The Protective Effects of α-Mangostin Attenuate Sodium Iodate-Induced Cytotoxicity and Oxidative Injury via Mediating SIRT-3 Inactivation via the PI3K/AKT/PGC-1α Pathway

It is well known that age-related macular degeneration (AMD) is an irreversible neurodegenerative disease that can cause blindness in the elderly. Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is a part of the pathogenesis of AMD. In this study, we evaluated the protective effect and mechanisms of alpha-mangostin (α-mangostin, α-MG) against NaIO3-induced reactive oxygen species (ROS)-dependent toxicity, which activates apoptosis in vivo and in vitro. MTT assay and flow cytometry demonstrated that the pretreatment of ARPE-19 cells with α-MG (0, 3.75, 7.5, and 15 μM) significantly increased cell viability and reduced apoptosis from NaIO3-induced oxidative stress in a concentration-dependent manner, which was achieved by the inhibition of Bax, cleaved PARP-1, cleaved caspase-3 protein expression, and enhancement of Bcl-2 protein. Furthermore, pre-incubation of ARPE-19 cells with α-MG markedly inhibited the intracellular ROS and extracellular H2O2 generation via blocking of the abnormal enzyme activities of superoxide dismutase (SOD), the downregulated levels of catalase (CAT), and the endogenous antioxidant, glutathione (GSH), which were regulated by decreasing PI3K-AKT-PGC-1α-STRT-3 signaling in ARPE-19 cells. In addition, our in vivo results indicated that α-MG improved retinal deformation and increased the thickness of both the outer nuclear layer and inner nuclear layer by inhibiting the expression of cleaved caspase-3 protein. Taken together, our results suggest that α-MG effectively protects human ARPE-19 cells from NaIO3-induced oxidative damage via antiapoptotic and antioxidant effects.


Introduction
Retinal pigmented epithelial cells (RPEs) are located between photoreceptor cells (cone and rod cells) and Bruch's membrane [1]. RPEs are highly metabolically active and sensitive to oxidative stress when the retina is exposed to many reactive oxygen species (ROS) [2]. However, abnormal RPEs are regarded as playing an important role in agerelated macular degeneration (AMD), a disease that results in irreversible center vision loss in the elderly population. This disease can generally be divided into two categories: dry AMD and wet AMD. The former is characterized by accumulation of drusen in the retina, thickening of Bruch's membrane, RPE cell and photoreceptor dysfunction, and severe abnormal neovascularization (angiogenesis), which grows into the central region of the retina, leading to retinal exudation, hemorrhage, and, eventually, serious impairment of vision [3,4]. At present, due to the clinical application of anti-vascular endothelial growth factor (VEGF) agents, wet AMD can be effectively controlled. However, the underlying mechanism of the pathology of dry AMD is still largely unknown. Therefore, there are currently no effective treatment options for dry AMD.
In cellular systems, the generation of reactive oxygen species (ROS) can cause cell damage and affect the structure and function of membrane lipids, proteins, and nucleic acids [5]. ROS is considered to be an important mechanism that leads to a variety of neurodegenerative diseases, such as AMD. Previous studies have demonstrated that inhibiting ROS-induced RPE cell damage may inhibit AMD progression [6][7][8]. Catalase, superoxide dismutase (SOD), and glutathione (GSH) are major enzymes that protect RPE cells through increased expression by effectively scavenging ROS and attenuating oxidative damage [9].
PGC-1α, PGC-1β, and PRC (PGC-1-related coactivator) belong to the peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family. They can regulate the biogenesis and respiratory function of mitochondria as well as targeting the mitochondrial antioxidant defense system [10]. The role of PGC-1α is to serve as a switch between mitochondrial biogenesis and oxidative damage by controlling the mitochondrial levels of ROS. Loss-of-function studies of PGC-1α have shown bursts of ROS and an increase in mitochondrial damage and degradation, whereas gain-of-function enhanced mitochondrial biogenesis and the expression of mitochondrial antioxidant defense system-related genes, such as SOD2 (superoxide dismutase 2) and TRX1 (thioredoxin) [10]. However, both pathways, mitochondrial biogenesis and ROS control, strive for the preservation of mitochondrial homeostasis. Post-translational modification of the energy sensors, AMPK (AMP-activated protein kinase), and SIRT-1/3 (sirtuin-1/3), which induce autophagy, are known to regulate PGC-1α activities [11]. In RPE cells, PGC-1α has been shown to drive mitochondrial biogenesis and to activate the antioxidant defense system [8,12]. Recent studies have reported that SOD2 is a major downstream signal of SIRT-3-mediated mitochondrial O2 •− reduction, and deacetylation of SOD2 by the SIRT-3 pathway regulates SOD2 enzymatic activity [13]. We therefore hypothesized that NaIO 3 might induce mitochondrial ROS in RPE cells and that scavenging mitochondrial ROS could be an effective strategy for preventing NaIO 3 -induced retinal toxicity.
Mangosteen (known as the "Queen of Fruit"), which is edible and also known to have medicinal properties, is generally cultivated in Southeast Asia [14]. The major bioactive secondary metabolites of mangosteen have been found to be xanthone derivatives, including α, β, and γ mangostin. Alpha-mangostin (α-MG) has antiinflammatory [15,16], antibacterial [17,18], antioxidant [19], anticancer [20,21], and cardioprotective [22] activities both in vitro and in vivo. α-MG possesses strong antioxidant activity that has gradually been confirmed in recent years. The researchers found that α-MG displayed the ability to scavenge ROS in each dose [23]. Márquez-Valadez and coworkers (2009) found that α-MG could be considered an accurate antioxidant/neuroprotective tool to be employed in further paradigms of neuronal cell damage [24]. Although α-MG has shown a variety of antioxidant and protective properties, its effects on retina health have not yet been clarified. In this study, we will evaluate whether α-MG may reduce or prevent NaIO 3 -induced retinal damage both in vitro and in vivo.

Cell Culture
The ARPE-19 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 µg/mL streptomycin and 100 U/mL penicillin (CSPC Pharmaceutical Group Ltd., Shijiazhuang, China) at 37 • C in an atmosphere containing 5% CO 2 . The cells were passaged every three days once grown to~90% confluence.

Cell Viability Assay
ARPE-19 cells (1.5 × 10 5 cells/well) were seeded into 24-well plates at 1 mL volume and incubated at 37 • C for 24 h. The culture medium was subsequently replaced by a medium containing various doses of α-MG (0, 3.75, 7.5, and 15 µM) alone or with a cotreatment of NaIO 3 for 24 h. For the cell viability assay condition and procedure, refer to [25].

Measurement of Intracellular ROS Production
The effect of different concentrations of α-MG (0, 3.75, 7.5, and 15 µM) on NaIO 3induced intracellular ROS generation in ARPE-19 cells was further confirmed by flow cytometry (BD Biosciences, San Jose, CA, USA), using DCFH-DA fluorogenic dye as a probe. Data analysis was performed using CellQuest.

Measurement of Mitochondrial Damage
For the analysis of mitochondrial status, the cells were incubated with JC-1 dye (2 µg/mL; Cayman Chemical, Ann Arbor, MN, USA) for 50 min. Two µL of Hoechst33342, a DNA-specific fluorescent dye, was added, and the cells were incubated in darkness at 37 • C for 10 min. For the conditions and procedure, refer to [25].

Measurements of Antioxidative Capacities and Extracellular H 2 O 2 Production
The effects of different concentrations of α-MG (pretreatment 0, 3.75, 7.5, 15 µM) on the NaIO 3 -induced antioxidative capacities and H 2 O 2 generation in ARPE-19 cells were further confirmed by ELISA kits. The activities of superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) were analyzed using ELISA kits from Cayman according to the manufacturer's instructions (Cat. 706002, 707002, and 703002, Cayman, Ann Arbor, MI, USA). The extracellular H 2 O 2 production was determined using a Biovision assay kit (Biovision Research Products, Milpitas, CA, USA) following the manufacturer's instructions.

Animal Model
Eight-week-old BABL/c mice were purchased from the Asia University Animal Care and Use Committee (IACVC No. 107-a51a-20, 01 August 2019) and housed in standard cages with a 12-h light-dark cycle. The mice were randomly divided into three groups with each group containing six mice:

1.
Mock group: Animals pretreated with an intraperitoneal (IP) injection of PBS and then a single intravenous (IV) injection of PBS.

2.
Vehicle-treated group: Animals pretreated with an IP injection of PBS and then a single IV injection of 40 mg/kg NaIO 3 [26].

3.
Experimental group: Animals pretreated with an IP injection of 20 mg/kg α-MG and then a single IV injection of 40 mg/kg NaIO 3 [27].

Histology and Immunohistochemistry
The mice were sacrificed, and both eyes were surgically collected and applied to the Davidson's solution (containing 10% formalin, 10% glacial acetic acid, and 4% formaldehyde) to fix the specimen for three days [28]. Eyeball tissues were embedded in paraffin before sectioned at 5 µm-thick and stained with hematoxylin and eosin (H&E) to capture the tissue section image via optical microscope (Olympus Optical, Tokyo, Japan). Different retinal thickness, i.e., whole retina, inner nuclear layer (INL), and outer nuclear layer (ONL) were randomly measured 6 times from the inferior and superior hemiretina in the range of 600-900 µm on nasal and temporal side of the optic nerve.
Besides the expression of caspase-3 in mice retina evaluated by Image J Immunohistochemistry Toolbox (National Institute of Health, Starkville, MD, USA), the tissue sections were incubated with cleaved caspase-3 (1:100, Cell Signaling Technology, Danvers, MA, USA) antibody in BondMax automated slide staining system (Vision BioSystems Ltd., Newcastle Upon Tyne, UK) and the tissue section image screenshotted.

Retinal Imaging
Optical coherence tomography (OCT) was performed using RTVue XR Avanti with AngioVue (Optovue Inc, Fremont, CA, USA). Briefly, OCT of a certain region of the retina was performed repeatedly, and the resultant scans were examined for changes [27].

Statistical Analysis
All grouping and subject selection in this study were decided completely at random. All data were analyzed in SPSS software. Multiple group comparisons were performed using one-way ANOVA followed by least significant difference (LSD) post hoc test. p < 0.05 indicated statistical significance for all tests. The values of the results were representative in terms of the mean ± standard deviation (SD).

α-Mangostin (α-MG) Protects ARPE-19 from NaIO 3 -Induced Damage
To confirm the cytotoxicity of α-MG on ARPE-19, the cells were pretreated with α-MG (0, 3.75, 7.5, and 15 µM) alone or with a co-treatment of NaIO 3 . After 24 h, the cell viability of ARPE-19 was assessed using an MTT assay. The cell survival rate was unaffected following the treatment of cells with < 15 µM α-MG ( Figure 1A). However, treatment with 20 µM α-MG significantly reduced the cell viability, with the effects being significantly different from the untreated controls (0 µM). For this reason, <15 µM α-MG was used for all subsequent experiments.
We also evaluated whether α-MG treatment could reduce ARPE-19 cell damage induced by NaIO 3 . Before being treated with NaIO 3 (6 mM), the ARPE-19 cells were pretreated with α-MG (3.75, 7.5, and 15 µM) for 1.5 h. Based on the results, significantly increased protective effects were found on cell death, compared with the group treated with NaIO 3 only ( Figure 1B).

Treatment with α-MG Suppressed NaIO 3 -Induced Mitochondrial Dysfunction
The JC-1 staining assay is used to evaluate the function of mitochondria [29]. JC-1 monomers emit green fluorescence and indicate damaged mitochondria, while JC-1 polymers emit red fluorescence and indicate healthy mitochondria. The treatment of ARPE-19 cells with NaIO 3 for 24 h caused the accumulation of JC-1 monomers in the mitochondria, which was reduced in a dose-dependent manner following treatment with various concentrations of α-MG (0, 3.75, 7.5, and 15 µM). (Figure 2A). Quantitative data revealed that the green/red fluorescence ratio and NaIO 3 -induced mitochondrial damage increased following treatment with NaIO 3 and improved following treatments with α-MG ( Figure 2B). The results indicated that NaIO 3 -induced mitochondrial dysfunction was improved via the inhibition of mitochondrial fission, but α-MG can alleviate this appearance.

α-MG Reduces NaIO 3 Induced Intracellular ROS and Extracellular H 2 O 2 Generation in ARPE-19 Cells
Previous research demonstrated that mitochondrial dysfunction induces the production of intracellular ROS [30]. We measured the levels of intracellular ROS using DCFH-DA (an intracellular ROS probe) and analyzed through flow cytometry. In agreement with the previous reports [8,25], treatment of the cultured cells with 6 mM of NaIO 3 caused approximately 60% cell death ( Figure 1B) and an approximately 200% increase in intracellular ROS production ( Figure 3A) respectively. Compared with the NaIO 3 -treated group, pretreatment with α-MG attenuated the NaIO 3 -induced intracellular ROS levels in the ARPE-19 cells ( Figure 3A). We then measured the level of extracellular H 2 O 2 using a commercial kit, and the results showed that the increased expression of H 2 O 2 in the NaIO 3 -induced group was significantly eliminated after treatment with α-MG ( Figure 3B). These results established α-MG as an effective antioxidant against NaIO 3 -induced oxidative stress by reducing ROS.

α-MG Improves NaIO 3 -Induced Decreasing Antioxidant Status in ARPE-19 Cells
In mammals, the cellular ROS generation major occurs in mitochondria, which are also considered as the center of cellular bioenergetics. SOD2 is a mitochondrial antioxidant that helps to eliminate O 2 •− and induce the production of H 2 O 2 . However, if H 2 O 2 cannot be metabolized, ROS will be produced. Antioxidant enzymes play a major role in ROS scavenging, and changes in their expression or/and activity are reported to be associated with AMD. As shown in Figure 4, the expressions of SOD activity were increased, but catalase and GSH were decreased in the NaIO 3 -induced group. α-MG (7.5 and 15 µM) treatment significantly reversed the reduced levels of catalase and GSH and the increased level of SOD, indicating that α-MG could modulate the activity of antioxidants. These results suggested that α-MG decreased NaIO 3 -induced oxidative stress by increasing intracellular antioxidant enzyme activity.

α-MG Protects NaIO 3 -Induced Apoptosis in ARPE-19 Cells
Oxidative stress can induce cell apoptosis. To further investigate this protective effect of α-MG on the cell viability of ARPE-19 cells, the morphological change and apoptosis rate of ARPE-19 cells, incubated with α-MG (3.75, 7.5, and 15 µM) for 1.5 h, and then exposed to 6 mM NaIO 3 for 24 h, were determined under a microscope ( Figure 5A) and by flow cytometry (Figure 5B), respectively. Consistently, exposure to 6 mM NaIO 3 led to a significantly higher rate of total apoptosis compared with that of the control group; however, pretreatment of ARPE-19 cells with α-MG at concentrations of 3.75, 7.5, and 15 µM before NaIO 3 exposure significantly decreased NaIO 3 -induced cell apoptosis. These results confirmed that α-MG inhibiting ROS levels could rescue NaIO 3 -induced retinal toxicity by apoptosis.

Effect of α-MG on Apoptotic-Related Protein Expression in NaIO 3 -Treated ARPE-19 Cells
To confirm the protective effect of α-MG against NaIO 3 -induced apoptosis in ARPE-19 cells, we further investigated the effects of α-MG on the expression of apoptosis-related molecules, i.e., Bcl-2, Bax, cleaved PARP-1, and cleaved caspase-3 by Western blotting (Figure 6). Compared with normal control cells, exposure to NaIO 3 induced lower levels of Bcl-2 and higher levels of Bax, cleaved PARP-1, and cleaved caspase-3. However, antioxidants of ARPE-19 cells with α-MG (3.75, 7.5 and 15 µM) for 1.5 h dose-dependently reversed this situation, as shown by the decreased expression of Bax, cleaved PARP-1, and cleaved caspase-3, as well as the increased expression of Bcl-2, which was statistically significant relative to those in NaIO 3 group.

α-MG Downregulates PGC-1α and SIRT-3 Expression through the Inhibition of PI3K/AKT Signaling Pathway in NaIO 3 -Treated ARPE-19 Cells
Sirtuin-3 (SIRT-3) is crucial for controlling mitochondrial metabolism and homeostasis and protects cells from death under conditions of stress [31]. Du et al. (2019) demonstrated that SIRT-3 was implicated in the effect of H 2 O 2 -induced RPE cell oxidative stress, autophagy, and apoptosis [32]. In this study, we assessed whether SIRT-3 was required for protection from ROS production in RPE cells during NaIO 3 treatment. To explore the protective mechanisms of α-MG, the PI3K/AKT signaling pathway and the expression of PGC-1α/ SIRT-3 were detected with western blot. The results suggested that the expression of PI3K and phosphorylation of AKT on ARPE-19 cells were significantly increased after 24 h of NaIO 3 treatment compared with the normal control group, which had been downregulated by α-MG ( Figure 7A,B). Simultaneously, as shown in Figure 7C,D, a statistically significant increase in PGC-1α and SIRT-3 protein expression was shown in the NaIO 3 group. Along with the result of α-MG on the depression of PGC-1α and SIRT-3 protein levels, we found that α-MG protected RPE cells against NaIO 3 -induced oxidative damage by reducing the SIRT-3 expression mediated by the PI3K/AKT/ PGC-1α signaling pathway.

α-MG Inhibition Preserves the Physiological Function of the Retina in Mice following NaIO 3 Treatment
To validate the role of α-MG in the pathogenesis of AMD in vivo, NaIO 3 -injected mice were used as an animal mode [8,25]. We then performed optical coherence tomography (OCT), hematoxylin, and eosin staining to assess geographic atrophy in mice. Optical coherence tomography was performed to detect the effects of α-MG on the retina injuries induced by NaIO 3 . The results showed that α-MG, at the end of the detection points, significantly decreased the thinning of the retina caused by NaIO 3 treatment ( Figure 8A,B).  To further prove this, hematoxylin and eosin (H&E) staining was performed to detect the effects of α-MG on the pathological changes of retina after NaIO 3 administration. Alterations in retinal morphology began to appear seven days after NaIO 3 injection in mice, with a reduction in the outer nuclear layer (ONL) and inner nuclear layer (INL) (Figure 9). In contrast, α-MG moderated the alterations in retinal morphology by preserving the ONL and INL thickness of the retina after NaIO 3 administration. As shown in the graph of H&E staining, compared with the mock group, the thickness of INL and ONL were decreased by about 10-30 µm after the induction of NaIO 3 , which could be reversed by about 10-20 µm with α-MG pretreatment. Collectively, our data show that α-MG mediates retinal degeneration in a dry AMD mouse model.

α-MG Reduced Apoptosis on Retinal Apoptosis Induced by NaIO 3 in Mice following NaIO 3 Treatment
Previous research has shown through in vitro studies that α-MG inhibiting ROS levels could rescue NaIO 3 -induced retinal toxicity by apoptosis. Therefore, we further explored whether α-MG could inhibit the protective effect of NaIO 3 -induced retinal cell damage by regulation cleaved caspase-3. We performed immunohistochemical staining on the cleaved caspase-3 after seven days of treated mice. The results showed that in the mock and α-MG groups, the expression level of cleaved caspase-3 was significantly lower in the NaIO 3 -treated group ( Figure 10). Figure 10. Effect of α-MG on NaIO 3 -induced retinal cell damage via apoptosis in mice. All retinal sections in three study groups were collected after seven days of NaIO 3 treatment and stained with cleaved caspase-3 by immunohistochemical. Red arrows represent the performance of cleaved caspase-3 (brown) in retinal pigment epithelial (RPE) layers. Compared with mock and experimental (NaIO 3 + α-MG) groups, the NaIO 3 group expressed a significantly large amount of cleaved caspase-3. Data were expressed by mean ± SD (n = 6). Values without a common superscript letter are significantly different (p < 0.05).

Discussion
NaIO 3 , as a kind of stable oxidant, has been shown to effectively induce retinal degeneration; this model is widely used because of its reproducibility and controllable degree of retinal damage [33]. NaIO 3 -induced cell death in RPE cells is a valuable in vitro model of AMD. Although many studies using this model demonstrate the death features and molecular events underlying the oxidative stress-mediated cellular responses mimicking the pathogenesis of AMD, it remains unclear regarding ROS-mediated signaling pathways, mitochondrial function, autophagy, and mitophagy, which are integrated to control cell viability in RPE [8,25].
Excessive mitochondrial ROS are known to induce apoptosis. It is well known that the activity of GSH, SOD, and catalase protects cells against ROS-induced oxidative damage [34]. As shown in Figure 4, the expressions of antioxidants except SOD were dramatically decreased in the NaIO 3 -induced group. α-MG treatment significantly reversed the reduced levels of catalase and GSH and the increased level of SOD, indicating that α-MG could modulate the activity of antioxidants. Previous research reported that NaIO 3 induced cytosolic ROS production, but not mitochondrial ROS production; furthermore, it activated ERK, p38, JNK, and protein kinase B (AKT) signaling pathways [35]. Consistently, in our study, the cytosolic ROS production in the ARPE-19 cells was demonstrated under the NaIO 3 -induced retinal degeneration, which was detected as intracellular ROS by DCFH-DA fluorescent probes ( Figure 3) and extracellular H 2 O 2 production. Additionally, we found that the induction of NaIO 3 also promoted mitochondrial damage via the relative intensities of green/red JC-1 fluorescence (Figure 2). These results indicate that α-MG could protect cells from NaIO 3 -mediated ROS injury by maintaining ROS levels and mitochondrial disruption. In addition, in vivo, we found that α-MG significantly retarded the atrophy of NaIO 3 -induced thickness of the total retina, INL, and ONL of NaIO 3 -treated mice at seven days ( Figure 9).
It is well known that PGC-1α participates in the regulation of mitochondrial function and redox state [36]. PGC-1α has been reported to be the upstream target gene of SIRT-1/3, indicating that PGC-1α can strongly stimulate SIRT-3 gene expression [37,38]. SIRT-3, a member of the sirtuin family, is localized in the mitochondria and plays pivotal roles in oxidative pathways by targeting several enzymes [39]. SIRT-3 plays important roles in the inhibition of oxidative stress-induced retinal cell death in early diabetic rats [40]. We observed that α-MG treatment decreased PGC-1α and SIRT-3 expression in NaIO 3 -induced RPE cells ( Figure 7C,D). With this result, we prove that PGC-1α and SIRT-3 are related to the effects of NaIO 3 -induced oxidative stress and apoptosis in RPE cells in vitro, but the treatment of α-MG can attenuate this appearance. Consequently, this study showed the capacity of α-MG on NaIO 3 -induced RPE cell oxidative stress and apoptosis and the importance of SIRT-3 on mitochondrial ROS regulation in ARPE-19 cells. Finally, we confirmed by the activated PI3K/AKT-PGC-1α signaling pathway that the expression of SIRT-3 was involved in the action of α-MG on RPE cell apoptosis.
In our previous studies, Chang et al. (2021) and Chan et al. (2019), we reported that NaIO 3 -induced PI3K/AKT activation was partially dependent on ROS production, eventually leading to cell death by apoptosis [25,41]. These results indicate that NaIO 3 induces apoptosis by increasing the cleaved form of caspase-3, PARP-1, and the Bax/Bcl-2 ratio in RPE cells. In addition, we found that α-MG significantly reduced the production of NaIO 3 -induced cleaved caspase-3 expression by in vivo (Figure 10).
In this study, we found that α-MG inhibited NaIO 3 -induced cell apoptosis by mediating PI3K/AKT inactivation (Figure 7), thereby downregulating the expressions of Bax, cleaved caspase-3, and cleaved PARP and upregulating Bcl-2 expression ( Figure 6). The results of this study show that α-MG significantly protected ARPE-19 cells against NaIO 3induced toxicity, high ROS levels, and apoptosis in vitro. Most importantly, the results of this study, for the first time, support a model whereby α-MG inhibits PI3K/AKT-PGC-1α signaling, which is required for the regulation of SIRT-3 protein expression ( Figure 11).

Conclusions
In summary, our in vitro and in vivo experimental results showed that α-MG could protect retinal cells (especially RPE cells) from oxidative stress damage via apoptosis. The protective function of α-MG on oxidative stressed RPE cells is expressed through its antiapoptotic effect. As a result, α-MG may be able to serve as a potent therapeutic agent for the treatment of retinal degeneration diseases.