LncRNA-COX2影响IRF-1调控小胶质细胞极化在实验自身免疫性葡萄膜炎中的作用及机制研究

Uveitis is a common seen ocular inflammation entity with high rate of blindness due to inflammatory damage to the retinal neurons. The disturbance of Th1/Th2/Th17 cells balance results from infection triggers the pathogenesis of uveitis. Microglia is the major immune surveillance cell in the retina, the M1-polarized form of microglia ignites the intraocular inflammation in early phase of uveitis following the excessively expressed Th1 signaling. IRF-1 is a key regulator of Th1/Th2 homeostasis and may participat in the potential autocrine circuit of endogenous IFN-γ production in microglia, which subsequently drives microglia toward M1 polarization. Recent studies showed that LncRNA-cox2 might suppress IRF-1 expression as a natural response of innate immunity during infection. This project is designed to explore the possibility that whether LncRNA-cox2 could exert an inhibitory function in the pathogenesis of experimental autoimmune uveitis (EAU) through regulating the valve effect of IRF-1 on Th1/Th2 balance, and thus switch the M1 polarization of microglia to a resting form to ultimately prevent the onset or alleviate the severity of EAU. This study may offer a better insight into the treatment of uveitis.

葡萄膜炎是常见的致盲性眼病，视网膜神经破坏是葡萄膜炎致盲的主要原因。小胶质细胞是视网膜中最重要的免疫细胞，我们前期研究表明，小胶质细胞在葡萄膜炎初期出现炎性极化，同时其抗原递呈及迁徙能力明显上调，提示小胶质细胞为葡萄膜炎发生的始动细胞之一。小胶质细胞极化同Th细胞失衡相关，IRF-1是免疫细胞中Th1/Th2信号的关键阀门因子，可诱导小胶质细胞内部形成IFN-γ自分泌环路促使其炎性极化。近年研究表明LncRNA-COX2可对IRF-1进行有效调控。本项目以实验自身免疫性葡萄膜炎模型为研究对象，探讨LncRNA-COX2对IRF-1基因转录及相关miRNA表达水平的影响，旨在阐明LncRNA-COX2参与IRF-1调控小胶质细胞Th1/Th2平衡的机制，由此逆转小胶质细胞的异常极化，阻止葡萄膜炎的发生及后续视网膜神经破坏。本研究可为葡萄膜炎的防治提供新的生物学标记和干预靶点。

本项目旨在阐明LncRNA-COX2、IRF-1 与小胶质细胞内部Th1/Th2 平衡及其极化机制之间的关系，想通过干预调控小胶质细胞的极化来探讨维护视网膜稳态的可能性，为感染相关的葡萄膜炎乃至其他神经视网膜炎症性疾病的防治探寻新的突破口。项目研究主要包含以下几个内容：1）：探讨LncRNA-COX2及IRF-1 在EAU 动物模型中的表达；2）：探讨过表达或干扰IRF-1后，LPS 刺激下的小胶质细胞表达lncRNA-COX2变化及其极性变化及内在机制；迄今，我们成功构建Lewis大鼠EAU模型并发现LncRNA-Cox2、IRF-1 在视网膜组织内mRNA表达水平与临床炎症严重程度呈正相关，在LPS刺激造BV2细胞炎症模型中，IRF-1及LncRNA-Cox2随着LPS刺激的浓度及刺激时间增加，表达水平升高，并与促炎因子IL-12的mRNA表达浓度呈正相关。。BV2细胞转染IRF-1过表达质粒后， LncRNA-Cox2、iNOS、IL-12、IFN-γmRNA表达升高，Arg-1、IL4 mRNA表达下降，细胞上清液内IL-12浓度升高，pNF-κB及pSTAT-1活性升高伴有pSTAT 3活性下调。BV2细胞转染IRF-1干扰质粒后（Sh-IRF1）pNF-κB及pSTAT-1表达明显下降，LncRNA-Cox2、IL-4及IFN-γ表达下调， Arg-1及IL-4表达上调。综上，体内及体外的实验发现IRF-1及LncRNA-Cox2参与EAU炎症全过程，其表达水平与临床炎症程度呈正相关，IRF-1通过调控NF-κB，STAT1及STAT3信号通路影响小胶质细胞极化进而影响着EAU模型炎症程度。