血浆激肽释放酶酶切NMDA受体1促进糖尿病视网膜神经损伤及其机制研究

Diabetic retinopathy (DR) is a common and sight-threatening complication of diabetes mellitus, and also the leading cause of new cases of blindness among adults. Plasma kallikrein contributes to excessive retinal vascular permeability and diabetic macular edema. In our preliminary results, we have shown that PK induced cortical neuronal cell death mediated by the cleavage at Arg323 extracellular domains of N-methyl-D-aspartate (NMDA) receptor 1 (NR1), which increase NMDA current and NMDA-induced Ca2+ influx. We also found that intravitreal (IVT) injections of activated PK in diabetic rats decreased Brn3a and increased caspase 3 expression in retinal extracts, and exacerbated electroretinography (ERG) defects. Based on these new preliminary data, we hypothesize that PK could induce retinal neuron degeneration and promote diabetic retinopathy through cleavage-dependent activation of the NR1 subunit of the NMDA receptor. This proposal will characterize the effects of plasma kallikrein on the neuroretina in diabetes using both gain-of-function (PK IVT injections) and loss-of-function approaches (PK deficiency mice and PK inhibitors ASP440); investigate the potential effects and mechanisms of PK on retinal ganglion cells (RGCs) death by using MTT-8, flow cytometry, whole cell patch clamp technique, and calcium imaging; and characterize the effect of truncated NR1 on RGCs apoptosis and neuroretinal function in vivo and vitro. In conclusion, the aim of this study is to reveal new mechanisms and find new potential target of PK for prevention and treatment of diabetic neuroretinal degeneration and dysfunction.

糖尿病视网膜病变是糖尿病严重的并发症之一。血浆激肽释放酶（plasma kallikrein， PK）增加糖尿病视网膜血管通透性和加重黄斑水肿。我们前期研究首次发现PK可以酶切截短N-甲基-D-天门冬氨酸（NMDA）受体1（NR1），导致NMDA和钙离子内流，促进大脑皮质神经元细胞凋亡；还发现PK眼球内注射降低糖尿病SD大鼠视网膜神经电位，增加视网膜casepase 3表达，降低Brn3a表达；这提示PK可通过酶切NR1调控糖尿病视网膜视神经损伤和变性。本课题研究外源性PK、PK缺失和PK抑制剂ASP440对糖尿病视网膜神经节细胞（RGCs）凋亡和神经功能的影响；采用MTT-8法、流式细胞仪、膜片钳，阐明PK促进RGCs凋亡的作用及机制；体内和体外实验探讨PK在过表达截短的NR1对糖尿病RGCs和视网膜神经功能的影响，揭示糖尿病视网膜神经变性新的发病机制，为防治开辟新靶点。

（1）糖尿病视网膜病变是糖尿病常见的并发症之一，导致视力受损，严重时致盲。在糖尿病视网膜病变中，血浆激肽释放酶/激肽系统（Plasma kallikrein-kinin system， KKS）激活，血浆激肽释放酶（plasma kallikrein, PK）表达上升，激活的PK会增加视网膜血管通透性和加重糖尿病黄斑水肿，但PK对糖尿病神经变性的影响和机制尚未阐明。本研究中采用链脲佐菌素（STZ）诱导的糖尿病模型，通过玻璃体腔内注射外源性PK，研究PK导致RGC凋亡的作用机制。结果表明：外源性PK注射导致视网膜结构破坏，细胞异质性增大，RGC数目减少，视网膜caspase3表达增加，brn3a表达降低；糖尿病小鼠全视网膜铺片免疫荧光取12个区域计数平均RGC数目：糖尿病组（n=10）（病程5个月）为842±63个，对照组（n=10）为917±146个；随糖尿病程进展，RGC呈下降趋势，糖尿病第7个月，糖尿病组(n=10)为781±69个，对照组(n=9)为810±93个。玻璃体腔内注射NMDA呈现浓度依赖性促进RGC凋亡，我们前期研究发现PK通过NMDA介导，增加NMDA内流动作电位和促进钙离子内流，从而导致神经元细胞死亡。采用CRISPR/Cas9靶向敲除KLKB1基因，获得KLKB1基因敲除小鼠。采用STZ腹腔注射建立糖尿病小鼠模型，发现玻璃体腔内注射NMDA后，RGC明显凋亡，PK缺失对糖尿病RGC的保护作用还在进一步研究中。我们的研究提示，PK在糖尿病视网膜神经变性中起一定作用，是糖尿病视网膜病变防治的潜在靶点。（2）基于全球疾病负担研究（Global Burden of Disease Study 2017）数据，我们研究发现：糖尿病、高空腹血糖和高体重指数导致严重的全球疾病负担，不同国家或地区之间有着显著差异，在中低、中、中高社会发展指数国家疾病负担更重。糖尿病视网膜病导致的视力损害在男女间存在差异，总体来说女性疾病负担大于男性。（3）我们研究发现：颈动脉粥样硬化、下肢周围动脉疾病、慢性肾脏疾病、代谢综合征增加2型糖尿病患者冠心病缺血性脑梗塞的风险。