Activation of Heme Oxygenase-1 by Mangiferin in Human Retinal Pigment Epithelial Cells Contributes to Blocking Oxidative Damage

Abstract

Mangiferin is a kind of natural xanthone glycosides and is known to have various pharmacological activities. However, since the beneficial efficacy of this compound has not been reported in retinal pigment epithelial (RPE) cells, this study aimed to evaluate whether mangiferin could protect human RPE ARPE-19 cells from oxidative injury mimicked by hydrogen peroxide (H₂O₂). The results showed that mangiferin attenuated H₂O₂-induced cell viability reduction and DNA damage, while inhibiting reactive oxygen species (ROS) production and preserving diminished glutathione (GSH). Mangiferin also antagonized H₂O₂-induced inhibition of the expression and activity of antioxidant enzymes such as manganese superoxide dismutase and GSH peroxidase, which was associated with inhibition of mitochondrial ROS production. In addition, mangiferin protected ARPE-19 cells from H₂O₂-induced apoptosis by increasing the Bcl-2/Bax ratio, decreasing caspase-3 activation, and blocking poly(ADP-ribose) polymerase cleavage. Moreover, mangiferin suppressed the release of cytochrome c into the cytosol, which was achieved by interfering with mitochondrial membrane disruption. Furthermore, mangiferin increased the expression and activity of heme oxygenase-1 (HO-1) and nuclear factor-erythroid-2 related factor 2 (Nrf2). However, the inhibition of ROS production, cytoprotective and anti-apoptotic effects of mangiferin were significantly attenuated by the HO-1 inhibitor, indicating that mangiferin promoted Nrf2-mediated HO-1 activity to prevent ARPE-19 cells from oxidative injury. The results of this study suggest that mangiferin, as an Nrf2 activator, has potent ROS scavenging activity and may have the potential to protect oxidative stress-mediated ocular diseases.

Keywords: Apoptosis; DNA damage; Mangiferin; Nrf2/HO-1; Reactive oxygen species.

Fig. 1

Mangiferin attenuated H₂O₂-induced reduction of cell viability in ARPE-19 cells. (A-C) Results of MTT assay analyzed after treating cells with various concentrations of H₂O₂ (A) or mangiferin (B) for 24 h or pre-treating cells with mangiferin (20 μM) for 1 h and then treating them with H₂O₂ (0.5 mM) for 24 h (C). (D) Representative morphological images of cells cultured under the same conditions (C, 200×). All data are represented as mean SD from triplicate experiments (***p<0.001 compared with the control group. ^###p<0.001 compared with the H₂O₂-treated group).

Fig. 2

Mangiferin protected H₂O₂-induced DNA damage in ARPE-19 cells. Cells were exposed to mangiferin (20 µM) for 1 h prior to treatment with H₂O₂ (0.5 mM) for 24 h. After treatment, the effect of mangiferin on H₂O₂-induced DNA damage was evaluated. Representative images of comet assay (A), 8-OHdG levels (B) and expression changes of γH2AX (C, D) were presented. All data are represented as mean SD from triplicate experiments (***p<0.001 compared with the control group. ^###p<0.001 compared with the H₂O₂-treated group).

Fig. 3

Mangiferin suppressed H₂O₂-induced apoptosis in ARPE-19 cells. Before treating the cells with H₂O₂ (0.5 mM) for 24 h, they were incubated in the presence or absence of mangiferin (20 µM) for 1 h. (A, B) Representative histograms (A) and quantitative results (B) of flow cytometry analysis by annexin V/PI staining. (C) Expression changes of the indicated proteins obtained through immunoblotting. (D) Differences in caspase-3 activity were presented in each treatment group. All data are represented as mean SD from triplicate experiments (***p<0.001 compared with the control group. ^###p<0.001 compared with the H₂O₂-treated group.)

Fig. 4

Mangiferin attenuated H₂O₂-induced mitochondrial impairment in ARPE-19 cells. Cells exposed with or without mangiferin (20 µM) for 1 h were treated with H₂O₂ (0.5 mM) for 24 h. (A, B) Representative histograms (A) and JC-1 monomer ratios (B) of flow cytometry using JC-1 staining in each treatment group. (C) The expression of cytochrome c in the mitochondrial and cytosolic fractions was investigated by immunoblotting. All data are represented as mean SD from triplicate experiments (***p<0.001 compared with the control group. ^###p<0.001 compared with the H₂O₂-treated group).

Fig. 5

Mangiferin ameliorated mtROS production and increased GSH/GSSG ratio in H₂O₂-treated ARPE-19 cells. Cells exposed with or without mangiferin (20 µM) for 1 h were stimulated with H₂O₂ (0.5 mM) for 1 h (A-C) or 24 h (D). (A, B) Representative histograms of flow cytometry (A) and the frequency of DCF-positive cells (B). (C) Representative fluorescence images of ROS production. (D) Bar chart indicated the GSH/GSSG ratio following the exposure to H₂O₂ and pretreatment with mangiferin. All data are represented as mean SD from triplicate experiments (***p<0.001 compared with the control group. ^###p<0.001 compared with the H₂O₂-treated group).

Fig. 6

Mangiferin abolished H₂O₂-induced production of mtROS and inactivation of MnSOD and PGx in ARPE-19 cells. Before treating the cells with H₂O₂ (0.5 mM) for 24 h, they were incubated in the presence or absence of mangiferin (20 µM) for 24 h. (A, B) Representative histograms of flow cytometry following MitoSOX staining (A) and the frequency of MitoSOX-positive cells. (C) Expression changes of MnSOD and PGx obtained through immunoblotting. (D, E) Changes in the enzymatic activity of MnSOD and PGx were obtained using commercially available assay kits. All data are represented as mean SD from triplicate experiments (***p<0.001 compared with the control group. ^#p<0.05, ^##p<0.01 and ^###p<0.001 compared with the H₂O₂-treated group).

Fig. 7

Activation of the Nrf2/HO-1 signaling by mangiferin in H₂O₂-treated ARPE-19 cells. Cells exposed with or without mangiferin (20 µM) for 1 h were stimulated with H₂O₂ (0.5 mM) for 24 h. Resveratrol (25 µM) was treated alone for 24 h and was used as a positive regulator of Nrf2 activation. (A, B) Expression changes of proteins presented in each treatment group were analyzed by immunoblotting using total proteins (A) or cytosolic and nuclear fractions (B). (C) The activity of HO-1 in each treatment group was expressed as a relative value. All data are represented as mean SD from triplicate experiments (***p<0.001 compared with the control group. ^###p<0.001 compared with the H₂O₂-treated group).

Fig. 8

Attenuation of the protective ability of mangiferin against H₂O₂-induced ROS production and apoptosis by ZnPP in ARPE-19 cells. Cells were treated with mangiferin (20 µM) and/or ZnPP (10 µM) for 1 h and then further treated with H₂O₂ (0.5 mM) for 24 h. (A, B) Representative flow cytometry results (A) and their average values (B) according to DCF-DA staining. (C, D) Representative histograms (C) and quantitative results (D) of flow cytometry analysis following double staining of annexin V/PI. (E) Cell viability was assessed using the MTT assay. All data are represented as mean SD from triplicate experiments (***p<0.001 compared with the control group. ^###p<0.001 compared with the H₂O₂-treated group. ^$$$p<0.001 compared with the mangiferin and H₂O₂-treated group).

Fig. 9

Schematic showing the protective effect of mangiferin against oxidative damage in RPE ARPE-19 cells. Mangiferin activated Nrf2/HO-1 signaling and suppressed H₂O₂-induced ROS generation, mitochondrial dysfunction, DNA damage and apoptosis in ARPE-19 cells.