Department of Ophthalmology, Asya Hospital, 34100 Istanbul, Turkey.
Department of Biophysics, Faculty of Medicine, Karamanoğlu Mehmetbey University, 70100 Karaman, Turkey.
Molecules. 2023 Aug 9;28(16):5961. doi: 10.3390/molecules28165961.
Diabetic retinopathy (DR), a complication of diabetes mellitus (DM), can cause severe visual loss. The retinal pigment epithelium (RPE) plays a crucial role in retinal physiology but is vulnerable to oxidative damage. We investigated the protective effects of selenium (Se) on retinal pigment epithelium (ARPE-19) and primary human retinal microvascular endothelial (ACBRI 181) cells against high glucose (HG)-induced oxidative stress and apoptotic cascade. To achieve this objective, we utilized varying concentrations of D-glucose (ranging from 5 to 80 mM) to induce the HG model. HG-induced oxidative stress in ARPE-19 and ACBRI 181 cells and the apoptotic cascade were evaluated by determining Ca overload, mitochondrial membrane depolarization, caspase-3/-9 activation, intracellular reactive oxygen species (ROS), lipid peroxidation (LP), glutathione (GSH), glutathione peroxidase (GSH-Px), vascular endothelial growth factor (VEGF) and apoptosis levels. A cell viability assay utilizing MTT was conducted to ascertain the optimal concentration of Se to be employed. The quantification of MTT, ROS, VEGF levels, and caspase-3 and -9 activation was accomplished using a plate reader. To quantitatively assess LP and GSH levels, GSH-Px activities were utilized by spectrophotometer and apoptosis, mitochondrial membrane depolarization, and the release of Ca from intracellular stores were evaluated by spectrofluorometer. Our investigation revealed a significant augmentation in oxidative stress induced by HG, leading to cellular damage through modulation of mitochondrial membrane potential, ROS levels, and intracellular Ca release. Incubation with Se resulted in a notable reduction in ROS production induced by HG, as well as a reduction in apoptosis and the activation of caspase-3 and -9. Additionally, Se incubation led to decreased levels of VEGF and LP while concurrently increasing levels of GSH and GSH-Px. The findings from this study strongly suggest that Se exerts a protective effect on ARPE-19 and ACBRI 181 cells against HG-induced oxidative stress and apoptosis. This protective mechanism is partially mediated through the intracellular Ca signaling pathway.
糖尿病性视网膜病变（DR）是糖尿病（DM）的一种并发症，可导致严重的视力丧失。视网膜色素上皮（RPE）在视网膜生理中起着至关重要的作用，但易受到氧化损伤。我们研究了硒（Se）对高糖（HG）诱导的氧化应激和凋亡级联反应对视网膜色素上皮（ARPE-19）和原代人视网膜微血管内皮（ACBRI 181）细胞的保护作用。为了实现这一目标，我们使用不同浓度的 D-葡萄糖（范围从 5 到 80mM）诱导 HG 模型。通过测定 Ca 超载、线粒体膜去极化、半胱天冬酶-3/-9 激活、细胞内活性氧（ROS）、脂质过氧化（LP）、谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GSH-Px）、血管内皮生长因子（VEGF）和凋亡水平来评估 ARPE-19 和 ACBRI 181 细胞中的 HG 诱导的氧化应激和凋亡级联反应。通过 MTT 细胞活力测定来确定要使用的 Se 的最佳浓度。使用平板读数器测定 MTT、ROS、VEGF 水平以及 caspase-3 和 -9 的激活。通过分光光度计定量评估 LP 和 GSH 水平，通过分光光度计评估 GSH-Px 活性以及线粒体膜去极化和细胞内储存的 Ca 释放。我们的研究表明，HG 诱导的氧化应激显著增加，通过调节线粒体膜电位、ROS 水平和细胞内 Ca 释放导致细胞损伤。用 Se 孵育可显著减少 HG 诱导的 ROS 产生，减少细胞凋亡和 caspase-3 和 -9 的激活。此外，Se 孵育可降低 VEGF 和 LP 的水平，同时增加 GSH 和 GSH-Px 的水平。这项研究的结果强烈表明，Se 对 ARPE-19 和 ACBRI 181 细胞具有保护作用，可抵抗 HG 诱导的氧化应激和细胞凋亡。这种保护机制部分是通过细胞内 Ca 信号通路介导的。
