基于RAGE靶标研究生地山茱萸环烯醚萜苷类成分干预糖尿病肾病的分子机制

The project, which is based on preliminary research, Combined traditional Chinese medical literature, from the whole traditional Chinese medical thought which is "active components to targets", proposes that AGEs is the main initial substance of "heat damage" Pathogenesis which is narrated in traditional Chinese medical diabetic nephropathy, AGEs -RAGE signaling pathway is the core mechanism of leading to diabetic nephropathy.The basic of subject preparatory work discoveries, the catalpa, acteoside, loganin, morroniside which are the main activity Iridoid Glycosides components in Radix Rehmanniae and Cornus Officinalis in intervening AGEs-RAGE targets, they are the majory form to embody the Chinese therapeutic method of "Ziyin and Qingre" in treating Diabetic Nephropathy by the way.The project would choose the human mesangial cell, human podocyte cell, human umbilical veins endothelium cell for the research object on this base. Modern biomedical technology such as surface plasmon resonancing, gene protein silencing,gene protein overexpressing will be used to carry out the downstream signaling pathways arrounding the RAGE targets and targeted study the action links,action targets intervened by the Iridoid Glycosides components in Radix Rehmanniae and Cornus Officinalis and expoud its molecular mechanisms.The findings of the project lay an experimental and theoretical foundation for the contact of pathological mechanisms of diabetic nephropathy between Chinese medicine and Western medicine .And it has important scientific significance that they reveal diabetic nephropathy in the treatment method of Chinese medicine.

本项目在良好的前期研究基础上，结合中医药文献，从"有效成分群对靶标群"的中医药整体思想出发，创新性提出了AGEs是糖尿病肾病中医所述"内热"病机的主要初始物质，AGEs-RAGE信号通路是形成糖尿病肾病的核心机制。课题前期工作基础发现，生地山茱萸中环烯醚萜苷类成分梓醇、毛蕊花糖苷、马钱苷、莫诺苷是干预AGEs-RAGE信号通路的主要活性成分，是治疗糖尿病肾病的中医"滋阴清热"治法的主要体现形式。本项目在此基础上拟选择人源性肾系膜细胞、足细胞及脐静脉内皮细胞为研究对象，利用表面等离子共振、基因蛋白沉默、基因蛋白过表达等现代生物医学技术，围绕RAGE靶标下游信号通路深入开展和靶向研究生地、山茱萸环烯醚萜苷类成分干预的作用环节、作用靶点，阐明其分子机制。本项目研究结果为糖尿病肾病中医与西医病机的有机桥接和联系奠定理论和实验基础，对揭示糖尿病肾病中医治法具有重要的科学意义。

课题以人源性肾系膜细胞株(HGMCs)、人源性脐静脉内皮细胞株（HUVEC）、小鼠足细胞株（MPC）为研究对象，建立了AGEs致HGMCs、HUVEC、MPC损伤的体外细胞模型，采用基因沉默、基因过表达、生物膜层干涉等技术围绕AGEs-RAGE及其下游的信号通路深入靶向研究生地、山茱萸环烯醚萜苷成分干预DN的主要作用靶点。实验结果：（1）马钱苷、莫诺苷、梓醇、毛蕊花糖苷均可抑制HGMCs的增殖及分泌LDH、FN、COL-IV、ROS，其中以马钱苷和莫诺苷作用最突出。在AGEs/RAGE信号通路蛋白上，马钱苷、莫诺苷、梓醇、毛蕊花糖苷均能降低RAGE下游信号蛋白的表达，在抑制PTK、MEK1/2、ERK1/2、SphK1、S1P2方面，以马钱苷作用较突出；对抑制NOX4蛋白表达，以马钱苷和梓醇较突出；对P38MAPK、TGF-β蛋白表达，以马钱苷、莫诺苷作用较强；对NF-κB磷酸化，以毛蕊花糖苷抑制作用较强，而在NF-κB转核方面，以莫诺苷的作用较突出。（2）马钱苷、莫诺苷、梓醇、毛蕊花糖苷均对AGEs损伤HUVEC具有显著的保护作用，表现为降低HUVEC凋亡，抑制HUVEC细胞产生MCP-1、VCAM-1、TGF-β，降低ET-1，升高NO。莫诺苷、马钱苷抑制MCP-1，莫诺苷、毛蕊花糖苷降低VCAM-1和TGF-β作用强；莫诺苷、马钱苷升高NO，马钱苷降低ET-1的作用强。过表达RAGE受体后加入SPHK、NF-κB等蛋白的抑制剂，可以削弱梓醇、马钱苷对HUVEC的保护效应，推测梓醇、马钱苷潜在的作用靶点分别为NF-κB、SPHK。（3）马钱苷、莫诺苷、梓醇、毛蕊花糖苷对AGEs诱导MPC的损伤有显著的改善作用，表现为降低MPC的凋亡率，下调bax、caspase3、上调bcl2、nephrin的表达，其中以马钱苷、梓醇的效果较优。在AGEs-RAGE的信号通路蛋白的抑制上，马钱苷抑制p38磷酸化作用较强、而梓醇降低Nox4表达效果更显著。（4）蛋白亲和力试验显示马钱苷与SphK1、莫诺苷与ERK1/2、梓醇与NF-κB、梓醇与RAGE的亲和力较强。综上研究证实生地山茱萸环烯醚萜苷代表成分对AGEs-RAGE轴及其下游通路的抑制各有侧重，通过多个环节、多个靶点协同增效而延缓DN的发生发展，也体现了生地-山茱萸药对应用于临床配伍的优势。