AChE调控糖尿病视网膜炎症反应和新生血管形成的作用及分子机制

Diabetic retinopathy (DR) is a leading cause of blindness with characteristics of chronic and subclinical inflammation. Previous studies indicate that Acytylcholinesterase (AChE) is closely associated with inflammatory response. We, for the first time, demonstrated that retinal AChE expression in mouse was markedly increased by intravitreous injection of pro-inflammatory cytokine-TNF-α. Genetical knockdown of AChE expression dramatically reduced TNF-α-induced retinal leukostasis and ICAM-1 expression. However, contribution of AChE to retinal inflammation and neovascularization during diabetes remains to be elucidated. Thus, in this study, animal model of streptozotocin-induced diabetes and oxygen-induced retinopathy (OIR) will be set up by using AChE heterozygous (AChE+/-) and wild type (AChE+/+) mice. Retinal expression and distribution of AChE will be detected by western-blot analysis and immunofluorescence. Diabetes-induced expression of adhesion molecules, retinal leukostasis, vascular leakage, formation of accellular capillaries and pericyte gosts will be examined in both AChE+/- and AChE+/+ mice. In addition, retinal vessel obliteration, neovascularization and revascularization will be compared in both AChE+/- and AChE+/+ OIR mice by Isolectin IB4 staining. Moreover, methylation and/or acetylation of histones associated with ACHE gene promoter in high-glucose treated retinal cells will be determined by immunoprecipitation (ChIP) and quantitative PCR (Q-PCR) to elucidate regulatory mechanisms of AChE during diabetes. Adenoviral vector expressing constitutive active AChE or mediating AChE siRNA will be constructed. Retinal pigment epithelial cell, rat Mȕller cell or human retinal vascular endothelial cells will be used for in vitro assay. Phosphorylation of NF-κB related kinases will be assessed by western-blot analysis. Would healing assay, transwell migration assay, matrigel tube formation and aorta ring assay will be used to determine endothelial angiogenic capacities. In addition, cellular VEGFR2 signaling and mobilization will be determined. Taken together, the overall goal of this project is to further elucidate the mechanism of inflammatory response and NV formation in DR and lay the theoretical and experimental basis for seeking novel therapeutic target of DR.

糖尿病视网膜病变（DR）是不可逆致盲眼病之一，其常表现出慢性及亚临床炎症特征。既往研究表明乙酰胆碱酯酶（AChE）可能参与调控组织的炎症反应，但具体机制尚未完全阐明。我们首次发现了玻璃体腔注射TNF-α可明显上调视网膜AChE表达，基因水平调控AChE明显抑制TNF-α诱导的视网膜炎症。本课题拟在前期工作的基础上，采用AChE基因缺陷小鼠建立DR相关动物模型，检测视网膜AChE表达定位并探讨AChE表达改变对视网膜炎症因子产生、血管渗漏及白细胞粘附、无细胞毛细血管和鬼影周细胞形成以及新生血管（NV）生成的影响；在此基础上，采用ChIP解析糖尿病状态下 AChE表达调控的表观遗传学机制并阐明调控AChE表达对NF-κB信号通路关键蛋白磷酸化的影响和对VEGFR2胞内信号传递的作用；以期揭示DR炎症反应及NV生成的分子机理；为丰富糖尿病视网膜病变的发病机制及寻找治疗新靶点奠定基础。

乙酰胆碱酯酶（Acetylcholinesterase, AChE）作为中枢神经系统炎症的标志物备受关注，但是其在视网膜炎症发生发展中作用尚不清楚。糖尿病视网膜病变被认为是低度炎症性疾病，因此本项目旨在通过建立糖尿病小鼠动物模型阐明AChE表达改变在调控视网膜炎症反应和新生血管生成中的作用机制，在此基础上揭示了糖尿病视网膜AChE表达调控的表观遗传学机制。在本项目支持下，所取得的研究结果如下；首先，在TNF-α诱导的视网膜炎症反应动物模型中，我们发现视网膜AChE表达上调，从基因水平敲除AChE表达或者给予AChE抑制剂显著抑制TNF-α诱导视网膜炎症因子ICAM-1表达，减轻血管处白细胞粘附及视网膜内白细胞浸润、缓解视网膜血管渗漏；体外机制研究表明，敲除AChE表达或者抑制AChE活性通过抑制IKK/IκB/NF-κB信号通路活化阻断TNF-α诱导的ICAM-1表达。其次，我们建立了链脲佐菌素诱导的糖尿病动物模型，发现AChE在野生型糖尿病小鼠视网膜表达上调并伴有ICAM-1水平增加、血管处白细胞粘附增多、血管渗漏及血管屏障功能破坏；这些改变在AChE基因缺陷型小鼠视网膜明显减轻或者可以被AChE抑制剂多奈哌齐阻断，进一步研究我们还发现多奈哌齐可以明显抑制NLRP3炎症小体的活化，提示我们抑制AChE减轻糖尿病视网膜炎症改变可能是通过调控NLRP3炎症小体活化实现的；此外，我们还建立了氧诱导的新生小鼠视网膜病变模型模拟糖尿病视网膜新生血管形成改变，结果证明多奈哌齐可明显降低视网膜新生血管形成，抑制HIF1-α活化及VEGF表达。最后，我们采用组蛋白去乙酰化酶抑制剂SAHA干预糖尿病小鼠，发现SAHA可增加小鼠视网膜H3K9乙酰化水平并降低视网膜AChE表达，提示我们糖尿病视网膜AChE表达上调可能受表观遗传学调控。在本项目资助下，已发表第一作者/通讯作者收录论文3篇，其中1篇为SCI论文，2篇为核心期刊论文；投稿会议论文3篇并在国际眼科学大会作英文发言，培养硕士研究生3名。