中性粒细胞胞外陷阱在糖尿病视网膜病变炎性微血管损伤中的作用及调控机制研究

Diabetic retinopathy (DR) is one of the most severe eye diseases which would irreversibly destroy visual function. Up to now, leukocyte adhesion and inflammation have been considered as the key elements for the pathogenesis of DR. However, the mechanism of how leukocytes adhere to endothelial cell and induce inflammation is still not clear. Recently, a novel mechanism for neutrophil inducing inflammation was described as neutrophil extracellular traps (NETs).Interestingly, our research found that NETs was related to DR, which provide a new direction for preventing and treating diabetic retinopathy. In the present project, we plan to do researches to explore the effect of NETs on leukocyte adhesion and inflammatory microvascular injury, also how it is regulated. Firstly, we plan to compare the existence of NETs in blood and eye between patients with and without diabetic retinopathy. Secondly, we plan to explore the effect of high glucose and advanced glycation end products（AGEs) on NETs formation ,and whether peptidyl arginase deaminase 4 (PAD4) are involved. Thirdly, we plan to build mice retinal microvascular endothelial cells (RMECs) and neutrophil co-culture model, to explore whether neutrophil adhere to RMECs mainly as NETs, to analyze the effect of NETs on RMECs viability and apoptosis by inhibiting NETs formation with PAD4 inhibitor or digesting NETs with DNase. We would also build RMECs barrier model, to explore the effect of NETs on RMECs barrier integrity and the expression of tight junction proteins, and to figure out whether this effect is elastase, histone and myeloperoxidase dependent by using antagonists. At the meantime, we plan to build diabetic retinopathy model in PAD4 wild type and PAD4 knock out mice, to explore the effect of PAD4 on NETs mediated neutrophil adhesion and inflammatory microvascular injury. This project would explore the role of NETs in microvascular barrier integrity and the pathogenesis of DR, would provide novel theoretical basis for developing better method to prevent and treat diabetic retinopathy, which is with scientific value and clinical significance.

糖尿病视网膜病变（DR）是严重损害视功能的最常见眼科疾病之一。白细胞粘附和炎性损伤是DR特征性表现，但具体发生机制尚不清楚。中性粒细胞胞外陷阱（NETs）是一种新发现的炎症作用形式，前期研究发现NETs与DR关系密切，为DR研究提供了新方向。本研究拟比较DR患者和正常人血及眼内的NETs表达差异；通过中性粒细胞和视网膜微血管内皮细胞共培养和屏障建立，明确NETs在中性粒细胞粘附中的角色，阐明NETs对微血管内皮细胞和微血管屏障的损伤作用及主要效应成分；建立小鼠DR模型,观察视网膜血管中NETs粘附情况及对血管功能的影响；通过对肽酰精氨酸脱亚氨酶-4（PAD4）表达的体内和体外干预，阐明PAD4调控NETs形成参与DR炎性损伤的机制及对DR病程的影响。本项目将明确NETs在DR炎性损伤中的作用，寻找NETs调控因子为开发有效防治DR的新药提供理论依据，具有重要意义和科学价值。

糖尿病视网膜病变（DR）是严重损害视功能的最常见眼科疾病之一。白细胞粘附和炎性损伤是DR特征性表现，但具体发生机制尚不清楚。中性粒细胞胞外陷阱（NETs）是一种新发现的炎症作用形式，前期研究发现NETs与DR关系密切。本研究比较了不同人群外周血中中性粒细胞以及NETs表达的差异，发现非增殖性糖尿病视网膜病变（NPDR）患者和增殖性糖尿病视网膜病变（PDR）患者外周血中性粒细胞百分比，中性粒细胞/淋巴细胞比例以及NETs含量均高于正常人群；但NPDR患者和PDR患者之间外周血中性粒细胞百分比，中性粒细胞/淋巴细胞比例以及NETs含量没有显著差异。进一步的，我们进行体外实验发现高糖刺激中性粒细胞形成NETs，并且这一过程受氧化应激以及NF-kB通路的调控。我们发现高糖刺激可以轻微促进ROS产生，而DPI（一种不可逆的NADPH oxidase 的抑制剂）可以抑制高糖诱导的NETs形成；此外，高糖刺激可以诱导NF-kB活化，而NF-kB抑制剂JSH-23可以减少高糖诱导的NETs形成。然而，PAD4不参与高糖诱导的中性粒细胞NETs形成，因为PAD4抑制剂在体外对高糖诱导的NETs形成没有明显影响。我们同时发现TLRs受体参与高糖诱导NETs形成的过程，TLR4激动剂明显促进高糖刺激导致的NETs生成，而TLR2和TLR4的拮抗剂可以抑制高糖刺激的NETs形成。并且，TLRs受体可能通过氧化应激和NF-kB活化途径调控高糖诱导的NETs形成。令人惊喜的是，我们发现高糖刺激后的中性粒细胞不单单粘附HUVEC数量明显增加，而且有抑制HUVEC的迁移作用，更重要的是这些作用主要通过NETs胞外途径发生。此外，高糖刺激后的中性粒细胞还主要通过胞外途径抑制了血管内皮细胞紧密连接蛋白的表达，破坏了血管内皮细胞屏障功能。在小鼠实验中，我们发现AIP1参与DR新生血管形成和血管通透性改变。与B6小鼠相比，AIP1-KO糖尿病小鼠的新生血管数量更密集，血管渗漏更明显，进一步的研究正在进行中。以上这些研究成果明揭示了糖尿病环境中NETs炎性损伤对于血管内皮细胞迁移和屏障功能的破坏作用。对于NETs形成的调控机制研究也为寻找调控NETs产生的目标分子提供了重要的理论依据。