转录因子X盒结合蛋白1参与视网膜色素上皮抗氧化防御机制

发布时间：2019-07-11 15:14

【摘要】：【目的】视网膜色素上皮(RPE)的氧化损伤在年龄相关性黄斑变性(ARMD)的发病机制中起着核心作用。本研究旨在探讨内质网应激及其重要的转录因子X盒结合蛋白1(XBP1)在丙烯醛引起的RPE氧化损伤中起到的作用,从而为阐明ARMD的发病机制提供思路。【方法】用75μmol/L丙烯醛分别处理人视网膜色素上皮细胞系(ARPE-19)2~24 h,观察内质网应激蛋白葡萄糖调节蛋白78(GRP78)及XBP1的表达。用XBP1 si RNA转染细胞,下调XBP1蛋白水平,用Western blot检测抗氧化基因Nrf2、SOD2在蛋白水平的表达;用DCF染色观察细胞活性氧产物(ROS)的产生;用TUNEL染色观察细胞凋亡改变。细胞用XBP1 si RNA或对照si RNA预处理后,加入丙烯醛,检测RPE细胞的凋亡情况。7只XBP1~(flox/flox)小鼠,一眼视网膜下注射Cre腺病毒,对侧眼视网膜下注射GFP腺病毒作对照。1周后取眼球。其中3只小鼠用TRIzol提取RPE的RNA,用实时RT-PCR法,检测XBP1的下游基因ERdj4和p58IPK在RNA水平的表达。另4只小鼠眼球作视网膜冰冻切片,观察腺病毒在视网膜下腔的表达,用免疫荧光染色法检测XBP1以及抗氧化基因Nrf2和SOD2在RPE的表达。【结果】丙烯醛分别处理RPE细胞2 h和4 h,GRP78的表达明显增加。处理6 h XBP1蛋白激活。XBP1表达的下调伴有抗氧化基因Nrf2和SOD2表达的减少,细胞内ROS的增加,并诱发细胞的凋亡。敲低XBP1以后丙烯醛对细胞的毒性作用明显增加。通过视网膜下注射Cre腺病毒成功转染XBP1~(flox/flox)小鼠RPE。与对照组相比,转染Cre腺病毒的小鼠RPE内的XBP1蛋白表达明显减少,RPE内的ERdj4和p58IPK RNA水平显著下调;抗氧化基因Nrf2和SOD2表达明显减少。【结论】丙烯醛作用于RPE细胞,激活内质网应激以及转录因子XBP1。抑制XBP1引起RPE内抗氧化基因表达的减少,ROS的产生增加,并且增加丙烯醛的细胞毒性。XBP1参与RPE细胞内抗氧化应激防御机制。
[Abstract]:[objective] oxidative damage of retinal pigment epithelial (RPE) plays a central role in the pathogenesis of age-related macular degeneration (ARMD). The purpose of this study was to investigate the role of endoplasmic reticulum stress and its important transcription factor X box binding protein 1 (XBP1) in the oxidative damage of RPE induced by acrolein, so as to provide a way to elucidate the pathogenesis of ARMD. [methods] Human retinal pigment epithelial cell line (ARPE-19) was treated with 75 渭 mol / L acrolein for 2 h, and the expression of endoplasmic reticulum stress protein glucose regulated protein 78 (GRP78) and XBP1 was observed. The XBP1 protein level was down-regulated by XBP1 si RNA, the expression of antioxidant gene Nrf2,SOD2 at protein level was detected by Western blot, the production of reactive oxygen species product (ROS) was observed by DCF staining, and the apoptosis was observed by TUNEL staining. After pretreatment with XBP1 si RNA or control si RNA, acrolein was added to detect the apoptosis of RPE cells. Seven XBP1~ (flox/flox) mice were injected with Creen adenoviruses subretinal in one eye and GFP adenoviruses were injected subretinal in the contralateral eyes as control. One week later, the eyeballs were taken. The RNA, extracted from RPE by TRIzol was used to detect the expression of downstream genes ERdj4 and p58IPK of XBP1 at RNA level by real-time RT-PCR. The expression of adenoviruses in the subretinal cavity was observed. The expression of XBP1 and antioxidant genes Nrf2 and SOD2 in RPE were detected by immunofluorescence staining. [results] the expression of GRP78 in RPE cells treated with acrolein for 2 h and 4 h was significantly increased. After 6 h treatment, the down-regulation of XBP1 protein expression was accompanied by the decrease of the expression of antioxidant genes Nrf2 and SOD2, the increase of intracellular ROS, and the apoptosis of XBP1 cells. The toxicity of acrolein to cells increased significantly after low XBP1. Successful transfer of XBP1~ (flox/flox) mouse RPE. by subretinal injection of Creenoviruses Compared with the control group, the expression of XBP1 protein in RPE was significantly decreased, the levels of ERdj4 and p58IPK RNA in RPE were significantly down-regulated, and the expression of antioxidant genes Nrf2 and SOD2 was significantly decreased. [conclusion] acrolein can activate endoplasmic reticulum stress and transcription factor XBP1. in RPE cells. [conclusion] acrolein acts on RPE cells and activates endoplasmic reticulum stress and transcription factor XBP1.. Inhibition of XBP1 decreased the expression of antioxidant genes in RPE, increased the production of ROS and increased the cytotoxicity of acrolein. XBP1 was involved in the intracellular antioxidant stress defense mechanism of RPE cells.
【作者单位】：中山大学中山眼科中心//眼科学国家重点实验室;西安交通大学医学院第一附属医院;美国俄克拉荷马大学医学院Harold
【基金】：国家自然科学基金青年项目(81200686) 高校博士学科点专项科研基金(20120171120108) 广东省自然科学基金博士启动项目(S2011040005378) 中山大学青年教师培育项目(11ykpy65)
【分类号】：R774

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