IL-17A经Müller细胞外泌体miR-92a-3p机制促进糖尿病性视网膜病变

Over the recent years, inflammation has been deemed as an important factor in promoting the occurrence and development of diabetic retinopathy (DR). Interleukin (IL)-17A, a proinflammatory cytokine, has been associated with autoimmune and inflammatory diseases. However, the role and involved mechanism of IL-17A in DR are still not well known. We have recently reported that IL-17A directly injures retinal Müller cell function and exacerbates DR, and more recently shown that IL-17A does not directly induce apoptosis of retinal ganglion cells. We hypothesized that retinal Müller cell secretion of exosomes is an important mechanism by which IL-17A inflammatory information is transmitted to retinal ganglion cells to accelerate DR development. Accordingly, we recently performed the sequencing analysis of microRNA (miRNA) in exosomes derived from retinal Müller cells. We found that IL-17A remarkable upregulated miR-92a-3p expression in the exosomes. Thus, on the basis of our reported work and pre-experimental results, in this project we firstly clarify that IL-17A damage to retinal ganglion cells depends on the mediation of Müller cells. Secondly, we reveal the mechanism by which Müller cell dysfunction induced by IL-17A results in retinal ganglion cell apoptosis and DR development. We will treat retinal ganglion cells with exosomes derived from Müller cells that have been treated with IL-17A under the high glucose condition or inject the exosomes into the vitreous cavity of DR mice. In addition, the Müller cell exosomes are treated with miR-92a-3p inhibitor and then the miR-92a-3p inhibitor loaded exosomes act on retinal ganglion cells or are injected into the vitreous cavity of DR mice. Subsequently, we demonstrate that the exosomes derived from Müller cells are taken up into ganglion cells and release miR-92a-3p in the ganglion cells to regulate its target gene WNT5A and affect DR. These studies will provide a new insight into the inflammatory pathogenesis mediated by IL-17A in DR and a novel target for treatment of DR with anti-inflammatory therapeutic strategies.

近年来炎症在糖尿病性视网膜病变（DR）中的作用受到了更多关注，但促炎细胞因子白介素-17A（IL-17A）的作用及机制仍知之甚少。我们已报道IL-17A促进Müller细胞功能紊乱及DR进展，最近研究还说明IL-17A没有直接引起神经节细胞凋亡。我们假设IL-17A通过Müller细胞分泌外泌体来传递炎性信息至神经节细胞以加速DR发展。据此我们抽提出Müller细胞外泌体进行了miRNA测序分析，发现IL-17A上调外泌体中miR-92a-3p表达。本项目拟在上述工作基础上，首先阐明IL-17A对神经节细胞的损伤作用依赖于Müller细胞的介导；然后揭示机制，将Müller细胞来源的外泌体及miR-92a-3p抑制剂负载的外泌体作用于神经节细胞或注射入DR小鼠玻璃体内，说明外泌体被神经节细胞摄取后释放出miR-92a-3p调节靶基因WNT5A及影响DR。为DR发病机制及治疗策略提供新视角。

糖尿病性视网膜病变（DR）是最常见的、严重的糖尿病并发症，也是导致青中年人视盲的主要原因。近十年来，我们致力研究促炎细胞因子白介素-17A（IL-17A）对DR发生发展的影响及其作用机制，希望对DR的炎性发病机制及治疗措施提供新的实验资料。在本项目中，首先我们发现IL-17A缺陷的Akita糖尿病小鼠的DR症状明显减轻，提示IL-17A参与DR的发病机制。有趣的是，视网膜神经节细胞不表达IL-17受体，IL-17A也不直接影响神经节细胞的幸存，而是依赖于视网膜Müller细胞的介导来发挥它对神经节细胞的损伤作用。于是，我们进一步探讨了Müller细胞介导IL-17A促进DR的分子机制。通过应用透射电子显微镜、纳米颗粒追踪分析及外泌体特异蛋白表达的技术，我们鉴定了Müller细胞释放的外泌体。接着，通过利用高通量测序服务及qRT-PCR检测，我们说明了IL-17A上调外泌体中miR-92a-3p的表达。在观察到Müller细胞分泌的外泌体可被神经节细胞所摄取后，我们通过生物信息学软件、qRT-PCR及双荧光素酶报告分析，确定了Notch-1是miR-92a-3p的直接靶基因。然后，我们将IL-17A或/和miR-92a-3p inhibitor/mimic处理Müller细胞后分离出来的外泌体作用于视网膜神经节细胞，结果表明Müller细胞释放的外泌体miR-92a-3p通过抑制其靶基因Notch-1传递了IL-17A对神经节细胞的损伤作用。进一步，我们观察到DR小鼠的视网膜神经节细胞在体内也可摄取Müller细胞来源的外泌体。然后，我们将IL-17A处理Müller细胞后分离出来的外泌体注射入DR小鼠的眼玻璃体内，发现视网膜Notch-1的表达下调，同时视网膜神经节细胞的凋亡及视网膜血管的退变加重；更重要的是，我们将miR-92a-3p inhibitor处理Müller细胞后分离出来的外泌体注射入DR小鼠的眼玻璃体内，阻抑了IL-17A外泌体的作用。本项目研究一方面说明了IL-17A参与DR的发生和发展，另一方面通过建立miRNA-mRNA信息传递链揭示了IL-17A促进DR进展的新颖机制。尤其是，我们提示了眼玻璃体内注射外泌体靶向调节miR-92a-3p--Notch-1传递链有可能减缓DR进程，这对于治疗DR具有潜在的应用前景。