miR-124调控糖尿病视网膜病变中小胶质细胞活化机制的研究

Pathogenesis of diabetic retinopathy (DR) has not been completely elucidated. Whereas the mechanisms of vascular and neuronal pathology in DR are not yet fully understood, increasing evidence indicates that inflammation plays a pivotal role in the pathogenesis of DR. The increased expression of adhesion molecules and proinflammatory cytokines, as well as microglial activation in the retina or vitreous, all demonstrate that local inflammation may represent the central pathway leading to DR. DR may be a chronic, low-grade inflammation has been revealed. Our recent studies have reported advanced glycation end products (AGEs) lead to microglial activation, and found that miR-124 levels significantly decreased and miR-126 levels significantly increased in DR activated microglia as compared with normal microglia. On current study, streptozotocin (STZ) induces mouse model of diabetes mellitus and RAGE siRNA recombinant plasmid-liposome complex intervents to RAGE knock-out in vivo. We detect rat retinal microglial cells activation by immunofluorescence double staining confocal laser scanning microscopy, and measure the levels of miR-124, miR-126, C/EBP-α, Ets-1, MCP-1, IκBα in mice retinal tissue expression by RT-PCR and Western blotting. TUNEL method was used to detect apoptosis of retinal ganglion cells. In vitro, AGEs induces activation of microglial cells, using RT-PCR and Western Blotting to measure the levels of miR-124, miR-126, Ets-1, C/EBP-α, MCP-1, IκBα expression by microRNAs mimics, microRNAs inhibitor. We identify microglial cells using CD11b, CD45 double immunostaining fluorescence by confocal laser scanning microscopy, observation of microglial cell morphology by fluorescence microscopy, the detection of microglial cell migration by Transwell cell migration assays, detection of retinal microglial release of IL-1 β, TNF-α by ELISA. Study of the regulation of miR-124 and miR-126 on retinal microglial activation in diabetic retinopathy reveals the advanced glycation end products ( AGEs ) and its receptor ( RAGE ) metabolic pathways through inflammation leading to neuronal degeneration mechanisms in diabetes, and provides a new theory and new drug targets to the effective prevention of DR.

糖尿病视网膜病变(DR)是一种慢性、低度炎性反应己逐渐被揭示。我们最近的研究已经发现，糖基化终末产物(AGEs)可诱导小胶质细胞活化，活化的小胶质细胞中miR-124表达下调、miR-126表达上调。本研究拟在前期工作基础上，采用链脲佐菌素诱导糖尿病视网膜病变小鼠模型，检测miR-124、miR-126、C/EBP-α、Ets-1以及IκBα等蛋白的表达和分布，并检测视网膜小胶质细胞活化以及神经节细胞凋亡情况，在体外，采用AGEs诱导活化小胶质细胞，检测细胞内miR-124、miR-126、C/EBP-α、Ets-1以及IκBα等蛋白的表达，培养液中MCP-1、IL-1β、TNF-α的分泌，并观察小胶质细胞的活化状态。探讨糖尿病视网膜病变中miR-124通过直接作用于各种转录因子调控小胶质细胞活化的机制，以及揭示miR-124通过间接作用于miR-126进而调控小胶质细胞活化的机制。

糖尿病视网膜病变 (Diabetic Retinopathy，DR)是糖尿病最常见的并发症，多年来，对DR发病机制的探讨大多集中于微血管病变研究上，近年来，炎症反应作为DR不同发生因素的共同的、终末致病途径己逐渐被揭示，因而越来越多的学者认为DR可能是一种慢性、低度炎性反应。研究表明小胶质细胞是目前已知的唯一一种视网膜抗原呈递细胞，小胶质细胞一般处于静止状态，糖尿病视网膜病变中活化的小胶质细胞不仅机械性吞噬神经元，还可释放白细胞介素、TNF-a、趋化因子等促炎症因子，加速RGCs的死亡。进一步了解糖尿病视网膜病变中小胶质细胞的活化机制，可更好地为糖尿病视网膜病变的预防和治疗开辟新的视角。. 一方面，我们的研究发现Amadori糖化蛋白诱导视网膜神经元源性的MCP-1的释放，进而诱导了小胶质细胞的活化，miR-124在此起到关键性作用，而中药黄芩素具有明显的抗炎作用，通过表观遗传学机制，抑制Amadori糖化蛋白对miR-124的抑制作用，进而减轻小胶质细胞活化，抑制DR的发生发展。另一方面，我们的研究发现Amadori糖化蛋白诱导小胶质细胞的活化释放TNF-a，是通过激活Rac1进行调控的，它激活Rac1，进而活化ROS，小胶质细胞活化释放TNF-a，此过程依赖miR-124的调控，因为Amadori糖化蛋白通过表观遗传学抑制小胶质细胞中miR-124的表达，进而研究表明Amadori糖化蛋白诱导小胶质细胞活化依赖miR-124的调控。最后，我们的研究拓展性的发现lncRNA MALAT1作为ceRNA通过对miR-124的竞争性结合，实现了lncRNA与mRNA之间的对话和交流，对DR中小胶质细胞的活化起调控作用。. 通过本研究一方面我们不仅探讨miR-124在Amadori糖化蛋白诱导视网膜神经元源性的MCP-1的机制，同时探讨黄芩素的抗炎作用，进而减轻小胶质细胞活化的机制，为今后临床中应用黄芩抑制DR的发生发展提供了理论依据，展现了单味中药在DR防治中能够有效控制的机理，为将来转化提供依据。另一方面，我们通过在玻璃体腔过表达miR-124可以减轻DR的发生发展，可以据此为今后DR的临床治疗提供一个新的思路，比如采用纳米颗粒携带miR-124进行玻璃体腔注射控制严重的糖尿病视网膜病变。