Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
National Clinical Research Center for Eye Diseases, Shanghai, China.
Cutan Ocul Toxicol. 2021 Mar;40(1):7-13. doi: 10.1080/15569527.2020.1860082. Epub 2020 Dec 17.
This study aimed to investigate the protective effects of quercetin on the tight junction proteins of human retinal pigment epithelial cells (ARPE-19 cells) suffering from oxidative stress injury and explore the possible mechanism. HO (300 μM) was used to establish an oxidative stress model of ARPE-19 cells. ARPE-19 cells were pretreated with different concentrations (0-80 μM) of quercetin before HO exposure. The expression and distribution of tight junction proteins and autophagy-related proteins were detected by Western blot and immunostaining. ARPE-19 cells were pretreated with 5 mM 3-methyladenine (3-MA). The cell viability weakened in the HO group compared with the control group. However, it was preserved after pretreatment with quercetin. It was observed that the expression levels of occludin, claudin-1 were decreased in the HO group. Quercetin treatment significantly enhanced the expression levels of them as compared to the HO group. HO alone strongly decreased the Zonula occludens protein 1 (ZO-1) expression in the cytomembrane. Quercetin supplementation enhanced the accumulation of ZO-1 in ARPE-19 cells. The expression levels of Beclin-1 and Microtubule associated protein light chain 3 II (LC-3II) increased, and that of P62 decreased in the quercetin protection group. The appearance of LC-3II, which examined by immunofluorescence experiments, enhanced in the quercetin protection group as compared with the control group. The expression levels of beclin-1 and LC-3II increased, and that of P62 increased in the autophagy-inhibited group compared with the quercetin protection group. The levels of occludin and claudin-1 also decreased. Quercetin prevents the loss of tight junction proteins by upregulating autophagy after oxidative stress in ARPE-19 cells.
本研究旨在探讨槲皮素对氧化应激损伤的人视网膜色素上皮细胞（ARPE-19 细胞）紧密连接蛋白的保护作用，并探讨其可能的机制。用 HO（300μM）建立 ARPE-19 细胞氧化应激模型，HO 暴露前用不同浓度（0-80μM）槲皮素预处理 ARPE-19 细胞，用 Western blot 和免疫染色检测紧密连接蛋白和自噬相关蛋白的表达和分布。用 5mM3-甲基腺嘌呤（3-MA）预处理 ARPE-19 细胞。与对照组相比，HO 组 ARPE-19 细胞活力减弱，但用槲皮素预处理后可保存。HO 组中 occludin 和 claudin-1 的表达水平降低，与 HO 组相比，槲皮素处理组表达水平明显升高。HO 组单独作用可明显降低细胞质膜中 Zonula occludens protein 1（ZO-1）的表达，槲皮素补充可增强 ARPE-19 细胞中 ZO-1 的积累。Beclin-1 和微管相关蛋白轻链 3 II（LC-3II）的表达水平升高，而 P62 的表达水平降低，在槲皮素保护组中。与对照组相比，免疫荧光实验检测到 LC-3II 的出现增强。与槲皮素保护组相比，自噬抑制组中 beclin-1 和 LC-3II 的表达水平升高，而 P62 的表达水平升高。occludin 和 claudin-1 的表达水平也降低。槲皮素通过上调自噬，防止 ARPE-19 细胞氧化应激后紧密连接蛋白的丢失。
