Biomolecules & Therapeutics 2024; 32(3): 329-340  https://doi.org/10.4062/biomolther.2023.175
Activation of Heme Oxygenase-1 by Mangiferin in Human Retinal Pigment Epithelial Cells Contributes to Blocking Oxidative Damage
Cheol Park1,†, Hee-Jae Cha2,†, Hyun Hwangbo3,4, EunJin Bang3,4, Heui-Soo Kim5, Seok Joong Yun6, Sung-Kwon Moon7, Wun-Jae Kim6,8, Gi-Young Kim9, Seung-On Lee10, Jung-Hyun Shim10,11,* and Yung Hyun Choi3,4,*
1Division of Basic Sciences, College of Liberal Studies, Dong-eui University, Busan 47340,
2Department of Parasitology and Genetics, Kosin University College of Medicine, Busan 49104,
3Anti-Aging Research Center, Dong-eui University, Busan 47340,
4Department of Biochemistry, College of Korean Medicine, Dong-eui University, Busan 47227,
5Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 46241,
6Department of Urology, Chungbuk National University College of Medicine, Cheongju 28644,
7Department of Food and Nutrition, Chung-Ang University, Ansung 17546,
8Institute of Urotech, Cheongju 28120,
9Department of Marine Life Sciences, Jeju National University, Jeju 63243,
10Department of Biomedicine, Health & Life Convergence Sciences, BK21 Four, College of Pharmacy, Mokpo National University, Muan 58554, Republic of Korea
11The China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan 450008, China
*E-mail: s1004jh@gmail.com (Shim JH), choiyh@deu.ac.kr (Choi YH)
Tel: +82-61-450-2684 (Shim JH), +82-51-890-3319 (Choi YH)
Fax: +82-61-450-2689 (Shim JH), +82-51-890-3333 (Choi YH)
The first two authors contributed equally to this work.
Received: October 4, 2023; Revised: November 4, 2023; Accepted: November 7, 2023; Published online: April 9, 2024.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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: Mangiferin, Reactive oxygen species, DNA damage, Apoptosis, Nrf2/HO-1
INTRODUCTION

Mangiferin, first isolated from the bark and leaves of the mango tree (Mangifera indica L.), is a natural xanthonoid polyphenol belonging to the xanthone family of secondary metabolites with various structures (Dutta et al., 2023; Mei et al., 2023). Although the absorption rate and oral bioavailability are too low, clinical application is not currently achieved, mangiferin has excellent potential as a nutraceutical material due to its various biological activities (Remali et al., 2022; Dutta et al., 2023). Accumulated studies have reported that mangiferin possesses a wide range of pharmacological properties, including antidiabetic, neuroprotective, cardiotonic, immunomodulatory, antiallergic, renoprotective, antithrombotic, memory enhancing and anti-cancer effects (Walia et al., 2021; Lum et al., 2022; Wang et al., 2022b). In particular, the improvement of chronic diseases by mangiferin is closely related to its strong antioxidant activity, which is mainly related to its ability to scavenge reactive oxygen species (ROS) through the regulation of antioxidant signaling pathways. For example, alleviation of heavy metal-induced mitochondrial dysfunction and apoptosis in hepatocytes by mangiferin was achieved by blocking ROS production through restoring activity of intracellular antioxidant enzymes (Agarwala et al., 2012; Pal et al., 2013). In addition, mangiferin showed anti-aging effects by blocking ROS-dependent matrix metalloproteinases activation and premature senescence induced by hydrogen peroxide (H2O2) in human skin keratinocytes and dermal fibroblast cells, respectively (Chae et al., 2011; Kanoi et al., 2021). Mangiferin also successfully inhibited nephrotoxicity and hepatotoxicity through reducing ROS production and increasing intracellular antioxidant defenses in streptozotocin-induced diabetic rats and liver tissue of arsenic-treated mice, respectively, thereby protecting renal and hepatic cells from apoptosis (Pal et al., 2014; Saha et al., 2016; Xu et al., 2017). Ding et al. (2018) reported that the anti-apoptotic effect of mangiferin in dexamethasone-treated osteoblasts was due to the inhibition of ROS generation accompanied by the activation of bone morphogenetic protein 2/Smad-1 signaling pathway. Hou et al. (2018) also demonstrated that high glucose-induced cardiac cell protection by mangiferin was associated with enhanced mitochondrial membrane potential (MMP) and autophagic flux associated with inhibition of the mTOR downstream signaling pathway by blocking ROS production. Similar to their results, the neuroprotective effect of mangiferin on amyloid b in cortical neurons was associated with the reduction of calcium load on mitochondria and inhibition of ROS generation by preservation of activity of endogenous enzymatic antioxidant enzymes such as superoxide dismutase (SOD) and catalase (Alberdi et al., 2018).

Moreover, after it was found that the protective role of mangiferin against D-galactosamine-induced hepatotoxicity was due to activation of the nuclear factor-erythrocyte 2-related factor 2 (Nrf2)-mediated antioxidant defense system (Das et al., 2012), a number of studies have been conducted on the role of Nrf2 in the antioxidant activity of mangiferin. As a transcription factor involved in antioxidant signaling pathways, Nrf2 has been documented to regulate defense mechanisms against oxidative stress and is critically involved in the transcriptional activity of several cytoprotective antioxidant enzymes in response to oxidative stress (Shaw and Chattopadhyay, 2020; Jenkins and Gouge, 2021). According to the results recently reported by Zhou et al. (2023), mangiferin reduced ROS levels and increased the levels of SOD and glutathione (GSH) in a Parkinson’s disease model, and was able to reduce oxidative stress by targeting the Nrf2 pathway. Mangiferin also protected ischemia/reperfusion-related events and helped restore hyperglycemia-induced impaired angiogenesis through Nrf2 signaling, which was associated with increased expression of heme oxygenase (HO)-1 (Gendy et al., 2022; Zhou et al., 2023). HO-1 is one of the representative downstream regulators of Nrf2 and contributes to the maintenance of redox homeostasis through its metabolites (Yu et al., 2018; Jenkins and Gouge, 2021). Although the importance of other antioxidant enzymes, including NADPH quinine oxidoreductase 1 (NQO-1), as a Nrf2-mediated down-regulator by mangiferin has also been reported, the role of HO-1 in terms of ROS scavenging has been further emphasized in its antioxidant activity (Saha et al., 2016; Al-Saeedi, 2021). Therefore, the Nrf2/HO-1 axis can be considered as the main defense pathway of mangiferin against oxidative stress, but the role of this signaling pathway in retinal pigment epithelial (RPE) cells has not been reported. In this study, it was investigated the role of the Nrf2/HO-1 pathway in the antioxidant capacity of mangiferin in RPE cells under conditions mimicking the oxidative stress induced by H2O2 treatment using the human RPE ARPE-19 cell line.

MATERIALS AND METHODS

Cell culture and treatment

ARPE-19 cells (CRL-2302™) were cultured in Dulbecco’s Modified Eagle’s Medium/F-12 medium containing 10% fetal bovine serum as previously described (Park et al., 2022). Mangiferin and H2O2 (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Thermo Fisher Scientific, Waltham, MA, USA) to prepare stock solutions, diluted to appropriate concentrations in culture medium, and then treated with cells. To trigger oxidative stress, cells were cultured in medium containing H2O2, and mangiferin and/or zinc protoporphyrin IX (ZnPP, Sigma-Aldrich) were added 1 h before H2O2 exposure.

Analysis of cell viability

Cell viability of ARPE-19 cells treated with mangiferin and H2O2 alone or with H2O2 for 24 h in the presence or absence of mangiferin and/or ZnPP was estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay following the methods of Cao et al. (2023). After treatment, morphological changes of the cells were observed using an inverted phase contrast microscope (Carl Zeiss, Oberkochen, Germany).

Comet assay

A Comet Assay Kit obtained from Trevigen, Inc. (Gaithersburg, MD, USA) was used to assess the degree of DNA damage in cells treated with H2O2 in the presence or absence of mangiferin. In brief, cells were mixed in low melting-point agarose (1%) and spread evenly on slides, according to the manufacturer’s instructions. After DNA denaturation, electrophoresis was performed and cells were immediately stained with an asymmetric cyanine dye, followed by visualization of fluorescent images under a fluorescence microscope (Carl Zeiss) at Core-Facility Center for Tissue Regeneration, Dong-eui University (Busan, Korea).

8-Hydroxyguanosine (8-OHdG) assay

To investigate the degree of oxidative DNA damage, the level of intracellular 8-OHdG, an RNA nucleoside that is an oxidized derivative of guanosine, was evaluated using the 8-OHdG Enzyme-Linked Immunosorbent Assay (ELISA) Kit (Abcam, Inc., Cambridge, UK). According to the manufacturer’s protocol, DNA isolated from H2O2-treated cells with or without mangiferin was digested using DNA Digestion Mix, and the level of 8-OHdG was measured by a competitive enzyme immunoassay. The optical density for each treatment group was recorded at 450 nm, and results were presented as ng of 8-OHdG/mL.

Western blot analysis

To analyze protein expression using Western blot analysis, total cellular proteins were extracted as previous described (Mukherjee et al., 2022). The mitochondrial, cytoplasmic and nuclear fractions were isolated using the Mitochondrial Fractionation Kit (Thermo Fisher Scientific) and Nuclear and Cytoplasmic Protein Extraction Kit (Sigma-Aldrich). After protein quantification, equal amounts of protein were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). The membranes were probed with primary antibodies and then incubated with horseradish peroxidase-conjugated secondary antibodies. Finally, the protein bands were visualized by a SuperSignal West Pico PLUS (Thermo Fisher Scientific). Actin, histone deacetylase 1 (HDAC1) and cytochrome c oxidase IV (COX IV) were used as housekeeping proteins for the total, nuclear and mitochondrial proteins, respectively. Primary and secondary antibodies were purchased from Thermo Fisher Scientific, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), Abcam, Inc. and Cell Signaling Technology (Beverly, MA, USA) (Table 1).

Table 1 List of antibodies used in this study

AntibodySpecies raisedDilutionProduct codeSource
γH2AXMouse monoclonal1:500MA1-2022Thermo Fisher Scientific
Bcl-2Mouse monoclonal1:1000sc-509Santa Cruz Biotechnology Inc.
BaxMouse monoclonal1:1000sc-7480Santa Cruz Biotechnology Inc.
Caspase-3Rabbit polyclonal1:1000#9662Cell Signaling Technology
PARPMouse monoclonal1:1000sc-8007Santa Cruz Biotechnology Inc.
Cytochrome cMouse monoclonal1:1000sc-13560Santa Cruz Biotechnology Inc.
MnSODRabbit polyclonal1:1000ab13533Abcam, Inc.
PGx1Rabbit polyclonal1:1000#63536Cell Signaling Technology
Nrf2Mouse monoclonal1:1000sc-518036Santa Cruz Biotechnology Inc.
p-Nrf2Rabbit polyclonal1:500PA5-67520Thermo Fisher Scientific
HO-1Mouse monoclonal1:1000sc-136960Santa Cruz Biotechnology Inc.
HDAC1Rabbit monoclonal1:500ab280198Abcam, Inc.
COX IVRabbit polyclonal1:1000#4844Cell Signaling Technology
ActinMouse monoclonal1:1000sc-47778Santa Cruz Biotechnology Inc.


Immunofluorescence

To examine the expression level of γH2AX in the nucleus, cells attached to the coverslip were treated with H2O2 in the presence or absence of mangiferin, fixed with 2% paraformaldehyde, and then permeabilized with 0.1% Triton-X-100 as previously described (Park et al., 2023). The cells were probed with anti-γH2AX antibody (Ser139, Thermo Fisher Scientific) and then reacted with Alexa Fluor 594-conjugated antibody (Cell Signaling Technology). After counterstaining the nuclei using 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), fluorescence images were acquired under a fluorescence microscope.

Measurement of apoptosis and MMP

Apoptosis was measured by flow cytometry using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Staining/Detection Kit (Abcam, Inc.) according to the manufacturer’s protocol. In brief, the collected cells were double-stained with annexin V and propidium iodide (PI) in the dark, and annexin V-positive cells were regarded as apoptosis-induced cells using a flow cytometer (BD Biosciences, San Jose, CA, USA). MMP was evaluated with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye following general manufacturer’s recommendations. The fluorescent intensities for JC-1 monomers and aggregates forms were analyzed by flow cytometry, and then percentages of JC-1 monomers were expressed to represent cells that lost MMP.

Caspase-3 activity assay

The activity of caspase-3 was determined using the Active Caspase-3 Staining Kit (Thermo Fisher Scientific), which is based on the spectrophotometric detection of p-nitroaniline (p-NA) cleaved from the substrate (N-acetyl-Asp-Glu-Val-Asp-p-NA). According to the manufacturer’s protocol, the cell lysates were obtained using reagents provided in the kit and then free p-NA was quantified using a microplate reader (Beckman Coulter Inc., Brea, CA, USA) at 405 nm. The activity of caspase-3 in each treatment group was determined relative to that in the control group.

ROS detection

A 2’,7’-dichlorofluorescein diacetate (DCF-DA) probe was used to detect the protective effect of mangiferin against H2O2-induced intracellular ROS levels. In brief, cells treated with H2O2 for 1 h with or without mangiferin were stained with DCF-DA (Thermo Fisher Scientific) according to the manufacturer’s instructions, and then ROS levels were quantitatively evaluated by DCF fluorescence representing the percentage of control cells through flow cytometry. Fluorescence images of cells in each treatment group were also captured with a fluorescence microscope to visually detect the difference in emitted DCF fluorescence intensity. In addition, MitoSOX™ Red mitochondrial superoxide indicator (Thermo Fisher Scientific) was used to measure mitochondrial superoxide. Briefly, after staining the cells with MitoSOX according to the manufacturer’s instructions, and the emitted fluorescence of MitoSOX was detected by flow cytometry.

Assessment of GSH/GSH disulfide (GSSG) ratio

The difference in the reduced GSH/oxidized GSSG ratio was quantified using the GSH/GSSG Ratio Assay Kit (Abcam, Inc.). In brief, after reacting the cells of each treatment group under the conditions recommended by the manufacturer, the concentrations of GSH and GSSG were measured based on the standard curve of GSH and GSSG.

Manganese SOD (MnSOD) and GSH peroxidase (GPx) activity assay

Colorimetric kits purchased from Abcam, Inc. were used to measure the activities of MnSOD and GPx, which are key mitochondrial oxidative stress enzymes, in cells exposed to H2O2 for 24 h with or without mangiferin. Mitochondrial fractions were prepared and the activity of each enzyme was presented as a relative value relative to the control group according to the manufacturer’s instructions.

HO-1 activity assay

The amount of bilirubin formed in the heme of cells was evaluated to detect HO-1 activity using an HO-1 ELISA kit (Abcam, Inc.). Briefly, the bilirubin level in each treatment group was quantified based on absorption at 510 nm using a microplate reader, according to the manufacturer’s method. HO-1 activity was expressed as a relative value to that of the control.

Statistical analysis

Data are expressed as mean ± standard deviation (SD). Statistical comparisons were performed using GraphPad Prism (Ver. 8.0, GraphPad Inc., San Diego, CA, USA). Statistical significance was set at *p<0.05, **p<0.01 and ***p<0.001 vs. control group; #p<0.05, ##p<0.01 and ###p<0.001 vs. H2O2-treated cells; $$$p<0.001 vs. mangiferin and H2O2 treatment group.

RESULTS

Protection from H2O2-induced decrease in cell viability by mangiferin in ARPE-19 cells

To investigate the protective effect of mangiferin against H2O2-induced cytotoxicity in ARPE-19 cells, the cell viability of ARPE-19 cells treated with H2O2 and mangiferin alone was first investigated. According to the MTT assay results, H2O2 decreased cell viability in a concentration-dependent manner at a treatment concentration of 0.25 mM or more, and mangiferin had a slight inhibitory effect on cell viability in the 30 and 40 μM treatment groups, but it was not significant (Fig. 1A, 1B). Therefore, the treatment concentration for mimicking oxidative damage by H2O2 was set to 0.5 mM, which showed a cell viability of about 60% compared to the control group, and the optimal pretreatment concentration for evaluating the protective effect of mangiferin was selected as 20 μM. And as a result of evaluating the protective effect of mangiferin against H2O2-mediated cytotoxicity, pretreatment with 20 μM mangiferin significantly increased the decrease in cell viability caused by 0.5 mM H2O2 treatment (Fig. 1C). In addition, mangiferin effectively inhibited morphological changes in flattened and thinned cells by H2O2 treatment (Fig. 1D).

Figure 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).

Inhibition of H2O2-induced DNA damage by mangiferin in ARPE-19 cells

To evaluate whether the protective effect of mangiferin against H2O2-mediated cytotoxicity was related to attenuation of DNA damage, the effects of mangiferin on H2O2-induced comet tail formation, increase in 8-OHdG content, and phosphorylation of histone H2AX (γH2AX) were investigated. As shown in Fig. 2, mangiferin pretreatment remarkably reduced the increase of these three DNA damage biomarkers by H2O2 treatment in ARPE-19 cells, demonstrating that the protective potential of mangiferin against genotoxicity mediated by oxidative stress, demonstrating that the protective potential of mangiferin against genotoxicity mediated by oxidative stress.

Figure 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).

Reduction of H2O2-induced DNA damage by mangiferin in ARPE-19 cells

Next, flow cytometry was performed according to annexin V/PI staining to investigate whether mangiferin inhibited H2O2-induced apoptosis. As depicted in Fig. 3A and 3B, apoptosis was significantly increased in H2O2-treated cells, but it was greatly abrogated by mangiferin pretreatment. Further, immunoblotting results indicated that H2O2 treatment downregulated Bcl-2 expression and upregulated Bax expression, which was related to activation of caspase-3 and degradation of poly(ADP-ribose) polymerase (PARP), and these changes by H2O2 treatment were effectively mitigated in the presence of mangiferin (Fig. 3C, 3D).

Figure 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.)

Suppression H2O2-induced mitochondrial damage by mangiferin in ARPE-19 cells

To examine whether the inhibitory effect of mangiferin on H2O2-induced apoptosis was related to the protective ability against mitochondrial dysfunction, MMP was measured. As shown in Fig. 4A and 4B, flow cytometric analysis by JC-1 staining showed that the frequency of JC-1 aggregates was down-regulated in H2O2-treated cells, whereas the frequency of JC-1 monomers was up-regulated. In addition, the expression of cytochrome c was increased in the cytosolic fraction by H2O2 treatment, but diminished in the mitochondrial fraction (Fig. 4C), indicating that cytochrome c was released from mitochondria into the cytosol due to disruption of mitochondrial membrane stability. However, these changes caused by H2O2 were all clearly abolished in cells pretreated with mangiferin.

Figure 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).

Inhibition of intracellular ROS generation and restoration of GSH/GSSG ratio by mangiferin in H2O2-treated ARPE-19 cells

Since the level of intracellular ROS and the ratio of GSH/GSSG reflect cellular oxidative stress, the effect of mangiferin on these changes by H2O2 treatment was investigated. As presented in Fig. 5A and 5B, the intensity of oxidized DCF, indicative of intracellular ROS generation, was about 10-fold higher in H2O2-treated cells compared to untreated cells, which was dramatically abrogated in mangiferin-pretreated cells. Concurrently, fluorescence microscopy of cells treated with H2O2 revealed a significant increase in DCF fluorescence intensity (green) compared to control cells (Fig. 5C), which was greatly reduced in the presence of mangiferin. In parallel, the GSH/GSSG ratio decreased by H2O2 treatment was significantly restored by mangiferin pretreatment (Fig. 5D), demonstrating that mangiferin lowered H2O2-induced intracellular oxidative stress, demonstrating that mangiferin lowered H2O2-induced intracellular oxidative stress.

Figure 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).

Attenuation of increased mitochondrial ROS (mtROS) production and restoration of decreased MnSOD and GPx activities by mangiferin in H2O2-treated ARPE-19 cells

Next, to explore whether the protective effect of mangiferin on the regulation of mitochondrial homeostasis is related to the inhibition of mtROS production, MitoSOX Red staining, a specific fluorescent probe for detecting mitochondrial superoxide production, was performed. As shown in Fig. 6A and 6B, H2O2 markedly promoted mitochondrial superoxide generation, but this effect was greatly mitigated by mangiferin pretreatment. To further evaluate the antioxidant activity of mangiferin, the roles of GPx and MnSOD were evaluated. As expected, H2O2 treatment greatly decreased the protein expression and activity of MnSOD and GPx (Fig. 6C-6E), indicating that the expression and activity of MnSOD and GPx were negatively correlated with ROS levels. However, mangiferin pretreatment counteracted their expression and enzymatic activity reduced by H2O2, indicating that mangiferin protected ARPE-19 cells against oxidative stress by attenuating excessive accumulation of intracellular and mtROS.

Figure 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).

Activation of Nrf2 signaling by Mangiferin in H2O2-treated ARPE-19 cells

To investigate the antioxidant capacity of mangiferin on Nrf2 and its downstream genes, ARPE-19 cells were treated with H2O2 in the presence or absence of mangiferin. The results showed that the total protein expression of Nrf2 and its phosphorylation level (p-Nrf2) were further increased in cells treated with H2O2 after mangiferin treatment than in cells treated with H2O2 alone (Fig. 7A). In particular, as in cells treated with resveratrol, a known Nrf2 activator, the expression of p-Nrf2 was predominantly expressed in the nucleus (Fig. 7B). Moreover, the protein expression and enzymatic activity of HO-1, a major downstream factor of Nrf2, were enhanced in cells co-treated with H2O2 and mangiferin compared with H2O2 exposure alone (Fig. 7A, 7C). On the other hand, the expression of Nrf2, p-Nrf2 and HO-1 and the activity of HO-1 were not induced in cells treated with mangiferin alone, indicating that mangiferin activated Nrf2/HO-1 signaling under oxidative stress conditions.

Figure 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).

Blockage of oxidative damage by mangiferin-induced Nrf2/HO-1 axis activation in H2O2-treated ARPE-19 cells

To further confirm that mangiferin protects ARPE-19 cells from H2O2-induced oxidative injury through activation of the Nrf2/HO-1 axis, the HO-1 inhibitor ZnPP was applied. As shown in Fig. 8A and 8B, ZnPP significantly abolished the protective effects of mangiferin on H2O2-induced ROS generation. Moreover, ZnPP dramatically abrogated the inhibitory effect of mangiferin against H2O2-induced apoptosis and cytotoxicity, as evidenced by flow cytometry and the MTT assay, respectively (Fig. 8C-8E), demonstrated that the Nrf2-mediated activation of HO-1 is at least involved in the regulation of ROS homeostasis by mangiferin in ARPE-19 cells.

Figure 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).
DISCUSSION

Although several previous studies have reported the potential application of mangiferin for the treatment of eye diseases (Liu et al., 2012; Kim et al., 2016), its efficacy against oxidative damage in RPE cells has not been properly evaluated. Therefore, in this study, the effect of mangiferin on oxidative damage in RPE ARPE-19 cells was evaluated and the results revealed that: 1) Mangiferin showed protective effects against oxidative stress-induced cytotoxicity, DNA damage, mitochondrial dysfunction and apoptosis in RPE cells; 2) These beneficial effects of mangiferin were due to inhibition of ROS production and restoration of damaged antioxidant systems; 3) Activation of the Nrf2/HO-1 signaling by mangiferin under conditions of oxidative stress was responsible for the protective effect of RPE cells (Fig. 9).

Figure 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.

High levels of ROS due to oxidative stress resulting from an imbalance in cellular redox homeostasis are directly involved in retinal damage and are therefore considered potential therapeutic targets for blocking retinal diseases (Wang et al., 2022a; Zhang et al., 2023). RPE cells exposed to a high oxidative stress environment are susceptible to defense against DNA damage, cellular senescence and apoptosis, and loss of antioxidant capacity underlies retinal degenerative diseases including age-related macular degeneration (Tong et al., 2022; Zhang et al., 2023). Mounting evidence has demonstrated that DNA and mitochondrial damage induced by oxidative stimuli closely accompany the induction of ROS-dependent apoptosis. Indeed, previous studies have shown that the cytotoxic effects of H2O2, known as the most stable ROS, on RPE cells are mostly related to mitochondrial dysfunction and apoptosis, which was associated with damage to intracellular macromolecules including DNA (Park et al., 2019; Hernandez et al., 2021; Park et al., 2022). In this study, H2O2 was used to mimic oxidative damage in RPE ARPE-19 cells, and mangiferin significantly blocked the H2O2-induced decrease in cell viability in ARPE-19 cells. Mangiferin was also able to block DNA damage caused by H2O2 in ARPE-19 cells, as evidenced by suppression of DNA tail formation and γH2AX expression, which are hallmarks of DNA double-strand breaks (Kopp et al., 2019; Cordelli et al., 2021), and normalized the level of 8-OHdG, a widely used biomarker for oxidative stress in nucleic acids (Hahm et al., 2022), increased by H2O2 treatment.

Consistent with previous findings (Park et al., 2022; Choi, 2023; Park et al., 2023), exposure to H2O2 increased the frequency of apoptosis-induced cells in ARPE-19 cells, with loss of MMP and accumulation of ROS. As is well known, abnormal accumulation of ROS due to oxidative stress contributes to mitochondrial membrane depolarization, resulting in MMP loss indicative of mitochondrial impairment (Bock and Tait, 2020; Tiwari et al., 2022). Loss of MMP in turn triggers the release of cytochrome c from mitochondria into the cytosol, where it activates the caspase cascade leading to the initiation of mitochondria-mediated intrinsic apoptotic pathway, ultimately resulting in the degradation of target proteins of effector caspases such as PARP for the induction of apoptosis. Moreover, proteins belonging to the Bcl-2 family are critically involved in the regulation of the intrinsic apoptosis pathway. Among them, pro-apoptotic proteins such as Bax play a key role in the formation of mitochondrial pores that disrupt mitochondrial membrane barrier stability, while anti-apoptotic proteins such as Bcl-2 play the opposite role (Lalier et al., 2022; Tiwari et al., 2022). Therefore, when the expression of Bcl-2 is relatively lower than that of Bax, mitochondrial membrane permeability increases and mitochondrial cytochrome c release is enhanced (Bock and Tait, 2020; Lalier et al., 2022). Similar to previous results (Clementi et al., 2022; Park et al., 2022), in the present study, cytosolic release of cytochrome c, an increase in the Bax/Bcl-2 expression ratio and cleavage of PARP by activation of caspase-3 were observed in ARPE-19 cells treated with H2O2. However, these effects were apparently eliminated in the presence of mangiferin, and these findings may be causally related, at least in part, to blockage of the caspase-3-dependent apoptotic pathway following repair of H2O2-induced MMP loss by mangiferin.

The level of ROS within cells is tightly regulated by a series of antioxidant enzymes, and exposure to pro-oxidants and dysfunction of antioxidant defenses lead to extensive cellular injury following ROS overaccumulation (Ahmadinejad et al., 2017; Pei et al., 2023). According to accumulated studies, since GSH, which acts as a cofactor for antioxidant enzymes in cells, removes ROS and electrophiles, the ratio of reduced GSH to oxidized GSSG is used as a measure of the redox state of cells (Enns and Cowan, 2017; Lou, 2022). Indeed, even in RPE cells, much evidence has been provided that increasing the ratio of GSH/GSSG reduces H2O2-induced cytotoxicity, whereas decreasing this ratio increases H2O2-induced apoptosis (Liu et al., 2016; Savion et al., 2019). According to the results of the current study, the GSH/GSSG ratio was reduced in ARPE-19 cells treated with H2O2, and the altered GSH/GSSG ratio was restored by mangiferin, which was related to quenching of ROS production. Mangiferin also increased the activity of GPx, which is involved in H2O2 detoxification (Ahmadinejad et al., 2017; Pei et al., 2023), suggesting that the preventive effect of mangiferin against oxidative stress is mediated by the attenuation of reactive oxygen intermediates. Mitochondria are organelles vulnerable to ROS and are also a main source of ROS in eukaryotic cells. H2O2 and superoxide are the major ROS formed in mitochondria, and since superoxide is converted to membrane permeable H2O2, they readily diffuse into cells easily (Munro and Treberg, 2017; Yan et al., 2020), suggesting that the preventive effect of mangiferin against oxidative stress is mediated by the attenuation of reactive oxygen intermediates. In additional experiments, mangiferin dramatically suppressed the production of mtROS by H2O2, which correlated with preservation of the expression and activity of MnSOD involved in suppression of mtROS generation (Palma et al., 2020; Liu et al., 2022). Although the molecular mechanisms for the correlation of the activities of GSH, MnSOD and GPx require further investigation, these results indicate that mangiferin protected ARPE-19 cells from DNA damage, mitochondrial dysfunction and induction of apoptosis under conditions of oxidative stress while exerting ROS scavenging activity.

To further identify the mechanism of antioxidant activity of mangiferin, the role of Nrf2, which enhances the antioxidant capacity by promoting the transcriptional activity of phase II detoxification enzymes, was investigated. Under physiological conditions, Nrf2 is located in the cytoplasm bound to Kelch-like ECH-associated protein 1 (Keap1) and is degraded through the ubiquitin-proteasome pathway (Shaw and Chattopadhyay, 2020; Jenkins and Gouge, 2021). To enhance the transcriptional activity of Nrf2-dependent antioxidant genes, Nrf2 must be phosphorylated for nuclear translocation after dissociation from Keap1. Among the downstream factors of Nrf2, HO-1 can break down heme into free iron, biliverdin and carbon monoxide. Subsequently, biliverdin is converted into bilirubin, thereby exerting strong antioxidant action (Yu et al., 2018; Jenkins and Gouge, 2021). Recently, it has been reported that Nrf2-dependent activation of HO-1 in RPE cells contributes to protection against mitochondrial damage-mediated apoptosis caused by oxidative stress (Clementi et al., 2022; Park et al., 2022), and the importance of the Nrf2/HO-1 axis as a mechanism for the antioxidant activity of mangiferin in numerous cell lines has been emphasized (Saha et al., 2016; Al-Saeedi, 2021; Gendy et al., 2022; Zhou et al., 2023). In this study, mangiferin not only increased the expression of Nrf2 protein but also its phosphorylation in ARPE-19 cells treated with H2O2, and they were expressed predominantly in the nucleus. Furthermore, mangiferin upregulated the expression and enzymatic activity of HO-1, demonstrating that mangiferin may increase HO-1 expression by acting as activators of Nrf2. In subsequent experiments using the HO-1 inhibitor ZnPP, the antioxidant potency of mangiferin to block apoptosis and cytotoxicity in H2O2-exposed ARPE-19 cells was largely offset, suggesting that HO-1 activation contributed to inhibition of H2O2-induced oxidative damage by mangiferin. Taken together, these findings indicate that activation of Nrf2/HO-1 axis by mangiferin contributes at least as one of the upstream signals for the inhibitory action of mangiferin on H2O2-induced cytotoxicity in ARPE-19 cells (Fig. 9). Therefore, it is imperative to explore in further studies whether mangiferin can control oxidative damage-mediated ocular diseases in vivo.

Taken together, the present data demonstrated that mangiferin could eliminate H2O2-induced cytotoxicity such as DNA damage and apoptosis by mediating ROS homeostasis to alleviate mitochondrial dysfunction in ARPE-19 cells. Moreover, mangiferin may contribute to blocking oxidative stress by enhancing the activity of the Nrf2/HO-1 axis, probably because H2O2-induced ROS accumulation was suppressed by HO-1 activation. To the best of my knowledge, this is the first report that mangiferin plays an important role in oxidative stress-induced RPE cell demise, and these findings support the preventive potential of mangiferin in oxidative injury-related ocular disease.

ACKNOWLEDGMENTS

This research was funded by the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korean government (2021R1A2C2009549) and an NRF grant from the Korean Government (MSIT) (2022R1A5A8033794).

CONFLICT OF INTEREST

The authors have no conflicts of interest relevant to this study to disclose.

AUTHOR CONTRIBUTIONS

Cheol Park: conceptualization, data curation, formal analysis, methodology, validation, visualization, writing–original draft, and editing. Hee-Jae Cha: conceptualization, data curation, methodology, validation, visualization, investigation, writing–original draft, writing–review, and editing. Hyun Hwangbo: conceptualization, data curation, formal analysis, methodology, validation, investigation. EunJin Bang: conceptualization, data curation, formal analysis, methodology, validation, investigation, software, resources. Heui-Soo Kim: conceptualization, data curation, formal analysis, methodology, validation, investigation, software, resources. Seok Joong Yun: formal analysis, methodology, validation, investigation, software, resources. Sung-Kwon Moon: conceptualization, data curation, formal analysis. Wun-Jae Kim: conceptualization, data curation, formal analysis, validation, investigation, software, resources. Gi-Young Kim: conceptualization, data curation, formal analysis, methodology, validation, investigation, software, resources. Seung-On Lee: data curation, formal analysis, validation, investigation, software. Jung-Hyun Shim: project administration, resources, supervision, and funding acquisition. Yung Hyun Choi: project administration, resources, supervision, funding acquisition. All the data were generated in-house. All authors agree to be accountable for all aspects of the work and to ensure their integrity and accuracy.

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