基于MMPs/TIMPs探讨益景汤干预早期糖尿病视网膜病变微血管损害的机制

Yijing decoction is an effective decoction for prevention and treatment of diabetic retinopathy summarized by the old Chinese Medicine expert professor Jin Weier and used nearly 20 years. Although our previous clinical and experimental studies have shown that it has positive effect on the damage of microvascularity in diabetic retinopathy, the underlying mechanism by which it prevents and treats diabetic retinopathy still remains to be further elucidated. Based on our previous studies, the research will be performed with the view angle of matrix metalloproteinase (MMPs) / tissue inhibitor of matrix metalloproteinases (TIMPs) to explore the critical pathologic changes of blood-retinal barrier (BRB) disruption in the early process of diabetic retinopathy. We will sing the methods of TUNEL, flow cytometry, immunohistochemical staining, western blot and RT-PCR in vivo and vitro studies to examine the effect of Yijing decoction on the damage of microvascularity, and observe the expression of VEGF, uPA, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, Caspase-3 and H-Ras which related to MMPs/TIMPs system and Collagen Ⅳ, Laminin, Occludin, claudin 5, Zonula occluden-1 which related to the structure of BRB, and access the changes of BRB structure. The objective of this study is to systematically evaluate the effect of Yijing decoction on prevention of BRB disruption in the early process of diabetic retinopathy, explore the underlying mechanism by which it prevents and treats diabetic retinopathy from molecular signal transmission pathway and gene expression level, and reveal the possible targets of action of this decoction.

益景汤是福建省名老中医金威尔教授总结用于糖尿病视网膜病变(DR)临床防治近20年的验方，前期临床和基础研究证实该方能有效干预DR微血管损害，但具体机制有待进一步明确。本课题拟在前期研究的基础上，针对DR早期发病过程中血-视网膜屏障（BRB）这一重要病理改变从MMPs/TIMPs角度进行探讨。拟在动物模型+细胞培养实验的基础上，采用益景汤进行干预，通过TUNEL、流式细胞仪、免疫组化、Western blot、RT-PCR等方法观察BRB相关结构的变化和MMPs/TIMPs相关因子（VEGF、uPA、MMP-2、MMP-3、MMP-9、TIMP-1、TIMP-2、Caspase-3、H-Ras）及BRB相关结构蛋白（CIV、LN、Occludin、claudin 5、ZO-1）等的表达，从分子信号传导和基因表达水平深化研究益景汤干预早期DR视网膜微血管损害的机制，探讨该方对DR的作用靶点。

糖尿病视网膜病变（DR）是糖尿病（DM）最常见的微血管并发症之一，是不可逆性致盲性眼病，开展DR早期防治研究是重要的课题。血-视网膜屏障（BRB）破坏是DR发生、发展的关键。益景汤是福建省名老中医金威尔教授用于DR防治近20年的验方，前期研究证实该方能有效干预DR微血管损害，但具体机制欠明确。本研究构建了视网膜微血管内皮细胞（ECs）和周细胞（RMPs）体外三维血管新生模型，采用益景汤含药血清作用于血管新生模型和益景汤作用于DM大鼠模型，通过光电镜、CCK-8、流式细胞仪、免疫组化、Wb、PCR等方法观察了BRB相关结构的变化和MMPs/TIMPs相关因子及BRB相关结构蛋白的表达，在体外内实验的基础上探讨了益景汤干预早期DR视网膜微血管损害的机制。细胞实验结果显示与高糖组相比，益景汤能够干预高糖抑制ECs增殖和诱导的ECs凋亡，减少高糖诱导的血管新生模型MMP-2、MMP-3、MMP-9、Caspase-3、H-Ras mRNA表达增加，降低高糖诱导的血管新生模型CIV、LN、VEGF-A、uPA蛋白表达增加，抑制高糖诱导的血管新生模型occludin、claudin-5、ZO-1蛋白表达降低（均P＜0.05）。动物实验结果显示16周时DM大鼠模型存在明显的视网膜微血管损伤，与DM对照组相比，益景汤能够减轻DM引起的视网膜微血管渗漏、RMPs凋亡和基底膜增厚，抑制DM大鼠视网膜组织中MMP-2、MMP-3、MMP-9蛋白表达的增强。同时，Wb检查显示益景汤能减少DM大鼠视网膜组织中VEGF-A、uPA蛋白表达（均P＜0.05），PCR显示益景汤能够抑制DM大鼠视网膜组织中Caspase-3、H-Ras mRNA的表达（均P＜0.05）。结果表明益景汤可以通过调控MMPs/TIMPs信号通路相关因子的表达，抑制高糖下BRB的损害。结果为临床推广应用益景汤防治DR提供了科学依据。