白藜芦醇防护糖尿病早期视网膜病变的作用及分子机制

Diabetic retinopathy (DR) is one of the leading causes of blindness worldwide. Although significant progress has been made, there is still no efficient treatment of this disease. One of the reasons may be that our knowledge of the mechanisms underlying early pathogenic processes is lacking. Our previous studies have indicated that structural and Functional damage to the retinal muller cells (RMCs) occur prior to the onset of vasculature abnormalities and ultimately lead to vascular disease. Therefore, it is crucial to find additional treatments that prevent or slow the damage of RMCs in DR. Resveratrol (Res) is a dietary polyphenol derived from grapes, peanuts, and other plant sources. Res has been shown to prevent DR in several studies. However, the protective effects of Res on RMCs' injury and its molecular mechanisms are not understood. MicroRNAs (miRNAs) are endogeneous small RNAs (less than 25 nt) that play key regulatory roles targeting mRNA for specific cleavage, mRNA deadenylation and decay, translational repression or activation. Recently, increasing evidence has indicated that miRNAs play important roles in DR. Some of these miRNAs may be the targets of Res anti-DR. In this project, we address the possible effects and underlying molecular mechanisms of Res on RMCs' damage in early phase of DR. In vivo, Sprague-Dawley (SD) rats with streptozotocin (STZ)-induced diabetes are used to determine the effects of Res on RMCs' damage and the associated mechanisms of miR-29b, RAX, PKR and NF-κB. In vitro, RMCs cultured in high glucose are used as the model. TUNEL, immuocytochemistry, laser confocal analytical assay, flow cytometry are used to study the straight protective effects of Res on RMCs' damage induced by high glucose. Meanwhile the associated molecular mechanisms of Res on miR-29b, RAX, PKR and NF-κB are deeply studied with siRNA, EMSA, qRT-PCR and western blotting method. Our studies reveal that the underlying mechanisms for the protective effects of Res on early damage of DR, which suggest a possible utility of Res as a adjuvant therapeutic agent in the management of early stage of DR.

糖尿病视网膜病变（DR）是世界范围内致盲的主要原因之一。申请人前期研究表明，视网膜muller细胞（RMCs）损伤发生在DR早期。探讨DR RMCs损伤的膳食防护具有重要意义。白藜芦醇（Res）作为天然植物多酚，其对DR的防护作用已被证实，但Res对RMCs损伤的作用及分子机制尚不清楚。微小RNAs（miRNAs）是新近发现的DR防治靶点，某些miRNAs可能在Res防护DR中发挥作用。本项目基于前期研究，采用STZ诱导SD大鼠动物模型，重点探讨Res对DR RMCs损伤的影响及其与miR-29b/RAX/PKR/NF-κB的关系；并结合体外高糖培养RMCs，运用特异性抑制剂、siRNA、荧光定量PCR等技术，深入研究Res通过影响miR-29b调节NF-κB凋亡通路防护DR早期RMCs损伤的分子机制。本研究旨在深入揭示Res防护DR早期病变的分子机制，为指导人们早期防治DR提供科学依据。

糖尿病视网膜病变是糖尿病最为常见的严重并发症，是造成成年人失明的主要原因。本项目以STZ诱导的SD大鼠糖尿病模型和体外高糖培养的SD大鼠视网膜Muller细胞为研究对象，利用苏木精-伊红染色、透射电镜、TUNEL、视网膜电图、实时荧光定量PCR技术、Western-blotting技术、免疫荧光双标、激光共聚焦显微技术、ELISA技术、体外细胞转染、流式细胞技术等研究技术，系统研究了糖尿病早期视网膜结构、功能的改变以及白藜芦醇对这些改变的影响，揭示了糖尿病早期视网膜内核层细胞异常改变发生的分子机制，以及白藜芦醇通过miR-29b抑制凋亡途径抗糖尿病早期视损伤的分子机制，为寻求糖尿病视网膜病变早期防护措施提供理论基础。研究结果表明，糖尿病引起大鼠视网膜内层病理结构、超微结构和内层视网膜功能发生改变，低剂量和高剂量白藜芦醇干预可明显改善视网膜上述改变，呈剂量效应、时间效应关系；糖尿病1月开始，视网膜内核层细胞出现凋亡，随时间延长，凋亡越明显，低剂量和高剂量白藜芦醇干预明显抑制糖尿病引起的视网膜内核层细胞凋亡，呈剂量效应关系，25 mmol/L高糖培养后，Muller细胞凋亡率明显增加，白藜芦醇干预可明显抑制高糖诱导的Muller细胞凋亡；糖尿病诱导视网膜组织和视网膜Muller细胞中miR-29b、SP1、RAX、P-PKR、GLAST及GS表达发生改变，白藜芦醇对上述表达改变有抑制效应；通过预先在细胞中转染了miR-29b mimic和miR-29b抑制剂进一步研究发现，白藜芦醇抑制凋亡途径作用及调节SP1表达作用受明显影响，初步提示白藜芦醇通过miR-29b发挥抗糖尿病早期视损伤作用；此外高糖培养对Muller细胞L-[2,3-3H]-谷氨酸的摄取能力及GS活性有明显影响，白藜芦醇干预对上述影响有明显抑制作用，提示白藜芦醇可能通过调节谷氨酸摄取和转运保护高糖引起Muller细胞损伤。这些研究为将来采取更有效措施防护糖尿病早期视网膜病变提供了理论依据，对糖尿病视网膜病变的早期防护具有重要意义。