[关键词]
[摘要]
目的:研究细胞自噬在缺氧条件下视网膜色素上皮(RPE)细胞表达炎性因子IL-1β和IL-6中的作用。
方法:将体外培养的人RPE细胞系随机分为缺氧组、3-甲基腺嘌呤(3-MA)+缺氧组、氯喹(CQ)+缺氧组和对照组。培养24h后采用Western blot法检测各组细胞的自噬关键蛋白微管相关蛋白1轻链3B(LC3B)、Beclin-1及p62的相对表达水平; 采用ELISA法检测各组细胞上清液中IL-1β和IL-6的浓度。
结果:缺氧组IL-1β和IL-6的浓度明显高于对照组、3-MA+缺氧组和CQ+缺氧组(均P<0.01)。缺氧组细胞中LC3B-Ⅱ/Ⅰ和Beclin-1的相对蛋白表达量均明显高于对照组和3-MA+缺氧组,缺氧组中LC3B-Ⅱ/Ⅰ的相对蛋白表达量低于CQ+缺氧组(均P<0.01)。缺氧组细胞中p62相对蛋白表达量明显低于对照组、3-MA+缺氧组和CQ+缺氧组(均P<0.01)。
结论:缺氧使RPE细胞LC3B-Ⅱ/Ⅰ、Beclin-1表达增强,p62表达减弱,并促进细胞分泌IL-1β和IL-6,自噬抑制剂3-MA和CQ能够减少RPE细胞表达IL-1β和IL-6。
[Key word]
[Abstract]
AIM:To investigate the role of cellular autophagy in the expression of IL-1β and IL-6 by retinal pigment epithelium(RPE)cells under hypoxia.
METHODS: A cultured human RPE cell line was randomly divided into the hypoxia group, 3-methyladenine(3-MA)+ hypoxia group, chloroquine(CQ)+ hypoxia group and the control group. Cells incubated in a hypoxic incubator were set as the hypoxia group. After culture for 24h, western blot was used to analyze the relative expression of autophagy-associated key proteins in each group, including microtubule associated protein 1 light chain 3B(LC3B), Beclin-1 and p62. Then, ELISA was applied to detect the concentration of IL-1β and IL-6 in the cell culture supernatants.
RESULTS: The concentration of IL-1β and IL-6 in the hypoxia group was significantly higher than that in the control group 3-MA+hypoxia group and CQ+ hypoxia group(all P<0.01). The relative expression of LC3B-II/I and Beclin-1 in the hypoxia group was significantly higher than that in the control group and 3-MA+hypoxia group. The relative expression of LC3B-II/I in the hypoxia group was significantly lower than that in the CQ+ hypoxia group(all P<0.01). The relative expression of p62 in the hypoxia group was significantly lower than that in the control group, 3-MA+hypoxia group and CQ+ hypoxia group(all P<0.01).
CONCLUSION: Hypoxia can enhance the expression of LC3B-II/I and Beclin-1 in RPE cells, weaken the expression of p62, and promote the secretion of IL-1β and IL-6. Autophagy inhibitors 3-MA and CQ can reduce the expression of IL-1βand IL-6 in RPE cells.
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[基金项目]
国家自然科学基金项目(No.81500726); 西安市科技局医学研究项目[No.201805097YX5SF31(4)]; 陕西省普通高校“青年杰出人才”支持计划