乙酰化调控Nrf2/ARE信号通路在视网膜缺血再灌注损伤中的作用及机制研究

The pathogenesis of retinal ischemia/reperfusion injury is complicated, and so far, there is a lack of effective treatments. The Nrf2/ARE pathway is one of the key endogenous anti-oxidant protection systems, and studies have shown that activation of Nrf2/ARE pathway protect retina from I/R injury. Our works have shown that histone deacetylase inhibitors(HDACIs) protect retina from I/R injury by reducing oxidative stress and endoplasmic reticulum stress, and our latest studies have shown the Nrf2 is remarkablely upregulated by HDACIs in retina after I/R injury. Thus, we hypothesize that HDACIs should protect retina from I/R injury via the Nrf2/ARE signaling pathway activated by acetylation. To validate the hypothesis, by using siRNA, immunofluorescence and other molecular biotechnology, we will investigate the effects of Nrf2 silence on retinal morphologic, functional changes, and cell apoptosis in Nrf2-deficient mice and retinal ganglion cells, to show the role of Nrf2 in the HDACIs-mediated protection. Then, we will analyze the effects of HDACIs on the activation, nuclear translocation of Nrf2, and activation of Nrf2/ARE pathway downstream genes SOD,HO-1, to identify the role and possible mechanisms of targeting Nrf2/ARE pathway by acetylation in retinal I/R injury. Our study will provide new strategies for the clinical treatment of retinal ischemic diseases and development of neuroprotective drugs.

视网膜缺血再灌注损伤发病机制复杂，目前尚缺乏有效的治疗措施。Nrf2/ARE通路是细胞抵抗氧化应激损伤重要的内源性通路之一。近年来研究显示该通路的激活对视网膜缺血再灌注损伤可发挥明显保护作用。我们前期研究发现去乙酰化酶抑制剂（HDACIs）具有抑制细胞氧化应激、内质网应激保护视网膜缺血再灌注损伤的作用，新近研究发现HDACIs可明显上调视网膜中Nrf2的表达。因此，我们推测：Nrf2在HDACIs治疗视网膜缺血再灌注损伤中起重要作用。本项目拟应用siRNA、免疫荧光等方法，在Nrf2-/-小鼠和视网膜神经节细胞中探讨HDACIs介导的乙酰化对视网膜中Nrf2的基因转录、蛋白表达和核转位的影响，及与Nrf2/ARE通路下游分子HO-1、SOD间的作用，明确Nrf2是否在HDACis治疗视网膜缺血再灌注损伤中发挥重要作用，并探讨其可能的分子机制，为缺血性眼病的防治及神经保护药物研发提供新视角。

本课题组前期研究发现缺血再灌注(Ischemia/Reperfusion,I/R)后视网膜中组蛋白乙酰化程度降低，去乙酰化酶抑制剂（HDACis）提高乙酰化程度，减轻氧化应激反应及细胞凋亡；且近期研究发现HDACis显著上调视网膜中Nrf2表达，而Nrf2/ARE通路是抗氧化应激重要的信号途径，本课题以这些发现为基础，应用Nrf2基因敲除小鼠前房加压缺血再灌注模型，且构建Nrf2 siRNA病毒载体，转染视网膜神经节细胞，首先在体内外实验中阐明Nrf2基因沉默对HDACis干预的视网膜I/R损伤后组织形态、功能、及细胞凋亡，氧化应激酶表达的影响, HDACis的干预减少Nrf2抑制因子Keap1的表达，且促进Keap1-Nrf2间的解离而增强Nrf2的核内转位。并且进一步证明HDACis通过乙酰化作用增强Nrf2表达，并且提高Nrf2与ARE间的结合力，通过Nrf2/ARE通路激活相关下游抗氧化因子的转录表达. 本项目阐述乙酰化调控Nrf2/ARE信号通路对视网膜I/R损伤的保护作用及其可能的分子机制，为今后的治疗提供新方向及防治的新靶点。