生地-山茱萸调控巨噬与肾固有细胞的交互应答干预糖尿病肾病的作用机制

Diabetic nephropathy (DN) is a kind of chronic metabolic inflammation disease. RAGE/RhoA/ROCK signaling pathway is the core part of the interaction between macrophages and renal cells. The inflammatory mediators produced during the process is the important basic material of “heat damage to Yin/Jin” of DN. The drug pair Rehmanniae Radix-Corni Fructus represents the therapy of "nourish Yin and clear heat". Through the previous research, Rehmanniae Radix-Corni Fructus’s effective component Catalpol and Loganin reduced RhoA and downstream protein expression, regulated macrophage migration and phenotypic transformation of M1/M2, delayed the progression of DN. In this project, the renal RAGE overexpression of spontaneous diabetic rats model, AGEs-induced macrophage-renal mesangial cells coculture model, AGEs-induced macrophage-renal endothelial cell coculture model and AGEs-induced macrophages-podocyte coculture model would be chosen and technology of gene silence and overexpression would be used to investigate the molecular mechanism and effects of the drug pair Rehmanniae Radix-Corni Fructus and its effective component Catalpol and Loganin basing the interaction between macrophages and renal cells. The study would provide a scientific basis of the drug pair Rehmanniae Radix-Corni Fructus and compatibility for the prevention and treatment of DN, establish foundation for the research and development of new drugs, and expound the scientific connotation of " nourish Yin and clear heat ".

糖尿病肾病（diabetic nephropathy, DN）是一种代谢性慢性炎症反应性疾病，其中RAGE/RhoA/ROCK信号通路是巨噬细胞与肾固有细胞交互应答的核心环节，产生的炎症介质是DN中医病机“内热伤阴/津”的重要物质基础。生地-山茱萸是DN“滋阴清热”治法的主要药对，前期工作基础发现，生地、山茱萸效应成分梓醇、马钱苷能通过RhoA及下游信号通路调控巨噬细胞迁移、浸润和M1/M2的表型转化，延缓DN病变的进程。本课题拟通过自发性糖尿病大鼠肾脏RAGE过表达模型、AGEs致巨噬细胞-肾系膜细胞、巨噬细胞-肾内皮细胞、巨噬细胞-肾足细胞共培养损伤模型，利用基因沉默、过表达等技术，以生地-山茱萸药对和效应成分梓醇、马钱苷及其配伍为研究对象，深入阐明巨噬与肾固有细胞交互应答的分子机制，为生地－山茱萸药对及其组方防治DN提供科学依据，为新药研发奠定基础，并阐述“滋阴清热”治法的科学内涵。

糖尿病肾病（diabetic nephropathy, DN）是糖尿病最常见且最难治的微血管并发症之一，也是产生终末期肾病的主要原因，炎症反应与DN的发生密切相关，在代谢性炎症反应过程中常伴随巨噬细胞的浸润及活化，其中非酶糖基化终末产物（AGEs）是炎症损伤的关键介质，AGEs可作用于肾脏固有细胞及巨噬细胞上的RAGE，激活细胞内的信号通路，产生的炎症介质是促进DN损伤的重要物质基础。生地-山茱萸药对是DN“滋阴清热”治法的主要药对，本课题通过建立RAGE过表达DN小鼠模型以及AGEs致肾脏固有细胞损伤模型，围绕AGEs-RAGE/RhoA/ROCK信号通路，深入阐明生地-山茱萸及其效应成分梓醇-马钱苷及其配伍调控巨噬细胞与肾固有细胞的交互应答的作用及机制。研究结果表明，生地-山茱萸药对及其有效成分梓醇-马钱苷可改善RAGE过表达后DN小鼠一般症状，改善肾脏固有细胞的损伤，也能抑制肾脏巨噬细胞的炎性浸润，下调炎症因子IL-6及趋化因子MCP-1等的分泌，下调 M1 型巨噬细胞极化标记蛋白 iNOS 及CD16/32的表达，上调 M2 型巨噬细胞极化标记蛋白 Arg-1 及CD206的表达，且生地-山茱萸药对组及梓醇-马钱苷配伍组作用最佳。体外实验结果表明，生地-山茱萸及梓醇-马钱苷可改善 AGEs 刺激巨噬细胞介导的肾固有细胞损伤标记蛋白的表达。体外机制研究表明，生地-山茱萸及梓醇-马钱苷均能显著抑制肾固有细胞或巨噬细胞的RAGE、RhoA、ROCK 蛋白的表达。另外，分别在固有细胞及巨噬细胞加入 RAGE、RhoA、ROCK 的抑制剂 FPS-ZM1、Rhosin hydrochloride、Y27632 后，生地-山茱萸及梓醇-马钱苷能通过抑制固有细胞的 RAGE/RhoA/ROCK 通路介导巨噬细胞 M1 型极化；也能通过抑制巨噬细胞的RAGE/RhoA/ROCK 通路介导固有细胞的损伤。因此，生地-山茱萸及梓醇-马钱苷可调控肾固有细胞RAGE/RhoA/ROCK 信号通路抑制巨噬细胞向肾固有细胞的迁移和M1/M2表型极化失衡，同时，也能通过调控巨噬细胞RAGE/RhoA/ROCK 信号通路，进一步抑制M1型活化及炎性因子的分泌，改善肾脏固有细胞的损伤。且药对组及梓醇-马钱苷配伍组效果最优，能更好的发挥中医药“滋阴清热，补益肝肾"的作用。