Abstract

Purpose: 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.

Methods: H₂O₂ (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 H₂O₂ 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).

Results: The cell viability weakened in the H₂O₂ 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 H₂O₂ group. Quercetin treatment significantly enhanced the expression levels of them as compared to the H₂O₂ group. H₂O₂ 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.

Conclusion: Quercetin prevents the loss of tight junction proteins by upregulating autophagy after oxidative stress in ARPE-19 cells.

摘要

目的：本研究旨在探讨槲皮素对人视网膜色素上皮细胞（ARPE-19细胞）氧化应激损伤的紧密连接蛋白的保护作用，并探讨其可能的机制。

方法：采用H ₂ O ₂ (300 μM)建立ARPE-19细胞氧化应激模型。ARPE-19 细胞在 H ₂ O ₂暴露之前用不同浓度（0-80 μM）的槲皮素预处理。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。ARPE-19 细胞用 5 mM 3-甲基腺嘌呤 (3-MA) 预处理。

结果：与对照组相比，H ₂ O ₂组细胞活力减弱。然而，它在用槲皮素预处理后得以保存。观察到H ₂ O ₂组中occludin、claudin-1的表达水平降低。与H ₂ O ₂组相比，槲皮素处理显着增强了它们的表达水平。H ₂ O ₂单独强烈降低细胞膜中封闭带蛋白 1 (ZO-1) 的表达。槲皮素补充剂增强了 ZO-1 在 ARPE-19 细胞中的积累。槲皮素保护组Beclin-1和微管相关蛋白轻链3II（LC-3II）表达水平升高，P62表达水平降低。通过免疫荧光实验检测的LC-3II的出现在槲皮素保护组中与对照组相比增强。与槲皮素保护组相比，自噬抑制组beclin-1和LC-3II表达水平升高，P62表达水平升高。occludin 和 claudin-1 的水平也降低了。

结论：槲皮素通过上调 ARPE-19 细胞氧化应激后的自噬来防止紧密连接蛋白的丢失。