Notch 信号通路在早期糖尿病视网膜病变发生中的作用和相关机制研究

Diabetic retinopathy (DR) is the most common complication of diabetes, and remains the leading cause of blindness among working-age individuals all over the world. Current treatments for DR (laser photocoagulation, intravitreal corticosteroids, intravitreal anti-VEGF agents and vitreoretinal surgery) are indicated in advanced stages of the disease, and are associated with significant adverse effects. Therefore, new pharmacological treatments for the early stages of DR are needed. DR is a microvascular disease of the retina. However, there is growing evidence to suggest that retinal neurodegeneration and inflammation are early events in the pathogenesis of DR. In the previous study we discovered that high glucose induces oxidative stress and activates E2f1, which mediate the cell cycle re-entry and apoptosis of retinal neurons. RNA sequcencing analysis of high glucose cultured retinal explants revealed that, high glucose-related deregulated genes (DEGs) include many members of Notch pathway including receptor, ligands, Fringe enzyme and downstream effector genes. Thus we hypothesize that Notch pathway activation and its reciprocal interaction with several other signal pathways including E2f1, Nrf2, Hif1a and Kdm5a may be the underlying mechanisms that lead to early stage of DR. We will employ retinal explant ex vivo culture system and spontaneous type II diabetic mouse model C57BL/KsJ-db/db to address how Notch pathway affects the pathogenesis of DR. Aim 1 the expression patterns of Notch pathway components, and their relationship with cell proliferation, cell apoptosis, retinal cell types, and blood vessels in WT and E2f1-/- retinal explants under 4 pathological situations (high glucose, oxidative stress, hypoxia, inflammation), and db/db mouse retina at different time points (8, 12, 16, 20 weeks). The mRNA and protein levels and their cellular location will be examined by RT-PCR, Western blot and immunohistochemistry or in situ hybridization. Aim 2 the effects of doxycycline-induced NICD1 expression on the expression patterns of Notch pathway components, and the status of cell proliferation, cell apoptosis and blood vessels in retinal explant under 4 pathological situations. Aim 3 the effects of shRNA and gamma-secretase inhibitor (GSI) on the expression patterns of Notch pathway components, and retinal function (ERG), vascular leakages (FFA), the status of cell proliferation, cell apoptosis and blood vessels in retinal explant under 4 pathological situations and db/db mouse retinas.

糖尿病视网膜病变（DR）是糖尿病最常见的并发症，虽然相关假说很多但其发病机制仍未完全明了。DR是一种微血管病变，但其早期改变主要为神经细胞凋亡和炎症。我们前期研究发现E2f1介导的神经元重新进入细胞周期和细胞凋亡是早期DR发生的重要机制、高糖激活了视网膜的Notch通路。我们认为高糖激活Notch通路、Notch通路和多个信号通路之间的正反馈环路是DR发生发展的重要机制。我们拟从3 个方面进行研究。1. 在野生型和E2f1-/-小鼠全视网膜培养DR模型和II型糖尿病db/db小鼠模型中，观察Notch通路成员的表达情况。2.通过药物诱导NICD1表达，测试上调Notch通路活性后视网膜细胞的反应，以及Notch通路是否和高糖、低氧、氧化应激、炎症刺激、E2f1激活有协同作用。3.通过RNA干扰技术和gamma-分泌酶抑制剂抑制Notch通路活性，观察下调Notch通路活性对早期DR的影响。

糖尿病视网膜病变（DR）是糖尿病最常见的并发症。早期DR主要表现为神经元凋亡、炎症及血管病变。晚期出现增殖型DR（PDR），表现为新生血管、视网膜下纤维化和视网膜变性。本项目主要研究了I型、II型糖尿病动物模型视网膜病变的发生机制及其和Notch等信号通路的关系。本研究的主要研究内容和结果如下：(1) 发现ROCK抑制剂12j可以抑制体外高糖培养的小鼠全视网膜DR模型的多个表型如细胞凋亡、胶质细胞增生、氧化应激和血管不退化。 (2)链脲佐菌素 (STZ) 诱导的新生大鼠视网膜病变主要由STZ的直接视网膜毒性导致的视网膜祖细胞生长停滞、凋亡引起，而不是高血糖；因为玻璃体腔注射的STZ并不导致血糖升高，但仍然导致新生大鼠视网膜病变（视网膜血管发育延迟、视网膜细胞凋亡、小胶质细胞增加及炎症）。因此STZ不适于检查新生啮齿动物糖尿病相关视网膜病变的机制研究。(3) II型糖尿病大鼠的DR及视网膜炎症和肠道菌群关系研究。II型糖尿病可导致肠道菌群失调，早期DR主要表现为视网膜神经病变。通过广谱抗生素灌胃可以减少成年大鼠视网膜小胶质细胞，缓解糖尿病性神经节细胞损伤。 (4) ZDF自发II型糖尿病大鼠表型，包括血管渗漏、神经节细胞减少和胶原细胞增生，胶质细胞TGF1/ Caveolin-1通路活化。 (5) 兔体内II型糖尿病诱导DR模型，表型包括视乳头水肿、血管渗漏、RPE下脂质沉淀、神经变性、血管增生、ERG异常，可能成为今后DR研究的新的平台；（6）建立了恒河猴视网膜下纤维化（SRF）模型，发现内层视网膜变薄和ERG震荡电位下降可能是SRF的早期特征；发现了4个SRF相关因子（GM-CSF, IL-10, TGF2 and VEGF），早期单次注射抗VEGF抗体可部分抑制SRF形成，VEGF可能是SRF发生重要促进因子。（7）发现p21cip1对异常分裂的视杆细胞具有保护作用。（8）发现Rb蛋白可以抑制Hif靶基因（如Epo, Vegfa, Vegfr2, Tie2, Bnip3基因），Rb/p107/Vhl三敲除小鼠是一个很好的视网膜新生血管模型。这些为阐明DR（包括晚期的PDR）的发病机制提供了新的证据，Rock激酶通路及Caveolin-1通路和Notch信号通路关系密切，它们和肠道菌群失调是今后DR治疗的潜在靶点。