Effect of Esculetin on Tert-Butyl Hydroperoxide-Induced Oxidative Injury in Retinal Pigment Epithelial Cells In Vitro

Abstract

Esculetin is a coumarin-derived compound with antioxidant and anti-inflammatory properties. The current study aims to evaluate the therapeutic implications of esculetin on retinal dysfunction and uncover the underlying mechanisms. Tert-butyl hydroperoxide (t-BHP) at a concentration of 300 μM was used to induce oxidative stress in human retinal pigment epithelial cell line (ARPE-19) cells. Esculetin at concentrations below 250 μM did not cause cytotoxicity to ARPE-19 cells. Cell viability analysis confirmed that t-BHP induced oxidative injury of ARPE-19 cells. However, ARPE-19 cells were protected from t-BHP-induced oxidative injury by esculetin in a concentration-dependent manner. As a result of the TUNEL assay to confirm apoptosis, esculetin treatment reduced the number of TUNEL-positive cells. Esculetin down-regulated the expression levels of Bax, Caspase-3, and PARP and up-regulated the expression level of Bcl2. Collectively, this study demonstrates that esculetin exerts potent antioxidant properties in ARPE-19 cells, inhibiting t-BHP-induced apoptosis under the regulation of apoptotic factors.

Keywords: age-related macular degeneration; apoptosis; esculetin; oxidative stress; retinal pigment epithelial cell.

Figure 1

Effect of esculetin on ARPE-19 cell injury. Cell viability of ARPE-19 cells exposed to different concentrations of esculetin (A), t-BHP (B), and t-BHP with esculetin (C) for 24 h. (D) Changes in ROS levels in ARPE-19 cells exposed to t-BHP with esculetin for 24 h were detected using DCFH-DA dye. (E,F) The intracellular levels of ROS were measured using CellROX green reagent. Scale bar = 25 μm. The values in the bar graphs represent the means ± SEM, n = 5. * p < 0.05 vs. control group, # p < 0.05 vs. t-BHP-treated group.

Figure 1

Effect of esculetin on ARPE-19 cell injury. Cell viability of ARPE-19 cells exposed to different concentrations of esculetin (A), t-BHP (B), and t-BHP with esculetin (C) for 24 h. (D) Changes in ROS levels in ARPE-19 cells exposed to t-BHP with esculetin for 24 h were detected using DCFH-DA dye. (E,F) The intracellular levels of ROS were measured using CellROX green reagent. Scale bar = 25 μm. The values in the bar graphs represent the means ± SEM, n = 5. * p < 0.05 vs. control group, # p < 0.05 vs. t-BHP-treated group.

Figure 2

Effect of esculetin on t-BHP-induced apoptosis in ARPE-19 cells. (A) Apoptosis of ARPE-19 cells exposed to t-BHP with esculetin for 24 h was detected using TUNEL staining. ×100 magnification. Scale bar = 50 μm. (B) The values in the bar graphs represent the means ± SEM, n = 5. * p < 0.05 vs. control group, # p < 0.05 vs. t-BHP-treated group.

Figure 2

Effect of esculetin on t-BHP-induced apoptosis in ARPE-19 cells. (A) Apoptosis of ARPE-19 cells exposed to t-BHP with esculetin for 24 h was detected using TUNEL staining. ×100 magnification. Scale bar = 50 μm. (B) The values in the bar graphs represent the means ± SEM, n = 5. * p < 0.05 vs. control group, # p < 0.05 vs. t-BHP-treated group.

Figure 3

Effect of esculetin on apoptosis-related signaling pathways in ARPE-19 cells. (A) Apoptosis-related protein assay. (B) The values in the bar graphs represent the means ± SEM, n = 4. * p < 0.05 vs. control group, # p < 0.05 vs. t-BHP treated group.

Figure 3

Effect of esculetin on apoptosis-related signaling pathways in ARPE-19 cells. (A) Apoptosis-related protein assay. (B) The values in the bar graphs represent the means ± SEM, n = 4. * p < 0.05 vs. control group, # p < 0.05 vs. t-BHP treated group.

Figure 4

Effect of esculetin on the expression of apoptosis-related proteins in ARPE-19 cells. (A) The protein expression levels of Bax, bcl-2, cleaved caspase-3, and cleaved PARP. (B) The values in the bar graphs represent the means ± SEM, n = 4. * p < 0.05 vs. control group, # p < 0.05 vs. t-BHP treated group.

Figure 5

Effect of esculetin on the expression of apoptosis-related mRNA in ARPE-19 cells. The levels of mRNA expression of Bax, bcl-2, caspase-3 and PARP. The values in the bar graphs represent the means ± SEM, n = 4. * p < 0.05 vs. control group, # p < 0.05 vs. t-BHP treated group.