Selenium Protects ARPE-19 and ACBRI 181 Cells against High Glucose-Induced Oxidative Stress

Abstract

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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ signaling pathway.

Keywords: ARPE-19 cells; high glucose; oxidative stress; selenium.

Figure 1

Effect of selenium (Se) on cell viability in ARPE-19 (A) and ACBRI 181 (B) cells (n = 5). Cells were incubated with increasing concentrations of Se (1 nM–1 mM) for various time points (0.5–48 h).

Figure 2

Calcium release from ARPE-19 cells exposed to selenium (Se) and different concentrations of D-glucose (5–80 mM) and their combinations (A). Fura-2, AM-loaded ARPE-19 cells were incubated for 45 min in a shaking water bath. Subsequently, cells were exposed to 100 µM H₂O₂ to induce stimulation. Time course chart recordings were taken to visualize the transient [Ca²⁺]_i levels in ARPE-19 cells. (B) Bar charts present the mean ± standard deviation data representing [Ca²⁺]_i concentration in H₂O₂-stimulated ARPE-19 cells (n = 6 for each group). A single asterisk indicates significant differences between the two groups (p < 0.001). ^a p < 0.001 vs. control, ^b p < 0.001 vs. Se, ^c p < 0.001 vs. 5 mM D-glucose, ^d p < 0.001 vs. 5 mM D-glucose + Se, ^e p < 0.001 vs. 20 mM D-glucose, ^f p < 0.001 vs. 20 mM D-glucose + Se, ^g p < 0.001 vs. 40 mM D-glucose, ^h p < 0.001 vs. 40 mM D-glucose + Se, ⁱ p < 0.001 vs. 80 mM D-glucose.

Figure 3

Calcium release from ARPE-19 cells exposed to selenium (Se), different concentrations of D-Glucose (5–80 mM), and their combinations after 2-APB incubation calcium release from ARPE-19 cells (A). Fura-2, AM-loaded ARPE-19 cells were incubated for 45 min in a shaking water bath. Subsequently, cells were exposed to 100 µM H₂O₂ to induce stimulation. Time course chart recordings were taken to visualize the transient changes in [Ca²⁺]_i levels in ARPE-19 cells. (B) Bar charts present the mean ± standard deviation data representing [Ca²⁺]_i concentration in H₂O₂-stimulated ARPE-19 cells (n = 6 for each group). A single asterisk (*) indicates significant differences between two groups (p < 0.05), and double asterisks (**) indicate significant differences between two groups (p < 0.001). ^a p < 0.001 vs. control, ^b p < 0.001 vs. Se, ^c p < 0.001 vs. Se + 2-APB, ^d p < 0.001 vs. 5 mM D-glucose, ^e p < 0.001 vs. 5 mM D-glucose + 2-APB, ^f p < 0.001 vs. 20 mM D-glucose, ^g p < 0.001 vs. 20 mM D-glucose + 2-APB, ^h p < 0.001 vs. 40 mM D-glucose, ⁱ p < 0.001 vs. 40 mM D-glucose + 2-APB, ^j p < 0.001 vs. 80 mM D-glucose.

Figure 4

Calcium release from ACBRI 181 cells exposed to selenium (Se), different concentrations of D-glucose (5–80 mM), and their combinations (A). Fura-2, AM-loaded ACBRI 181 cells were incubated for 45 min in a shaking water bath. Subsequently, cells were exposed to 100 µM H₂O₂ to induce stimulation. Time course chart recordings were taken to visualize the transient changes in [Ca²⁺]_i levels in ACBRI 181 cells. (B) Bar charts present the mean ± standard deviation data representing [Ca²⁺]_i concentration in H₂O₂-stimulated ARPE-19 cells (n = 6 for each group). A single asterisk indicates significant differences between the two groups (p < 0.001). ^a p < 0.001 vs. control, ^b p < 0.001 vs. Se, ^c p < 0.001 vs. 5 mM D-glucose, ^d p < 0.001 vs. 5 mM D-glucose + Se, ^e p < 0.001 vs. 20 mM D-glucose, ^f p < 0.001 vs. 20 mM D-glucose + Se, ^g p < 0.001 vs. 40 mM D-glucose, ^h p < 0.001 vs. 40 mM D-glucose + Se, ⁱ p < 0.001 vs. 80 mM D-glucose.

Figure 5

Calcium release from ACBRI 181 cells exposed to selenium (Se), different concentrations of D-Glucose (5–80 mM), and their combinations after 2-APB incubation calcium release from ACBRI 181 cells (A). Fura-2, AM-loaded ACBRI 181 cells were incubated for 45 min in a shaking water bath. Subsequently, cells were exposed to 100 µM H₂O₂ to induce stimulation. Time course chart recordings were taken to visualize the transient changes in [Ca²⁺]_i levels in ACBRI 181 cells. (B) Bar charts present the mean ± standard deviation data representing [Ca²⁺]_i concentration in H₂O₂-stimulated ARPE-19 cells (n = 6 for each group). A single asterisk (*) indicates significant differences between two groups (p < 0.05). ^a p < 0.001 vs. control, ^b p < 0.001 vs. Se, ^c p < 0.001 vs. Se + 2-APB, ^d p < 0.001 vs. 5 mM D-glucose, ^e p < 0.001 vs. 5 mM D-glucose + 2-APB, ^f p < 0.001 vs. 20 mM D-glucose, ^g p < 0.001 vs. 20 mM D-glucose + 2-APB, ^h p < 0.001 vs. 40 mM D-glucose, ⁱ p < 0.001 vs. 40 mM D-glucose + 2-APB.

Figure 6

Effects of selenium (Se) and high-glucose (HG) on intracellular ROS production of ARPE-19 (A) and ACBRI 181 (B) cells. Red bars indicate Se administered groups, and blue bars indicate only D-glucose administered groups. A single asterisk indicates significant differences between each group (p < 0.05). ^a p < 0.001 vs. control, ^b p < 0.001 vs. Se, ^c p < 0.001 vs. 5 mM D-glucose, ^d p < 0.001 vs. 20 mM D-glucose, ^e p < 0.001 vs. 40 mM D-glucose.

Figure 7

Effects of selenium (Se) and high-glucose (HG) on mitochondrial membrane depolarization of ARPE-19 (A) and ACBRI 181 (B) cells. Red bars indicate Se administered groups, and blue bars indicate only D-glucose administered groups. A single asterisk indicates statistically significant differences between each group (p < 0.05). ^a p < 0.001 vs. control, ^b p < 0.001 vs. Se, ^c p < 0.001 vs. 5 mM D-glucose, ^d p < 0.001 vs. 20 mM D-glucose, ^e p < 0.001 vs. 40 mM D-glucose.

Figure 8

Effects of selenium (Se) and high-glucose (HG) on apoptosis of ARPE-19 (A) and ACBRI 181 (B) cells. Red bars indicate Se administered groups, and blue bars indicate only D-glucose administered groups. A single asterisk indicates statistically significant differences between each group (p < 0.05). ^a p < 0.001 vs. control, ^b p < 0.001 vs. Se, ^c p < 0.001 vs. 5 mM D-glucose, ^d p < 0.001 vs. 20 mM D-glucose, ^e p < 0.001 vs. 40 mM D-glucose.

Figure 9

Effects of selenium (Se) and high-glucose (HG) on caspase-3 (A,C) and -9 (B,D) activation in ARPE-19 (left panels) and ACBRI 181 (right panels) cells. Red bars indicate Se administered groups, and blue bars indicate only D-glucose administered groups. A single asterisk indicates statistically significant differences between each group (p < 0.05). ^a p < 0.001 vs. control, ^b p < 0.001 vs. Se, ^c p < 0.001 vs. 5 mM D-glucose, ^d p < 0.001 vs. 20 mM D-glucose, ^e p < 0.001 vs. 40 m M D-glucose.