O-GlcNAc糖基化通过调节pSTAT3-VEGF通路导致糖网病BRB破坏机理

Diabetic retinopathy (DR) is one of the most common microvascular complications caused by diabetes mellitus，which is closely associated with the breakdown of blood-retinal barrier (BRB), but the mechanism underlying the disease is not well understood. Recently, we observed that it is associated with elevated O-linked β-N-acetyl glucosamine (O-GlcNAc) levels in DR. The augmented O-GlcNAc could cause overexpression of STAT3 phosphorylation, which is related with the increased expression of vascular endothelial growth factor (VEGF) and decreased occludin levels. Therefore, we suppose that inhibition of O-GlcNAc could downregulate pSTAT3-VEGF pathway to protect BRB function. We will use animal models and primary endothelia cells, to confirm that STAT3 are co-modified by O-GlcNAcylation and phosphorylation, to inspect the effects of O-GlcNAc on pSTAT3 expressions, to explore the regulatory mechanism of O-GlcNAc on BRB breakdown via pSTAT3-VEGF pathway by co-immunoprecipitation, virus infection, western blot, immunofluorescence and retinal serial sections. Furtherly, clinical samples will be used to confirm the relationship between the modification levels and BRB function. The study will help to illustrate the pathogenesis of BRB breakdown and provide new insights in the treatment of DR.

糖尿病视网膜病变（简称“糖网病”、DR）是糖尿病最常见微血管并发症之一，发病原因主要是血-视网膜屏障（BRB）破坏，但机制仍未完全阐明。我们前期研究发现DR中存在O-GlcNAc糖基化的过度修饰，致使血管内皮生长因子（VEGF）表达上调且BRB紧密连接蛋白丢失，这可能与O-GlcNAc促进STAT3磷酸化（pSTAT3）有关。据此我们推测抑制O-GlcNAc通过下调pSTAT3-VEGF通路发挥对BRB的保护作用。本项目拟采用原代内皮细胞及动物模型，结合Co-IP、病毒转染、WB、免疫荧光、视网膜铺片等技术验证STAT3存在O-GlcNAc与磷酸化共修饰，探讨O-GlcNAc对pSTAT3的修饰及调控作用，从而阐明抑制O-GlcNAc下调pSTAT3-VEGF通路保护BRB的分子机制；并通过临床样本进一步验证。该项目将为DR发病提出新的机理，并为探索DR治疗新靶点提供思路。

糖尿病视网膜病变（Diabetic retinopathy, DR）是糖尿病主要并发症之一，随着糖尿病发病人数的不断增加，DR已成为工作人群视力丢失的主要原因。DR引起视力下降的主要原因是糖尿病性黄斑水肿及增殖性糖尿病视网膜病变。虽然抗VEGF药物的出现及玻璃体视网膜手术为缓解DR患者视力丢失带来希望，但由于DR的病因错综复杂，目前仍无有效手段从病因上抑制DR发生、发展。我们前期研究发现高血糖环境引起O-GlcNAc糖基化修饰水平升高，参与VEGF 表达上调及新生血管形成的多个环节，其中信号转导及转录激活因子3（Signal transducer and activator of transcription3，STAT3）是关键调控者之一。本项目将以PDR患者玻璃体临床标本为研究对象，检测糖基化修饰水平及STAT3的升高情况。壁腹膜下注射链脲佐霉素构建糖尿病大鼠模型，高糖培养基（25mM）培养人血管内皮细胞，检测结果显示STAT3的表达水平明显升高，其升高程度与O-GlcNAc糖基化修饰水平相一致。通过玻璃体腔注射Thiamet G过表达O-GlcNAc 糖基化，及Alloxan抑制O-GlcNAc 糖基化，检测O-GlcNAc 糖基化对STAT3表达的关键作用。CO-IP结果显示，STAT3存在糖基化及磷酸化共修饰，协同调节pSTAT3Y705 表达的表达水平，使pSTAT3Y705表达水平处于平衡状态；但竞争性抑制STAT3S727 位点。进一步研究结果还显示O-GlcNAc糖基化通过STAT3磷酸化调节VEGF表达，减少高血糖环境诱导的血管内皮细胞凋亡，对血视网膜屏障起保护作用。该项目的研究为阐明DR发病机制提供了证据，为早期干预DR提出了可行性的治疗靶点。