Melatonin inhibits NaIO3-induced ARPE-19 cell apoptosis via suppression of HIF-1α/BNIP3-LC3B/mitophagy signaling

Age-related macular degeneration (AMD) leads to gradual central vision loss and eventual irreversible blindness. Melatonin, an endogenous hormone, exhibits anti-inflammatory and antitumor effects; however, the role it plays in AMD remains unclear. Herein, we investigated the anti-AMD molecular mechanism of melatonin after sodium iodate (NaIO3) treatment of ARPE-19 cells in vitro and in animal models with the goal of improving the therapeutic effect. The in vitro results showed that melatonin protected against NaIO3-induced cell viability decline, mitochondrial dysfunction and apoptosis in ARPE-19 cells, and melatonin also alleviated NaIO3-induced reactive oxygen species (ROS) production, mitochondrial dysfunction and mitophagy activation. Melatonin reduced NaIO3-induced mitophagy activation through HIF-1α-targeted BNIP3/LC3B transcription, whereas ROS inhibition realized with N-acetylcysteine (NAC, a ROS inhibitor) combined with melatonin reduced the effect of NaIO3 on mitophagy. An animal model of AMD was established to confirm the in vitro data. Mouse tail vein injection of NaIO3 and melatonin was associated with enhanced repair of retinal layers within 7 days, as observed by optical coherence tomography (OCT) and hematoxylin and eosin (H&E) staining. A reduction in BNIP3 and HIF-1α levels, as determined by immunohistochemistry (IHC) assay, was also observed. These results indicate that melatonin attenuated NaIO3-induced mitophagy of ARPE-19 cells via reduction in ROS-mediated HIF-1α targeted BNIP3/LC3B signaling in vitro and in vivo. Melatonin may be a potential therapeutic drug in the treatment of AMD.


Introduction
Age-related macular degeneration (AMD) is the major cause of vision damage and blindness in elderly people in developed countries [1]. The most important risk factors associated with AMD are age, oxidative stress, inflammation, and genetic factors [2]. Additionally, retinal pigment epithelium (RPE) cells, also known as monolayer pigmented cells, play an important role in providing nutrients to the retina and overall health to photoreceptors of the eyes [3]. Abnormalities in physiological function and reactive oxygen species (ROS) production in RPE cells contribute to vision damage and subsequently to AMD; however, the pathophysiology of AMD remains unclear [4,5].
Sodium iodate (NaIO 3 ) is a strong oxidizing agent that has been extensively used in preclinical experimental models of RPE dystrophy in vivo and in vitro. NaIO 3 animal models have been used to investigate the mechanism associated with AMD pathogenesis because NaIO 3 effectively produces large quantities of ROS in RPE cells in various animal species, including mice, sheep, and rabbits [6,7]. Previous reports showed that NaIO 3 -induced ROS production affected the function of photoreceptors and the choriocapillaris, contributing to RPE cell damage [8]. Moreover, a previous study showed that several natural remedies (e.g., glycyrrhizin) relieved RPE cell damage induced by NaIO 3 treatment. Glycyrrhizin attenuated NaIO 3 -induced RPE and retinal injury through AKT and Nrf2/HO-1 signaling in vitro and in vivo [9]. In addition, αB crystallin, a biomarker of advanced AMD [10], effectively protected against NaIO 3 -induced retinal degeneration [11]. Therefore, NaIO 3 has been widely used to study the molecular mechanism of RPE cell death in AMD.
Melatonin is an important hormone secreted by the pineal gland and regulates physiological circadian rhythms in humans [12]. Melatonin has antioxidant [13], anti-inflammatory [14], anti-proliferative [15], antimetastatic [16] and pro-apoptotic [17] effects during tumorigenesis and in several diseases. Melatonin that is produced in the retina [18], and may play a key role in retinal homeostasis. n clinical research, 6 months of treatment with melatonin (3 mg/day), a patient's AMD was reversed due to retinal protection and macular regeneration with no significant side effects [19]. Previous research in our laboratory demonstrated that melatonin repressed EGF-induced cathepsin S expression in a cell model of proliferative vitreoretinopathy (PVR), [20], which is considered to be a predecessor of AMD. Melatonin has been hypothesized to rebuild telomeres via activation of telomerase in the retina of patients with AMD [21], suggesting an important role for melatonin in AMD treatment.
ROS, including free radicals, are primary risk factors for AMD. Increased ROS levels in RPE cells strongly promoted oxidative damage in mitochondrial DNA (mtDNA) [22], causing extensive mtDNA damage that induced eye disease in animal models. [23]. Furthermore, ROS enhanced the expression of HIF-1α by mediating increased binding activity of NF-κB at the HIF-1αpromoter. Other factors, including VEGF [24], ANGPT [25] and MMPs [26,27] directly contributed to AMD and have been associated with HIF targets. However, the relationships among ROS, HIF-1α, and AMD remains unclear. In this study, the ARPE-19 human RPE cell line was co-treated with NaIO 3 and melatonin to investigate the in vitro antioxidant and antiapoptotic effects of melatonin. In addition, a NaIO 3 -induced AMD-like animal model was established to analyze the mechanism associated with RPE and photoreceptor cell death in vivo.

Melatonin decreases NaIO 3 -induced ARPE-19 cell death.
For effectively treating AMD-like cells in culture with NaIO 3 [28,29], an MTT assay was performed to determine optimal NaIO 3 concentrations (2.5, 5, 10, 15 and 20 mM) and eliminate those that might cause cytotoxicity in ARPE-19 cells. The results showed that NaIO 3 treatment of 15 and 20 mM led to significant toxicity in ARPE-19 cells (Fig. 1A). In addition, our previous study showed that melatonin at lower concentrations (0 ~ 2 mM) exerted a protective effect on ARPE-19 cells by attenuating the abnormal progression without inducing cytotoxicity. To explore the protective effect of melatonin against NaIO 3 -induced cell injury, cells were pretreated with low concentrations of NaIO 3 (0.5, 1 and 2 mM) for 2 h and then treated with a higher NaIO 3 concentration (15 mM) for an additional 22 h. The results of this combinatory treatment showed that melatonin strongly inhibited NaIO 3 -induced cell death (Fig. 1B) and cell morphology changes (Fig. 1C). A colony formation assay was performed to confirm the protective effect of the combination treatment on ARPE-19 cells. Cotreatment with melatonin increased the proliferation of ARPE-19 cells (Fig. 1D). These results indicated that melatonin significantly reduced the NaIO 3 -induced ARPE-19 cell death rate.

Melatonin inhibits NaIO 3 -induced apoptosis in ARPE-19 cells
An Annexin-V/PI assay and measurement of mitochondrial membrane potential were performed to investigate the mechanism through which NaIO 3 treatment induced cell death. Treatment with melatonin increased the cell survival rate via the inhibition of NaIO 3 -induced apoptosis ( Fig. 2A). Mitochondrial depolarization was also observed. Melatonin treatment reduced the proportion of ARPE-19 cells with depolarized mitochondria (Fig. 2B). The levels of apoptosis-associated proteins were also examined. Following administration of cotreatment, melatonin reduced the protein levels of cleavedcaspase-9, cleaved-caspase-3, cleaved-PARP, and total cytochrome c, which had been activated by NaIO 3 treatment (Fig. 2C). However, we found increased expression of mitochondrial cytochrome c in ARPE-19 cells treated with NaIO 3 combined with melatonin (Additiional file 1: Fig S1). Taken together, these data demonstrated that Fig. 1 The effects of NaIO 3 alone or in combination with melatonin on the proliferation of ARPE-19 cells. A ARPE-19 cells were treated with NaIO 3 (0, 2.5, 5, 10, 15, and 20 mM) for 24 h; cell viability was measured by MTT assay. B ARPE-19 cells were cotreated with NaIO 3 (15 mM) and melatonin at a series of concentrations (0, 0.5, 1, and 2 mM) for 24 h; cell viability was evaluated by MTT assay. C The morphology of ARPE-19 cells cotreated with NaIO 3 (15 mM) and melatonin at a series of concentrations (0, 1 and 2 mM) for 24 h. D The cell proliferation rate was determined from the results of a colony formation assay. All of the data are presented as the mean ± SEM of three independent experiments. **, P < 0.01 compared with the control; #, P < 0.05, ##, P < 0.01 compared with the NaIO 3 treatment group melatonin inhibited NaIO 3 -induced cell apoptosis via inactivation of apoptosis-associated proteins in ARPE-19 cells.

Melatonin reduces NaIO 3 -induced cell apoptosis through inhibition of HIF-1α expression in ARPE-19 cells
NaIO 3 -induced RPE cell death in a previous study [30]; however, the mechanism through which NaIO 3 induced ARPE-19 cell death and can be inhibited by melatonin was unclear. To identify the proteins involved in the effects of melatonin and NaIO 3 treatment, treated cells were collected for further human apoptosis-related protein array analysis. NaIO 3 treatment increased the protein level of HIF-1α, and cotreatment with melatonin reduced HIF-1α expression (Fig. 3A). Similar results were observed through western blot analysis, nuclear fraction real-time PCR and immunofluorescence assays ( Fig. 3B-D). To assess HIF-1α involvement in NaIO 3 -induced cell apoptosis, a transfection assay using short interfering RNA (siRNA) against HIF-1α was performed. Knockdown of HIF-1α in combination with melatonin treatment further suppressed NaIO 3 -induced cell apoptosis and mitochondrial depolarization (Fig. 3E, F). Knockdown of HIF-1α in conjunction with the combination Fig. 2 Melatonin reduced NaIO 3 -induced apoptosis in ARPE-19 cells. After combination treatment with NaIO 3 and melatonin, an Annexin-V/PI assay and MitoPotential kit were used to detect A cell apoptosis. The Y-axis presents the cell viability (%) of normal healthy cells (LL), early and apoptotic cells (LR/UR) and necrotic cells (UL), and B the mitochondrial membrane potential, (upper left): percentage of dead cells with intact mitochondrial membrane; (lower left): percentage of live cells with depolarized mitochondrial membranes. C Cell lysates were analyzed and cleaved caspase-9, cleaved caspase-3, cleaved PARP and cytochrome C was detected by using western blotting (β-actin was used as the internal control). All of the data are presented as the mean ± SEM of three independent experiments. **, P < 0.01 compared with control group, and #, P < 0.05 compared with the NaIO 3 treatment group treatment significantly inhibited the expression of cleaved caspase-9, cleaved caspase-3, cleaved PARP, and cytochrome c (Fig. 3G), suggesting a key role for HIF-1α in NaIO 3 -induced cell apoptosis.

Mechanism of HIF-1α action in melatonin-inhibited NaIO 3 induces apoptosis in ARPE-19 cells
HIF-1α activation is necessary for triggering NaIO 3 -induced apoptosis, and HIF-1α is considered to be the transcription factor of BNIP3 [31]. In addition, BNIP3 has been demonstrated to be involved in mitophagy signaling [32], resulting in mitophagy and combined activation of LC3B on the mitochondrial membrane and leading to enhanced signaling downstream. The expression of BNIP3 and LC3B was detected after cotreatment was administered. The protein levels of BNIP3 and LC3B were similar to those of HIF-1α (Fig. 4A). The results of immunofluorescence staining showed that melatonin suppressed BNIP3 and LC3B activation after NaIO 3 treatment (Fig. 4B). An immunoprecipitation assay was performed to examine the interaction between BNIP3 and LC3B. The immunoblot results suggested that melatonin decreased the binding ability of BNIP3 and LC3B, which had been enhanced via NaIO 3 treatment (Fig. 4C). A mitophagy detection kit was used to quantify mitophagy activation after treatment. Melatonin effectively reduced NaIO 3 treatment-induced accumulation of mitophagosomes in ARPE-19 cells (Fig. 4D). To assess mitophagy activity through immunofluorescence, a mitochondria-targeted red fluorescent protein Keima (mt Keima)-Parkin [33] was used. We observed that NaIO 3 -induced PINK1-dependent mitophagy, which was rescued in melatonin-treated cells (Additional file 1: Fig. S2). To confirm the function of BNIP3 in NaIO 3 -induced cell apoptosis, we transfected BNIP3 siRNA into cells. Knockdown of BNIP3 combined with melatonin treatment profoundly suppressed NaIO 3 -induced cell apoptosis and mitochondrial depolarization. Western blotting demonstrated that knockdown of BNIP3 combined with melatonin treatment inhibited the expression of cleaved-caspase-9, cleaved-caspase-3, cleaved-PARP, and cytochrome c ( Fig. 4E-G). Chromatin immunoprecipitation was performed to confirm the relationship between HIF-1α and BNIP3. Melatonin reduced promoter binding by HIF-1α, which had been enhanced by NaIO 3 treatment (Fig. 4H). Taken together, these results suggest that melatonin inhibits NaIO 3 -induced ARPE-19 cell apoptosis via suppression of the HIF-1α/BNIP3-LC3B axis in mitophagy signaling.

Effect of melatonin inhibits NaIO 3 -induced ROS-mediated mitophagy signaling pathway
ROS are considered to be causes of AMD [33]. A NaIO 3 cell model was reported to promote the ROS accumulation [28]. Performing a staining assay with the fluorescent ROS probe DCFH-DA, NaIO 3 combined with melatonin was found to cause a significant decrease in ROS production compared with the effect of NaIO 3 treatment alone (Fig. 5A); therefore, we used the ROS inhibitor N-acetylcysteine (NAC) combined with NaIO 3 and melatonin to examine the role played by ROS. NAC in combination with NaIO 3 and melatonin significantly protected ARPE-19 cells against NaIO 3 -induced injury (Fig. 5B, C). NAC combined with melatonin suppressed NaIO 3 -induced cell apoptosis and mitochondrial depolarization (Fig. 5D, E). The western blot results demonstrated that NAC combined with melatonin inhibited the expression of cleavedcaspase-9, cleaved-caspase-3, cleaved-PARP, BNIP3, and LC3B (Fig. 5F). In addition, hypoxia causes a gradient of oxidative stress mediated by H 2 O 2 , promoting various ocular diseases, such as retinopathy and age-related macular degeneration [34,35]. The results showed that ARPE-19 cells treated with H 2 O 2 exhibited significantly decreased cell viability; this effect was rescued by melatonin treatment (Additional file 1: Fig. S3A). Additionally, melatonin suppressed H 2 O 2 -induced (Additional file 1: Fig. S3B) and apoptosis (Additional file 1: Fig. S3C). However, the western blot findings suggested that melatonin significantly reduced the H 2 O 2 -induced expression of HIF-1α, BNIP3, and LC3B in ARPE-19 cells (Additional file 1: Fig. S3D). H 2 O 2 combined with melatonin suppressed H 2 O 2 -induced mitophagy (Additional file 1: Fig. S4A) and mitochondrial Keima-Red expression in Fig. 3 The role played by HIF-1α in NaIO 3 -induced cell apoptosis. A Whole-cell lysates of ARPE-19 cells were treated with NaIO 3 and melatonin and then analyzed with a human apoptosis array. B Western blot and quantification of HIF-1α in the nuclear fraction and total lysate of ARPE-19 cells. Lamin B was used as the nuclear fraction control, and β-actin was used as a total cell-lysate control. C The mRNA expression of HIF-1α was measured by real-time PCR. GAPDH was used as the internal control. D HIF-1α expression was analyzed by immunofluorescence. Scale bar = 50 μm Transfection with HIF-1α siRNA in ARPE-19 cells treated with NaIO 3 and melatonin. Then, apoptosis E and mitochondrial membrane potential F were analyzed by flow cytometry assay. G Total protein lysate of cells with HIF-1α siRNA transfection was used to explore apoptosis-associated protein levels by western blot assay. β-actin was used as the internal control. All of the data are presented as the mean ± SEM of three independent experiments. ** P < 0.01 compared with si-control group, # P < 0.05 compared with the si-control + NaIO 3 treatment group, ## P < 0.05 compared with the si-HIF-1α + NaIO 3 Fig. S4B). These results suggested that melatonin inhibits NaIO 3 -induced ROSmediated mitophagy via HIF-1α targeting that inhibits BNIP3/LC3B signaling pathway.

Protective effects of melatonin on retinal degeneration in NaIO 3 -treated mice
To confirm our results of cell NaIO 3 -induced damage observed in vitro, a retinal degeneration mouse model Immunoprecipitation (IP) data after western blotting were used to investigate the relationship between the BNIP3 and LC3B proteins (β-actin was used as the internal control). All of the data are presented as the mean ± SEM of three independent experiments. ** P < 0.01 compared with the si-control group, # P < 0.05 compared with the si-control + NaIO 3 treatment group, and ## P < 0.05 compared with the si-BNIP3 + NaIO 3 treatment group was established. Oral treatment of the mice with melatonin (25 or 50 mg/kg) for 7 days was followed by NaIO 3 injection into the tail vein (40 mg/kg) and then oral treatment with melatonin was reinitiated and maintained for 7 days. After treatment, fundus photographs and optical coherence tomography (OCT) were performed to observe the thickness of the whole retina, inner nuclear layer (INL) and outer nuclear layer (ONL). Significant pigmentary changes in the RPE layer and loss of retinal lamination were observed in the NaIO 3 group (Fig. 6A).  The effects of melatonin on retinal degeneration in NaIO 3 -treated mice. C57BL/6 mice were pretreated with melatonin (25, 50 mg/kg) for 7 days and cotreated with NaIO 3 via intravenous injection. A After intravenous injection, changes in the optical coherence tomographic (OCT) images in the day 7-group mice were collected using a Micron IV camera. B Retinal histology for day 7 mice was detected by H&E staining and IHC staining. C The lengths of the ONL and IS/OS were quantified on the basis of retinal histology data. D Normal organ tissues were examined by H&E staining. E Proposed mechanism by which melatonin alleviates NaIO 3 -induced ROS stress-mediated mitophagy through HIF-1α/BNIP3 modulation of LC3 expression in vitro and in vivo. All of the data are presented as the mean ± SEM of three independent experiments. **P < 0.01 compared with the control group and # P < 0.05 compared with the NaIO 3 treatment group However, photographs and OCT taken after high-dose melatonin treatment revealed a fundus similar to that of the control group. In addition, Fig. 6B displays a tissue slice stained with hemoxylin and eosin (H&E) and immunohistochemical dye suggested that the high-dose melatonin group exhibited reduced NaIO 3 -induced overexpression of BNIP3 and HIF-1α in the RPE layer (Fig. 6B). The thicknesses of the ONL, IS and OS were then quantified (Fig. 6C). Melatonin treatment restored retinal thickness, and no indication of damage to the lung, liver, heart, kidney, or spleen was observed (Fig. 6D). These results suggest that oral treatment with melatonin effectively reduced NaIO 3 -induced retinal injury.

Discussion
AMD remains a major cause of blindness in the elderly population. An effective treatment to reduce disease progression has not been identified to date [36]. Melatonin is a naturally occurring and important hormone that has been reported to protect RPE cells from oxidative stress [37]. Melatonin has been shown to inhibit EGF-induced proliferation and motility of ARPE-19 cells by activating the AKT/mTOR pathway [20]. We explored the protective effect of melatonin on ARPE-19 cells and RPE cell after NaIO 3 -induced cell injury to further identify treatment options for AMD. First, we found that a low dose of melatonin effectively reduced cell death through inactivation of apoptosis signaling in NaIO 3 -AMD cell models.
In addition, knockdown of BNIP3 and HIF-1α directly enhanced the efficiency of melatonin treatment. Melatonin disrupted the binding between BNIP3 and HIF-1α, resulting in inhibition of NaIO 3 -induced mitophagy. Upstream HIF-1α activation and ROS production were decreased after treatment, suggesting that melatonin regulated NaIO 3 -induced cell apoptosis in RPE cells via ROS activation. In an AMD animal model, melatonin treatment alleviated NaIO 3 -induced retinal dysfunction and RPE cell injury (Fig. 6E); thus, melatonin may be a potential treatment for patients with AMD. HIF-1α is a critical protein that mediates adaptive responses to hypoxia by regulating the expression of various genes [38]. A recent study reported that HIF-1α can block mitochondrial respiration and electron transport chain (ETC) activity by regulating miR-210 expression in various cell types [39]. Furthermore, hypoxia-induced mitochondrial autophagy is required for HIF-1α targeting BNIP3 [40] and LON [41] expressions. Suppression of the mitochondrial ROS/ HIF-1α pathways through direct inhibition of HIF-1α induced ETC complex I dysfunction and metabolic pathways in leukemia and lymphoma [42]. Autophagy may be involved in the mechanism of oxidative stress activation. Since the transport of cargo was compromised in degenerative RPE cells in patients with AMD, the activation of the autophagy pathway may have been delayed or blocked, leading to increased oxidative stress and causing irreversible injury to RPE cells [43]. Autophagy is important to the metabolism of RPE cells. Knockdown of the ATG7 or BECN1 gene in H 2 O 2 -treated ARPE-19 cells increased ROS generation. In addition, rapamycin treatment promoted autophagy signaling, causing decreased ROS production [44]. These results suggest that autophagy confers RPE cell resistance to oxidative stress and may alleviate AMD.
Mitophagy is a kind of autophagy that is characterized by removal damaged mitochondria through selective pathways. Recent studies have reported that aging promotes the accumulation of damaged mitochondria since aging decreases the efficacy of mitophagy [45], promoting the accumulation of mitochondrial ROS and oxidative damage. A previous stud showed that aged RPE cells exhibit more significant mtDNA damage [23]. These results suggest that mitophagy plays an essential role in AMD. Additionally, melatonin has been reported to protect cells from oxidative damage via mitophagy repression [46], suggesting that melatonin regulates ROS and mitophagy. Melatonin effectively reduced retinal injury in AMD, as demonstrated by previous in vivo animal and in vitro RPE cell models [47,48]. Melatonin protected RPE cells via inactivation of oxidative stressinduced apoptosis and activation of autophagy [49]. The molecular mechanisms related to AMD include oxidative stress and inflammation, but the underlying mechanisms remain unclear. In the present study, a human apoptosis protein array analysis demonstrated that HIF-1α is a vital protein associated with angiogenesis, and its abundance increased after NaIO 3 treatment; when cotreated with melatonin, the expression of HIF-1α was decreased.
Additionally, BNIP3 expression changed in parallel with HIF-1α expression changes, since HIF-1α regulated BNIP3 levels via transcription [31]. Moreover, based on the current study results, HIF-1α/BNIP3 signaling plays an important role in oxidative stress-induced mitophagic cell death. Therefore, HIF-1α/BNIP3 might be a target in AMD treatment. Additionally, decreased HIF-1α expression in NaIO 3 -treated ARPE-19 cells was associated with BNIP3 knockdown, which was particularly intriguing. Upregulation of HIF-1α induced apoptosis of the hUSLF cells through the increased expression of BNIP3 [50]. Other reports demonstrated that knockdown of BNIP3 markedly attenuated HIF-1α inhibition of human HCN-1A cell apoptosis and induced autophagic cell survival [51].

Conclusion
Our study demonstrates that melatonin reduced NaIO 3 -induced ROS-mediated mitophagy in ARPE-19 cells through the suppression of HIF-1α targeting of BNIP3 signaling. HIF-1α/BNIP3 should be considered a novel therapeutic target for AMD.

Cell culture and drugs treatment
The ARPE-19 human retinal pigment epithelia cell line was obtained from the Bioresources Collection and Research Center (BCRC), Food Industry Research and Development Institute (Hsinchu, Taiwan). ARPE-19 cells were cultured in Dulbecco's modified Eagle's medium/ nutrient mixture F-12 Ham (DMEM/F12) containing 10% FBS and 1% penicillin/streptomycin antibiotic at 37 °C with 5% CO 2 . For drug treatment, ARPE-19 cells were treated with melatonin (2 mM) in the absence or presence of NaIO 3 (15 mM). For inhibitor treatment, ROS inhibitor NAC (1 mM) was pre-treated with melatonin (2 mM) for 2 h, and then added with or without of NaIO 3 (15 mM).

Cell viability assay
Cell viability was detected by MTT assay. ARPE-19 cells were seeded in 24-well culture plates (4 × 10 4 cells/well) and treated with melatonin and NaIO 3 or NAC for 24 h. Fresh medium containing MTT (0.5 mg/ml) was used to incubate the treated cells for 4 h, and then, 0.8 ml of isopropanol was added to dissolve the purple formazan. Absorbance was measured at 570 nm with a microplate reader (Labsystems, Helsinki, Finland).

Colony formation assay
ARPE-19 cells were seeded in 6-well culture plates (5 × 10 3 cells/well) and treated with melatonin (0.5, 1 and 2 mM) with or without NaIO 3 (15 mM) for 2 weeks. The colonies were washed twice with PBS, fixed with methanol and stained with PBS containing 5% v/v Giemsa solution for 4 h. Colonies were measured and photographed.

Annexin-V/PI assay and measurement of mitochondrial membrane potential (MMP)
For Annexin-V/PI detection and MMP analysis, ARPE-19 cells were seeded in 6 cm dishes (4 × 10 5 cells) and treated with melatonin (1 and 2 mM) with or without NaIO 3 (

Monitoring of mitophagy
Treatment cells were cultured on 8-well Lab-Tek Chambered Coverglass (2 × 10 4 cells) for 24 h, washed with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized with PBS containing 0.1% Triton X-100 for 10 min. DAPI was incubated with 2% bovine serum albumin at room temperature for 2 h, and the cells were stained using a Dojindo Mitophagy Detection Kit (Dojindo EU GmbH, Munich, Germany). After staining, the cells were visualized with a Zeiss LSM 510 META confocal microscope (Heidelberg, Germany) and analyzed. Keima was used for mitochondria detection, and cells were transfected with a pMitophagy Keima-Red mPark2 plasmid using TurboFect transfection reagent for 6 h; the medium was then replaced with fresh medium, and the cells were incubated for 18 h. The cells were then treated with melatonin and NaIO 3 . After staining, the change in mt-Keima fluorescence was analyzed with a Zeiss LSM 510 META confocal microscope (Heidelberg, Germany) according to the manufacturer's protocols.

Human apoptosis array analysis
ARPE-19 cells were seeded in 10 cm dishes (1.2 × 10 6 cells) and treated with melatonin and NaIO 3 for 24 h. After drug treatment, the cells were lysed using lysis buffer containing protease inhibitor, sonicated and centrifuged. The supernatant was collected for the analysis of apoptosis or antiapoptotic protein levels using a Human Apoptosis Array Kit (ARY009).

Mitochondria lysate and nuclear fraction preparation
ARPE-19 cells were seeded in 10 cm dishes (1.2 × 10 6 cells) and treated with melatonin or a combination of the aforementioned drugs for 24 h. After washing twice with PBS, mitochondria and nuclear lysates were isolated using an AllPure Mammalian Mitochondria Isolation Kit and Nuclear Protein Isolation Kit following the respective manufacturer's instructions.

Cell lysate preparation and Western blot analysis
After drug treatment, cell pellets were lysed with lysis buffer containing protease inhibitor and sonicated on ice. Protein samples were centrifuged for 30 min at 13,000 rpm, and the concentration was measured by the Bradford method (Bio-Rad). Equal amounts (20 μg) of protein sample were separated by 10-12% SDS-PAGE for 2 h and transferred to PVDF membranes for 2 h. The membranes were blocked with Tris-buffered saline containing 0.1% v/v Tween-20 (TBST) containing 5% v/v nonfat milk for 1 h. The primary antibodies were incubated with the membrane overnight at 4 °C, and the secondary antibodies were incubated with the membrane at room temperature. The results were detected with an Luminescent Image Analyzer LAS-4000 mini.

Isolation of RNA and real-time qRT-PCR
The total RNA of the ARPE-19 cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA), and the cDNA was reverse transcribed using GoScript ™ Reverse Transcription Mix (Promega Corporation). Gene expression was detected with GoTaq qPCR Master Mix reagents (Promega Corporation) in an ABI PRISM 7700 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The real-time PCR primer pairs were as follows: for GAPDH, 5′-CAT CAT CCC TGC CTC TAC TG-3′ (forward) and 5′-GCC TGC TTC ACC ACC TTC -3′ (reverse); for BNIP3, 5′-GCC ATC GGA TTG GGG ATC TAT-3′ (forward) and 5′-GCC ACC CCA GGA TCT AAC AG-3′ (reverse); and for HIF-1α, 5′-GAA CGT CGA AAA GAA AAG TCTCG-3′ (forward) and 5′-CCT TAT CAA GAT GCG AAC TCACA-3' (reverse). GAPDH was used as the internal control. All of the gene levels were normalized to the level of the GAPDH bene, and fold change was calculated by the 2 −ΔΔCt method.

Chromatin immunoprecipitation (ChIP)
ARPE-19 cells were seeded in 10 cm dishes (1.2 × 10 6 cells) and treated with melatonin and NaIO 3 for 24 h. Cells were crosslinked with 4% paraformaldehyde for 10 min and incubated with 125 mM glycine for 5 min at room temperature. Then, the lysates were sonicated and immunoprecipitated with antibody against HIF-1α or with mouse IgG. Samples were incubated at 65 °C overnight, RNase was added for 1 h at 37 °C, and proteinase K was added for 2 h at 45 °C. After purification, DNA as dissolved in 20 μl nuclease-free water. The ChIP primers used for the real-time PCR were as follows: for BNIP3, 5′-CTT CCC TGC ACG TCC TCA C-3′ (forward) and 5′-CCG GGT TCT CCT TTG AAG GG-3′ (reverse). The data were collected using an ABI PRISM 7700 real-time PCR system.

Immunofluorescence staining
ARPE-19 cells were seeded in 8-well Lab-Tek Chambered Coverglass (2 × 10 4 cells) and treated with melatonin and NaIO 3 for 24 h. Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, permeabilized with PBS containing 0.1% Triton X-100 for 10 min, and blocked with 2% bovine serum albumin for 2 h. Primary antibodies against HIF-1α and BNIP3 were incubated in 2% bovine serum albumin at 4 °C overnight, and secondary antibodies were incubated in 2% bovine serum albumin at room temperature for 2 h. DAPI reagent was used for counterstaining the cell nucleus. The data were visualized with a Zeiss LSM 510 META confocal microscope (Heidelberg, Germany) and analyzed.

In vivo animal model and immunohistochemistry analysis
This protocol of the animal experiment was approved by the Institutional Animal Care and Use Committee of Chung Shan Medical University (IACUC number: 2400). 5-week-old male C57BL/6JNarl mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Before NaIO 3 treatment, the mice were assigned to a 7-day group, and this group was divided into 5 subgroups (n = 5 per subgroup) and treated orally with melatonin (25 and 50 mg/kg) for 7 days. Melatonin was administered orally before the room lights were switched off for 1 h. After melatonin pretreatment for 7 days, NaIO 3 was injected into the tail vein (40 mg/kg), and then, the mice were given melatonin orally for the final 7 days of the experiment. After melatonin treatment mice in the 7-day group were subjected to OCT imaging and sacrificed. Lung, liver, spleen, kidney and eye tissues were harvested, fixed in formalin, and processed for H&E staining. The retina in the eyes was analyzed by BNIP3 and HIF-1α staining.

In vivo mice optical coherence tomography
Mice were anesthetized, and then, the pupils were dilated using a drop of 1.0% tropicamide (Alcon Laboratories, Inc.). Photography and fluorescein angiography of the retina in each eye were performed with a Micron IV camera (Phoenix Research Laboratories, Inc.). Optical coherence tomography images were taken with an image-guided tomographer (Micron IV-OCT2; Phoenix Research Laboratories, Inc.).

Statistical analysis
All of the data as are presented as the mean ± SEM of at least three independent experiments. The significance of the differences between datasets was assessed by t test or one-way ANOVA (GraphPad Prism 6). Differences were considered significant when P < 0.05 or P < 0.01.