of great significance. In diabetic state, a great amount of advanced glycation end products (AGEs) emerge and tie with receptors to activate the inflammation path, a large number of chemokine macrophage infiltrate, and inflammatory effect is amplified and damage results, which constitutes the basis of the disease. The main content of this project is, based on the inflammation path, to build a "Multi-target Molecular Indicators Evaluation System", to explore molecular mechanism of "qi and yin deficiency, collateral stasis" pathogenesis for early diabetic nephropathy. That is, following the holistic concept of TCM, through the replication of early diabetic nephropathy animal model, to observe multi-targets, lipid disorders and hemodynamics, hemorheology and other indicators on AGEs / RAG E/NF- κB / MCP -1 inflammation path; using analysis methods as repeated measure analysis of variance and clustering and principal component data analysis, to analyze the relationship between the various indicators, and the difference of each indicator at different stages, before and after drug intervention; integrated with the clinical manifestations and pathological changes, to reveal the material basis of early diabetic nephropathy "qi and yin deficiency, collateral stasis" pathogenesis，and provide a scientific basis for TCM treatment based on syndrome differentiation.

早期糖尿病肾病的中医主要病机是"气阴两虚、络脉瘀阻"，指导临床辨证论治取得较好疗效。目前认为 "炎症"是糖尿病肾病发病的关键因素。糖尿病状态下，糖基化终末产物（AGEs）产生并与受体（RAG E）结合激活NF- κB，使MCP -1增加，巨噬细胞浸润，引起炎症反应。本项目的主要内容是基于该炎症路径构建"多靶点分子指标评价体系"，探究早期糖尿病肾病"气阴两虚、络脉瘀阻"病机分子机制。即遵循中医整体观念，通过复制早期糖尿病肾病动物模型，观察AGEs/ RAG E/NF- κB /MCP -1炎症路径上多靶点、糖脂代谢紊乱及血流动力学、血液流变学等指标，运用重复测量方差分析及聚类和主成分数据分析方法，分析各指标间的相互关系，不同阶段和药物干预前后各指标的差异，结合临床表现、病理变化，共同揭示早期糖尿病肾病"气阴两虚、络脉瘀阻"病机物质基础，为中医辨证论治提供科学依据。

本项目复制早期糖尿病肾病（DN）大鼠模型，观察AGEs/RAGE/NF-KB/MCP-1 炎症路径上多靶点、糖脂代谢紊乱及血流动力学、血液流变学指标变化；运用方差分析及主成分数据分析方法分析不同阶段和药物干预前后各指标的差异，指标间的相互关系、并结合一般表现、病变，揭示早期DN“气阴两虚、络脉瘀阻”的物质基础。.方法：高糖高脂饮食结合低剂量链尿佐菌素（STZ）静脉注射复制早期DN大鼠模型，从多角度来反映DN大鼠存在“气阴两虚、脉络瘀阻”的病机；通过HE、PAS染色及免疫组化技术检测肾病变及巨噬细胞浸润；运用Western blot、ELISA及实时荧光定量-PCR等技术检测AGEs和RAGE，NF-κB、TNF-α、IL-1β、MCP-1等指标；运用鸟枪法研究脂质代谢情况；运用方差分析和主成分数据分析方法分析各指标间的相关性、DN大鼠不同发展阶段和药物干预后各指标的差异。.结果：成功复制早期DN大鼠模型：模型组大鼠精神萎靡、懒动、体形消瘦，多饮、多食、多尿症状，毛色枯黄，竖立；模型组大鼠出现肾功能异常及肾的早期病变，血液流变学和微循环异常。肾组织炎症因子表达增加，巨噬细胞浸润；AGEs和RAGE升高。随着病程的延长肾功能及肾的早期病变加重，血液流变学和微循环异常加重。糖肾方治疗可改善DN大鼠肾功能及其病变，可改善肾血液流变学和微循环及其炎症相关指标。通过相关性分析显示，血脂、血粘滞度与内皮细胞的功能指标、尿蛋白的排出、血清炎症因子的含量及肾组织的炎症因子的表达呈正相关；肾组织炎症因子的表达水平与尿蛋白的排出呈正相关；AGE与RAGE呈正相关；RAGE与尿蛋白的排出呈正相关；RAGE、AGE水平与肾组织炎症因子的表达呈正相关。.结论：早期DN大鼠可能存在“气阴两虚、络脉瘀阻”病机变化；DN大鼠肾脏组织炎症因子表达增加，巨噬细胞浸润，而糖肾方可抑制炎症因子的表达和巨噬细胞的浸润，提示炎症在糖尿病肾损伤中起着重要的作用；早期DN肾组织中AGE及RAGE的表达比正常组明显增高，糖肾方干预后AGE及RAGE的表达下降，提示AGE-RAGE与早期DN“气阴两虚、络脉瘀阻”的病机可能具有相关性。