Melatonin antagonizes oxidative stress-induced mitochondrial dysfunction in retinal pigmented epithelium cells via melatonin receptor 1 (MT1)

Abstract

High energy-consumption in retinal pigmented epithelium (RPE) cells poses oxidative stress (OS) and contributes to mitochondrial dysfunction (MD) for retinal degeneration-associated diseases. In the present study, we evaluated the protective role of Melatonin, a natural antioxidant, against the hydrogen peroxide (H₂O₂)-induced damage to RPE cells. The cellular viability, apoptosis, the expression of apoptosis-associated proteins and mitochondrial function were examined in the retinal ARPE-19 cells, post the treatment with H₂O₂ or (and) with Melatonin. The regulation by Melatonin receptor 1 (MT1) on the Melatonin-mediated protection was also examined via MT1 knockdown with siRNA. Results demonstrated that Melatonin significantly ameliorated cell viability reduction, reduced apoptosis and downregulated the apoptosis-associated proteins in H₂O₂-treated ARPE-19 cells. The H₂O₂-induced mitochondrial dysfunction was also significantly blocked by the Melatonin treatment, presenting as a reduced accumulation of reactive oxygen species (ROS) and mitochondrial superoxide and an ameliorated reduction of mitochondrial membrane potential (MMP). In addition, the knockdown of MT1 with MT1-specific siRNA inhibited the Melatonin-mediated protection against OS damage in ARPE-19 cells. In summary, we confirmed the protective role of Melatonin against H₂O₂-induced mitochondrial dysfunction in RPE cells. MT1 knockdown blocked such protective role of Melatonin. It is implied that Melatonin exerts a protective role against oxidative stress via Melatonin-MT1 signaling in RPE cells.

INTRODUCTION

Due to the extremely high workload of retinal pigment epithelial (RPE) cells, enriched mitochondrial population and robust metabolic activity were required to meet the high-energy need in RPE cells (Plafker et al., 2012; Boulton et al., 2001). High local reactive oxygen species (ROS) and an amplified oxidative phosphorylation produce during the robust metabolic activity in RPE cells (King et al., 2004). Sustained high ROS production will eventually overcome host neutralizing capability, and then cause cytosolic or mitochondrial damage, and even result in cell apoptosis. Oxidative stress (OS), particularly, chronic intracellular OS, constantly threatens the structural and functional integrity of the RPE cells (Plafker et al., 2012). Particularly, age-related macular degeneration (AMD), which is a leading cause of blindness in the elderly population (Congdon et al., 2004; Mehta, 2015; Randolph, 2014), has been increasingly attributed to the intracellular ROS accumulation (Rózanowska et al., 1995; Jin et al., 2001). Therefore, countermeasures to antagonize the OS-mediated damage to RPE cells or to eliminate ROS might be alternative measures for AMD prevention and treatment.

ROS mainly targets mitochondrium, destroys its membrane integrity, causes a collapse in the mitochondrial membrane potential (ΔΨm, MMP), and thus results in mitochondrial dysfunction. Released mitochondrial mediators even activate apoptosis-associated caspases, such as cytochrome c (Cyt c) and induce cell apoptosis (Lieven et al., 2003; Sano et al., 2006; Jiang et al., 2005; Balaban et al., 2005). A reduced MMP, an inactivated complex IV and the release of Cyt c were found in the RPE cells under OS (Liu et al., 2015; Hanus et al., 2015a). Autophagy is a self-degradation process for non-essential or damaged cellular constituents, to keep the balance between organelle biogenesis, protein synthesis and their clearance. (De Duve, 1963; De Duve and Wattiaux, 1966). There is cross-talk with ROS in both cell signaling and protein damage, in response to ROS-mediated damage (Lee et al., 2012a). Mitochondrial ROS and oxidized lipids in mitochondria regulate autophagy (Kissová et al., 2006), and can be antagonized by antioxidants (Kissová et al., 2006). H₂O₂ also plays a regulatory role in autophagy in response to nutrient starvation (Djavaheri-Mergny et al., 2006).

In recent years, several antioxidants have been identified to antagonize OS in RPE cells, such as Melatonin (Ozdemir et al., 2014), Gossypol Acetic Acid (Hanus et al., 2015b), Lycium barbarum polysaccharides (Liu et al., 2015) and Epigallocatechin gallate (EGCG) (Cia et al., 2014). In the present study, we examined the antioxidant effect of Melatonin in retinal pigment epithelial (RPE) cells under oxidative stress. Then the induction of autophagy and the regulation by Melatonin on autophagy was also investigated. Our study indicated a promising anti-oxidative stress effect of Melatonin in retinal pigment epithelial cells.

MATERIALS AND METHODS

Cell culture and treatment

Human RPE cell line ARPE-19 (ATCC, Manassas, VA, USA) was cultured with Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Rockville, MD, USA), adding 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) and 2 mM L-glutamine solution (Sigma-Aldrich, St. Louis, MO, USA) at 37°C with CO₂ 5%. For Melatonin treatment, ARPE cells with 85% confluence were updated with DMEM + 1% FBS, in which, Melatonin was added with a final concentration of 0, 20, 50, 100 or 200 μM for 0, 12, 24, 48 or 72 hr. For H₂O₂ treatment, the medium was added with 0 or 400 μM H₂O₂ (Thermo Scientific, Rockford, IL, USA) (Tsai et al., 2017; Xie et al., 2017). For the knockdown of Melatonin receptor 1 (MT1), ARPE cells with 85% confluence were transfected with 40 or 80 nM siRNA 1-MT1 or siRNA 2-MT1 (GenePharma, Shanghai, China), with Scramble RNA as negative control for 48 hr. The transfection was performed via Lipofectamine RNAiMax (Invitrogen), according to reagent manual.

Protein samplepreparation and western blotting

ARPE cells were scratched and were collected for the protein isolation of mitochondrial and cytosol fractions with Mitochondria/Cytosol Fractionation Kit (BioVision, Milpitas, CA, USA), according to the kit’s manual. Protein concentration was titered using the BCA protein assay reagent (Sigma-Aldrich). Protein samples were separated with 12% SDS-PAGE gel, and were transferred to a PVDF Transfer Membrane (Thermo Fisher Scientific, Waltham, MA, USA). The membrane was inoculated with a rabbit polyclonal antibody to cytochrome c, caspase 3, caspase 9, Cox4l1 or β-actin (either with 1:1000, all from Sigma-Aldrich) for the antigen-antibody binding. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Pierce, Rockford, IL, USA)and enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech, Amersham, UK) were utilized to visualize the Ag-Ab binding.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining

TUNEL staining was performed for apoptosis assay with TUNEL Apoptosis Detection Kit (Promega, Madison, WI, USA). ARPE-19 cells were fixed with 4% Paraformaldehyde, were washed with phosphate buffered solution (PBS)for three times, and then were incubated for 1 hr at room temperature 26°C with TUNEL and DAPI (4’, 6-Diamidino-2-Phenylindole, Dihydrochloride) solution respectively for segmented DNA and nucleus. Cells were observed and imaged with confocal laser microscopy (Olympus, Tokyo, Japan).

Measurement of mitochondrial ROS, superoxide and MMP

Cytosolic ROS was stained with DCFH-DA (2’,7’-Dichlorodihydro-fluorescein diacetate) (Invitrogen), which was converted to DCF (2,7-dichlorofluo-rescein) in the presence of ROS, according to the reagent manual. 2,7-dichlorofluo-rescein (DCF) fluorescence was measured with a luminescence spectrometer (Ex/Em = 488/530 nm). The mitochondrial superoxide was stained with MitoSOX^TM Red (Invitrogen Molecular Probes, Carlsbad, CA, USA), which highly selectively binds to the mitochondrial superoxide. MitoSOX Red fluorescence was detected with a luminescence spectrometer (Ex/Em = 510 / 580 nm). MMP was quantified with MitoSensor red (Abcam, Cambridge, UK) according to the kit’s manual. MitoSensor red (Pierce) attaches to the mitochondrial membrane in live cells, presenting red in monomeric form, and detaches when the mitochondrial membrane is broken, presenting green in aggregated form. MitoSensor red fluorescence was observed with a luminescence spectrometer (Ex/Em = 579 / 599 nm) and was indicated as an OD value.

Statistical Analysis

Quantitative results are presented as mean ± S.D. of the results from at least triple independent experiments. Student’s t test or one-way ANOVA followed by Newman-Keuls test was performed to compare results from two or more groups. A p value < 0.05 or less was considered statistically significant.

RESULTS

Cytotoxity of Melatonin in retinal pigmented epithelium ARPE-19 cells

Melatonin is chemically known as N-acetyl-5-methoxy tryptamine (Singh and Jadhav 2014) (Fig. 1A). It mainly regulates sleep and wakefulness in animals (Hardeland et al., 2006), and antagonizes OS in plants (Tan et al., 2012). Firstly, we examined the cytotoxic effect of Melatonin in ARPE-19 cells with MTT assay. It is shown in Fig. 1B that the treatment with 20, 50, 100 or 200 μM Melatonin did not downregulate the viability of ARPE-19 cells post the incubation for 48 hr. However, the cellular viability was markedly higher in the ARPE-19 cells with the treatment with 100 μM Melatonin for 48 hr (Fig. 1A). We then repeated the assay for various time points with 100 μM Melatonin; as indicated in Fig. 1C, significant cellular viability increase was found at 48 or 72 hr post Melatonin treatment. Therefore, Melatonin upregulates the viability of retinal pigmented epithelium ARPE-19 Cells with a concentration of 100 μM.

Fig. 1

Cytotoxicity of Melatonin in retinal pigmented epithelium ARPE-19 cells. A: Chemical structure of Melatonin. B: Cell viability of the ARPE-19 cells, which were treated with increasing concentrations of Melatonin or control for 24 hr; C: Cell viability of the ARPE-19 cells, which were treated with 0 or 100 μM Melatonin for 0, 12, 24 or 48 hr;. Cell viability was assessed with the 3- (4,5-cimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay. Data represent the means and SD of three independent experiments.

Melatonin blocks the H₂O₂-induced apoptosis in ARPE-19 cells

To explore the influence of Melatonin on oxidative stress-induced damage to retinal pigmented epithelium cells, we examined the viability and apoptosis induction in the H₂O₂-treated ARPE-19 cells. It was found that H₂O₂ treatment significantly reduced the cellular viability of ARE-19 cells with a concentration of 400 μM, at 12, 24 or 48 hr post treatment (p < 0.05, p < 0.01 or p < 0.001, Fig. 2A). However, such viability reduction was blocked by 200 μM Melatonin at 24 or 48 hr post treatment (p < 0.05 or p < 0.01, Fig. 2A), and even by 100 μM Melatonin at 48 hr post treatment (p < 0.05, Fig. 2A). Moreover, there was a significant difference in such blockage between 100 and 200 μM Melatonin (p < 0.05, Fig. 2A).

Fig. 2

MTT assay for viability and TUNEL staining for apoptosis in the H₂O₂- or (and) Melatonin-treated ARPE-19 cells. A: Relative vitality of ARPE-19 cells, post the treatment with 0 or 400 μM H₂O₂, or (and) with 0, 100 or 200 μM Melatonin for 0, 12, 24 or 48 hr, via MTT assay. B: Apoptosis in the four groups (0 μM H₂O₂ + 0 μM Melatonin, 400 μM H₂O₂ + 0 μM Melatonin, 400 μM H₂O₂ + 100 μM Melatonin, 400 μM H₂O₂ + 200 μM Melatonin, treatment for 48 hr for each group of cells) of ARPE-19 cells, by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Nucleus was stained with DAPI (4’,6-Diamidino-2-Phenylindole, Dihydrochloride), and two types of staining was merged. C: Relative apoptotic ARPE-19 cells, presenting as percentage of TUNEL-stained to total cells, with the percentage of apoptotic cells in H₂O₂ treatment group as 100%. * p < 0.05, ** p < 0.01 or **** p < 0.0001, ns: no significance.

To further investigate the inhibition by Melatonin on the H₂O₂-induced damage to ARPE-19 cells, we performed TUNEL staining for apoptosis in the H₂O₂- or (and) Melatonin-treated ARPE-19 cells. Compared to normal ARPE-19 cells (First column, Fig. 2B), there were dramatically higher amounts of TUNEL-positive nuclei in the H₂O₂-treated (48 hr) ARPE-19 cells (Second column, Fig. 2B; p < 0.0001, Fig. 2C). We also examined the influence of Melatonin on the H₂O₂-induced apoptosis in ARPE-19 cells. It was found that 100 μM Melatonin reduced the H₂O₂-promoted TUNEL-positive cells (48 hr), however not significantly (Third column, Fig. 2B). Such reduction of H₂O₂-promoted TUNEL-positive cells was significant when 200 μM Melatonin (48 hr) (Fourth column, Fig. 2B; p < 0.01, Fig. 2C).

In addition, western blotting was performed to evaluate apoptosis-associated proteins in H₂O₂- or (and) Melatonin-treated ARPE-19 cells. Compared to mitochondrial cytochrome c (Cyto c) (Cox4I1 as loading control for mitochondria) (Fig. 3A and 3B), significantly more cytochrome c (Cyto c) was released in cytosol in response to H₂O₂ treatment (400 μM) (p < 0.001, Fig. 3C). Such promotion was markedly blocked by 100 or 200 μM Melatonin (p < 0.01 or p < 0.001), dose-dependently (p < 0.01). The H₂O₂-induced promotion to cleaved caspase 3 (Cleaved CASP 3) and Cleaved CASP 9 was also significant (p < 0.0001, Fig. 3D and 3E), and was also blocked by Melatonin treatment (p < 0.001 or p < 0.0001), dose-dependently (p < 0.001). Taken together, these results indicate that Melatonin blocks the H₂O₂-induced apoptosis in ARPE-19 cells.

Fig. 3

Effect by Melatonin on the expression of apoptosis-related proteins in the H₂O₂-treated ARPE-19 cells. A: Western blot analysis of cytochrome c in mitochondria and in cytosol, of pro-caspase 3/9 and cleaved caspase 3/9 in the ARPE-19 cells, post the treatment (for 48 hr) with or without 400 μM H₂O₂, with 0, 100 or 200 μM Melatonin; B-D: Relative levels of cytosol / mitochondria cytochrome c (cyto / Mito cyt c) (B), Cleaved / Pro CASP 3 (C) and Cleaved / Pro CASP 9 (D) in the four groups of ARPE-19 cells. Experiments were independently repeated for triple times. ** p < 0.01, *** p < 0.001 or **** p < 0.0001.

Melatonin antagonizes H₂O₂-induced mitochondrial dysfunction in ARPE-19 cells

Retinal pigmented epithelium cell is one type of high energy-consuming cell, in which the mitochondrion is the energy plant (Barot et al., 2011). Accumulating evidence supports the contribution of mitochondrial dysfunction to many retinal degenerations (Barot et al., 2011). To evaluate the oxidative stress-exerted damage to and a possible protection by Melatonin to such damage in retinal pigmented epithelium cells, we examined mitochondrial function in the H₂O₂-treated ARPE-19 cells, with or without Melatonin treatment. As indicated in Fig. 4A, compared with normal ARPE-19 cells, more DCFH-DA was oxidized to highly fluorescent compound DCF, presenting green under fluorescence microscope, in the ARPE-19 cells, post H₂O₂ treatment (400 μM for 48 hr) (p < 0.0001, Fig. 4B). Though the Melatonin treatment (100 μM for 48 hr) did not pose a significant effect on the conversion of DCFH-DA to DCF in normal ARPE-19 cells, markedly less DCF was found in the ARPE-19 cells, post the treatment with both 400 μM H₂O₂ and 100 μM Melatonin, than in the ARPE-19 cells, post the treatment only with 400 μM H₂O₂ (p < 0.01, Fig. 4A and 4B). In addition, theH₂O₂-promoted (400 μM for 48 hr) mitochondrial superoxide (p < 0.001, Fig. 4C) was inhibited by Melatonin (100 μM for 48 hr) (p < 0.01) in ARPE-19 cells. The H₂O₂-mediated MMP reduction and the Melatonin-mediated blockage of such reduction were also observed in ARPE-19 cells (p < 0.01 or p < 0.001, Fig. 4D and 4E). Taken together, our results confirmed Melatonin’s antagonism of the H₂O₂-induced mitochondrial dysfunction in ARPE-19 cells.

Fig. 4

Mitochondrial function in the H₂O₂- or (and) Melatonin-treated ARPE-19 cells. A: Conversion of DCFH-DA (2’,7’-Dichlorodihydro-fluorescein diacetate) to DCF (2,7-dichlorofluo-rescein) in the four groups of ARPE-19 cells (0 μM H₂O₂ + 0 μM Melatonin as control, 400 μM H₂O₂ + 0 μM Melatonin, 0 μM H₂O₂ + 100 μM Melatonin, 400 μM H₂O₂ + 100 μM Melatonin, treatment for 48 hr for each group of cells); B: Percentage of DCF fluorescence-positivity (to total cells); C: Percentage of mitochondrial superoxide (to control (0 μM H₂O₂ + 0 μM Melatonin)) in the four groups of ARPE-19 cells, with nucleus stained as blue with DAPI; D and E: MitoSensor red staining for (D) and the percentage of Mitochondrial membrane potential (MMP) (to control) in the four groups of ARPE-19 cells. Experiments were performed in triplicate independently. ** p < 0.01, ***p < 0.001 or ****p < 0.0001, ns: no significance.

Influence of the knockdown of MT1 melatonin receptor on the Melatonin-mediated mitochondria protection in ARPE-19 cells

To reconfirm the Melatonin-mediated mitochondria protection in ARPE-19 cells, we knocked down the MT1, and then re-evaluated the mitochondrial function in the ARPE-19 cells, post treatment with H₂O₂ or (and) Melatonin. We firstly evaluated the MT1 expression in the H₂O₂-treated ARPE-19 cells, and the relative MT1 mRNA level to β-actin was not significantly regulated by the H₂O₂ treatment (Fig. 5A). Figure 5B demonstrated that the siRNA-mediated knockdown of MT1 was significant in ARPE-19 cells, with of concentration of 40 or 80 nM for 48 hr (p < 0.05 or p < 0.001 for 40 nM; p < 0.01 or p < 0.0001 for 80 nM); and siRNA 2-MT1 was more efficient than siRNA 1-MT1 (either p < 0.01 for 40 or 80 nM). Moreover, the Melatonin-mediated inhibition to the H₂O₂-mediated mitochondrial dysfunction (DCF conversion, mitochondrial superoxide accumulation and MMP reduction) was blocked by the MT1 knockdown. There was a significant difference in the relative DCF level (p < 0.05 for siRNA 2-MT1 with 40 nM, p < 0.05 for siRNA 1-MT1 and p < 0.01 for siRNA 2-MT1 with 80 nM, Fig. 5C), in the relative mitochondrial superoxide accumulation (p < 0.05, p < 0.01 or p < 0.001, Fig. 5D) and in the relative MMP reduction (p < 0.05, p < 0.01 or p < 0.001, Fig. 5E) between siRNA-transfected (siRNA 1-MT1 and siRNA 2-MT1) and scramble RNA-transfected ARPE-19 cells, post the treatment with both the treatment with H₂O₂ and Melatonin. Moreover, there was marked difference in the influence on both mitochondrial superoxide accumulation and MMP reduction between siRNA 1-MT1 and siRNA 2-MT1 (either p < 0.05 for relative mitochondrial superoxide or relative MMP, with 80 nM, Fig. 5D or 5E), being consistent with the knockdown efficiency of both siRNAs. Therefore, Melatonin antagonizes the H₂O₂-induced mitochondrial dysfunction in ARPE-19 cells.

Fig. 5

Mitochondrial function in the H₂O₂- or (and) Melatonin-treated ARPE-19 cells, post Melatonin receptor 1 (MT1) knocked down. A: Relative MT1 mRNA level in the ARPE-19 cells, post the treatment with 0, 200 or 400 μM H₂O₂ for 12 or 48 hr; B: Relative MT1 mRNA level in the ARPE-19 cells, post the transfection with 40 or 80 nM siRNA 1-MT1, siRNA 2-MT1 or scramble RNA for 48 hr; C-E: Percentage of DCF fluorescence-positivity (to Scramble) (C), percentage of mitochondrial superoxide (to Scramble) (D) and relative mitochondrial membrane potential (to Scramble) (E) in the H₂O₂-treated (400 μM for 48 hr) and Melatonin-treated (100 μM for 48 hr) ARPE-19 cells, post the transfection with 40 or 80 nM siRNA 1-MT1, siRNA 2-MT1 or scramble RNA. Experiments were performed in triplicate independently. * p < 0.05, ** p < 0.01, ***p < 0.001 or ****p < 0.0001, ns: no significance.

DISCUSSION

Melatonin, chemically named as N-acetyl-5-methoxytryptamine, is a natural antioxidant with a circadian secretion pattern (Colin-Gonzalez et al., 2015). Deregulation of Melatonin with a disrupted circadian rhythm is associated with oxidative stress and inflammatory processes for several central nervous system (CNS) disorders (Colin-Gonzalez et al., 2015; Espino et al., 2012). It was found in multiple organs (Reiter, 1991), including in the retina, particularly during night (Tosini et al., 2012). An important role of Melatonin has recently been recognized in retinal physiology and patho-physiology (Tosini et al., 2012; Wiechmann and Sherry, 2013). Melatonin protects such retinal cells as retinal pigment epithelial cells and photoreceptors against oxidative stress- or ischemia-induced cell death (Osborne et al., 1998; Liang et al., 2004; Fu et al., 2012). Daily melatonin administration also protects the retina against progression of retinal age-related macular degeneration (AMD) (Rosen et al., 2009). Knockout of MT1 accelerates the age-related loss of photoreceptors cells (Baba et al., 2009). In the present study, oxidative stress model in RPE cells was constructed by the treatment with 400 μM H₂O₂ on ARPE-19 cells. The H₂O₂ treatment markedly reduced the cellular viability, induced apoptosis and promoted apoptosis-associated proteins in ARPE-19 cells. However, these pathological changes were blocked by the melatonin with 100 μM, such concentration was also confirmed to be protective in neural cells in previous study (Su et al., 2015). Thus, we confirmed the protective role of Melatonin against H₂O₂ treatment. However, it is not known whether such concentration was physiologically and pharmacologically safe for treatment.

The mitochondrion is the energy plant (Barot et al., 2011) for the high energy-consuming retinal pigmented epithelium cells. And mitochondrial dysfunction has been implicated by accumulating evidence in many retinal degeneration-associated diseases (Barot et al., 2011). Chronic overproduction of ROS has been found in the retina and results in abnormal mitochondrial functions in diabetes (Kowluru, 2005; Madsen-Bouterse and Kowluru, 2008), in Glaucoma (Lee et al., 2012b; Kong et al., 2009) and in age-related macular degeneration (AMD) (Hageman et al., 2005). In our study, we are interested in whether the mitochondrion is the target for Melatonin against oxidative stress-posed damage to ARPE-19 cells. The mitochondrial dysfunction-associated pathological changes, such as the accumulation of ROS and superoxide in mitochondria and the MMP reduction, were found in H₂O₂-treated ARPE-19 cells, however, were inhibited by Melatonin. Therefore, we speculated that the mitochondrion may be the target for Melatonin against oxidative stress in RPE cells.

Melatonin receptor (MT) is located widely in multiple organs, including the retina (Uz et al., 2005). MT1 and MT2 are expressed in many types of cells within eye (Tosini et al., 2013). It was found that mice lacking MT1 receptors have elevated intraocular pressure (IOP), significantly reduced retinal ganglion cells, implying dysfunctional melatonin signaling as a possible risk factor in the pathogenesis of glaucoma (Tosini et al., 2013). It has also been confirmed that Melatonin modulates visual function in the mouse retina via the MT1 melatonin receptor (Baba et al., 2009). It has been confirmed that the Melatonin-MT1 signaling involves in the anti-oxidative stress procession in many types of cells or organs, such as the heart (Yu et al., 2014), germ cells (Mukherjee et al., 2015) and corneal fibroblasts (Choi et al., 2011). However, it is not clear whether the Melatonin-MT1 signaling is involved in the Melatonin-mediated protection against oxidative stress in RPE cells. We reconfirmed the protective role of Melatonin in RPE cells with gene knockdown method. The MT1-specific siRNA markedly blocked the Melatonin-mediated protection against oxidative stress in RPE cells. The Melatonin-mediated amelioration of mitochondrial dysfunction in the H₂O₂-treated ARPE-19 cells was blocked by the MT1 knockdown.

Up to now, the antagonization by melatonin against oxidative stress was not well understood. Melatonin reduces reactive oxygen species formation in normal human epidermal keratinocytes (NHEK), not only by directly scavenging ROS, but also by promoting anti-oxidative enzyme activity (Kleszczyński et al., 2016). The direct scavenging effect against ROS was also observed in aged seed (Deng et al., 2017). We did not examine a possible direct anti-oxidative stress effect in RPE cells. However, the loss-of-function results confirmed the MT-dependence, indicating the involvement of Melatonin-MT signaling in such effect. The activated signaling pathway then could mediate the translocation of the nuclear erythroid 2-related factor 2 resulting in activation of phase-2 antioxidant enzymes (γ-GCS, HO-1, NQO1) (Kleszczyński et al., 2016), and triggering the transcriptional upregulation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and CHOP.

Though various studies confirmed the protective effect of melatonin against oxidative damage and mitochondrial damage (Suofu et al., 2017; Ding et al., 2014), such kind of antagonizing role of melatonin has never been reported against oxidative stress-induced mitochondrial dysfunction in retinal pigmented epithelium cells. However, in the present study, we did not know about the downstreamer involvement, post the MT1 activation.

In summary, we confirmed the protective role of Melatonin against H₂O₂-induced apoptosis and mitochondrial dysfunction in RPE cells. MT1 knockdown blocked such protective role of Melatonin against the H₂O₂-mediated damage to RPE cells. This suggests that Melatonin exerts a protective role against oxidative stress via Melatonin-MT1 signaling in RPE cells.

ACKNOWLEDGEMENT

The present study was supported by the grant from Yantai Science and Technology Development Plan (Grant No. 2018SFGY105 and 201307).

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES

• Baba, K., Pozdeyev, N., Mazzoni, F., Contreras-Alcantara, S., Liu, C., Kasamatsu, M., Martinez-Merlos, T., Strettoi, E., Iuvone, P.M. and Tosini, G. (2009): Melatonin modulates visual function and cell viability in the mouse retina via the MT1 melatonin receptor. Proc. Natl. Acad. Sci. USA, 106, 15043-15048.
• Balaban, R.S., Nemoto, S. and Finkel, T. (2005): Mitochondria, oxidants, and aging. Cell, 120, 483-495.
• Barot, M., Gokulgandhi, M.R. and Mitra, A.K. (2011): Mitochondrial dysfunction in retinal diseases. Curr. Eye Res., 36, 1069-1077.
• Boulton, M., Rózanowska, M. and Rózanowski, B. (2001): Retinal photodamage. J. Photochem. Photobiol. B, 64, 144-161.
• Choi, S.I., Dadakhujaev, S., Ryu, H., Im Kim, T. and Kim, E.K. (2011): Melatonin protects against oxidative stress in granular corneal dystrophy type 2 corneal fibroblasts by mechanisms that involve membrane melatonin receptors. J. Pineal Res., 51, 94-103.
• Cia, D., Vergnaud-Gauduchon, J., Jacquemot, N. and Doly, M. (2014): Epigallocatechin gallate (EGCG) prevents H2O2-induced oxidative stress in primary rat retinal pigment epithelial cells. Curr. Eye Res., 39, 944-952.
• Colin-Gonzalez, A.L., Aguilera, G., Serratos, I.N., Escribano, B.M., Santamaria, A. and Tunez, I. (2015): On the Relationship Between the Light/Dark Cycle, Melatonin and Oxidative Stress. Curr. Pharm. Des., 21, 3477-3488.
• Congdon, N., O’Colmain, B., Klaver, C.C., Klein, R., Muñoz, B., Friedman, D.S., Kempen, J., Taylor, H.R. and Mitchell, P.; Eye Diseases Prevalence Research Group. (2004): Causes and prevalence of visual impairment among adults in the United States. Arch. Ophthalmol., 122, 477-485.
• De Duve, C. (1963): The lysosome. Sci. Am., 208, 64-72.
• De Duve, C. and Wattiaux, R. (1966): Functions of lysosomes. Annu. Rev. Physiol., 28, 435-492.
• Deng, B., Yang, K., Zhang, Y. and Li, Z. (2017): Can antioxidant’s reactive oxygen species (ROS) scavenging capacity contribute to aged seed recovery? Contrasting effect of melatonin, ascorbate and glutathione on germination ability of aged maize seeds. Free Radic. Res., 51, 765-771.
• Ding, K., Wang, H., Xu, J., Lu, X., Zhang, L. and Zhu, L. (2014): Melatonin reduced microglial activation and alleviated neuroinflammation induced neuron degeneration in experimental traumatic brain injury: possible involvement of mTOR pathway. Neurochem. Int., 76, 23-31.
• Djavaheri-Mergny, M., Amelotti, M., Mathieu, J., Besançon, F., Bauvy, C., Souquère, S., Pierron, G. and Codogno, P. (2006): NF-kappaB activation represses tumor necrosis factor-alpha-induced autophagy. J. Biol. Chem., 281, 30373-30382.
• Espino, J., Pariente, J.A. and Rodríguez, A.B. (2012): Oxidative stress and immunosenescence: therapeutic effects of melatonin. Oxid. Med. Cell. Longev., 2012, 670294.
• Fu, Y., Tang, M., Fan, Y., Zou, H., Sun, X. and Xu, X. (2012): Anti-apoptotic effects of melatonin in retinal pigment epithelial cells. Front. Biosci., 17, 1461-1468.
• Hageman, G.S., Anderson, D.H., Johnson, L.V., Hancox, L.S., Taiber, A.J., Hardisty, L.I., Hageman, J.L., Stockman, H.A., Borchardt, J.D., Gehrs, K.M., Smith, R.J., Silvestri, G., Russell, S.R., Klaver, C.C., Barbazetto, I., Chang, S., Yannuzzi, L.A., Barile, G.R., Merriam, J.C., Smith, R.T., Olsh, A.K., Bergeron, J., Zernant, J., Merriam, J.E., Gold, B., Dean, M. and Allikmets, R. (2005): A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc. Natl. Acad. Sci. USA, 102, 7227-7232.
• Hanus, J., Anderson, C. and Wang, S. (2015a): RPE necroptosis in response to oxidative stress and in AMD. Ageing Res. Rev., 24 (Pt B), 286-298.
• Hanus, J., Zhang, H., Chen, D.H., Zhou, Q., Jin, P., Liu, Q. and Wang, S. (2015b): Gossypol Acetic Acid Prevents Oxidative Stress-Induced Retinal Pigment Epithelial Necrosis by Regulating the FoxO3/Sestrin2 Pathway. Mol. Cell. Biol., 35, 1952-1963.
• Hardeland, R., Pandi-Perumal, S.R. and Cardinali, D.P. (2006): Melatonin. Int. J. Biochem. Cell Biol., 38, 313-316.
• Jiang, S., Moriarty-Craige, S.E., Orr, M., Cai, J., Sternberg, P. Jr. and Jones, D.P. (2005): Oxidant-induced apoptosis in human retinal pigment epithelial cells: dependence on extracellular redox state. Invest. Ophthalmol. Vis. Sci., 46, 1054-1061.
• Jin, G.F., Hurst, J.S. and Godley, B.F. (2001): Hydrogen peroxide stimulates apoptosis in cultured human retinal pigment epithelial cells. Curr. Eye Res., 22, 165-173.
• King, A., Gottlieb, E., Brooks, D.G., Murphy, M.P. and Dunaief, J.L. (2004): Mitochondria-derived reactive oxygen species mediate blue light-induced death of retinal pigment epithelial cells. Photochem. Photobiol., 79, 470-475.
• Kissová, I., Deffieu, M., Samokhvalov, V., Velours, G., Bessoule, J.J., Manon, S. and Camougrand, N. (2006): Lipid oxidation and autophagy in yeast. Free Radic. Biol. Med., 41, 1655-1661.
• Kleber, A., Kubulus, D., Rössler, D., Wolf, B., Volk, T., Speer, T. and Fink, T. (2014): Melatonin modifies cellular stress in the liver of septic mice by reducing reactive oxygen species and increasing the unfolded protein response. Exp. Mol. Pathol., 97, 565-571.
• Kleszczyński, K., Zillikens, D. and Fischer, T.W. (2016): Melatonin enhances mitochondrial ATP synthesis, reduces reactive oxygen species formation, and mediates translocation of the nuclear erythroid 2-related factor 2 resulting in activation of phase-2 antioxidant enzymes (γ-GCS, HO-1, NQO1) in ultraviolet radiation-treated normal human epidermal keratinocytes (NHEK). J. Pineal Res., 61, 187-197.
• Kong, G.Y., Van Bergen, N.J., Trounce, I.A. and Crowston, J.G. (2009): Mitochondrial dysfunction and glaucoma. J. Glaucoma, 18, 93-100.
• Kowluru, R.A. (2005): Diabetic retinopathy: mitochondrial dysfunction and retinal capillary cell death. Antioxid. Redox Signal., 7, 1581-1587.
• Lee, J., Giordano, S. and Zhang, J. (2012a): Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling. Biochem. J., 441, 523-540.
• Lee, S., Sheck, L., Crowston, J.G., Van Bergen, N.J., O’Neill, E.C., O’Hare, F., Kong, Y.X., Chrysostomou, V., Vincent, A.L. and Trounce, I.A. (2012b): Impaired complex-I-linked respiration and ATP synthesis in primary open-angle glaucoma patient lymphoblasts. Invest. Ophthalmol. Vis. Sci., 53, 2431-2437.
• Liang, J., Wessel, J.H. 3rd, Iuvone, P.M., Tosini, G. and Fukuhara, C. (2004): Diurnal rhythms of tryptophan hydroxylase 1 and 2 mRNA expression in the rat retina. Neuroreport, 15, 1497-1500.
• Lieven, C.J., Vrabec, J.P. and Levin, L.A. (2003): The effects of oxidative stress on mitochondrial transmembrane potential in retinal ganglion cells. Antioxid. Redox Signal., 5, 641-646.
• Liu, L., Lao, W., Ji, Q.S., Yang, Z.H., Yu, G.C. and Zhong, J.X. (2015): Lycium barbarum polysaccharides protected human retinal pigment epithelial cells against oxidative stress-induced apoptosis. Int. J. Ophthalmol., 8, 11-16.
• Luthra, S., Fardin, B., Dong, J., Hertzog, D., Kamjoo, S., Gebremariam, S., Butani, V., Narayanan, R., Mungcal, J.K., Kuppermann, B.D. and Kenney, M.C. (2006): Activation of caspase-8 and caspase-12 pathways by 7-ketocholesterol in human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci., 47, 5569-5575.
• Madsen-Bouterse, S.A. and Kowluru, R.A. (2008): Oxidative stress and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives. Rev. Endocr. Metab. Disord., 9, 315-327.
• Mehta, S. (2015): Age-Related Macular Degeneration. Prim. Care, 42, 377-391.
• Mukherjee, A., Haldar, C. and Vishwas, D.K. (2015): Melatonin prevents dexamethasone-induced testicular oxidative stress and germ cell apoptosis in golden hamster, Mesocricetus auratus. Andrologia, 47, 920-931.
• Osborne, N.N., Nash, M.S. and Wood, J.P. (1998): Melatonin counteracts ischemia-induced apoptosis in human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci., 39, 2374-2383.
• Ozdemir, G., Ergün, Y., Bakariş, S., Kılınç, M., Durdu, H. and Ganiyusufoğlu, E. (2014): Melatonin prevents retinal oxidative stress and vascular changes in diabetic rats. Eye (Lond.), 28, 1020-1027.
• Plafker, S.M., O’Mealey, G.B. and Szweda, L.I. (2012): Mechanisms for countering oxidative stress and damage in retinal pigment epithelium. Int. Rev. Cell Mol. Biol., 298, 135-177.
• Randolph, S.A. (2014): Age-related macular degeneration. Workplace Health Saf., 62, 352.
• Reiter, R.J. (1991): Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr. Rev., 12, 151-180.
• Richter, C. (1998): Oxidative stress, mitochondria, and apoptosis. Restor. Neurol. Neurosci., 12, 59-62.
• Rosen, R., Hu, D.N., Perez, V., Tai, K., Yu, G.P., Chen, M., Tone, P., McCormick, S.A. and Walsh, J. (2009): Urinary 6-sulfatoxymelatonin level in age-related macular degeneration patients. Mol. Vis., 15, 1673-1679.
• Rózanowska, M., Jarvis-Evans, J., Korytowski, W., Boulton, M.E., Burke, J.M. and Sarna, T. (1995): Blue light-induced reactivity of retinal age pigment. In vitro generation of oxygen-reactive species. J. Biol. Chem., 270, 18825-18830.
• Sano, Y., Furuta, A., Setsuie, R., Kikuchi, H., Wang, Y.L., Sakurai, M., Kwon, J., Noda, M. and Wada, K. (2006): Photoreceptor cell apoptosis in the retinal degeneration of Uchl3-deficient mice. Am. J. Pathol., 169, 132-141.
• Singh, M. and Jadhav, H.R. (2014): Melatonin: functions and ligands. Drug Discov. Today, 19, 1410-1418.
• Su, L.Y., Li, H., Lv, L., Feng, Y.M., Li, G.D., Luo, R., Zhou, H.J., Lei, X.G., Ma, L., Li, J.L., Xu, L., Hu, X.T. and Yao, Y.G. (2015): Melatonin attenuates MPTP-induced neurotoxicity via preventing CDK5-mediated autophagy and SNCA/α-synuclein aggregation. Autophagy, 11, 1745-1759.
• Suofu, Y., Li, W., Jean-Alphonse, F.G., Jia, J., Khattar, N.K., Li, J., Baranov, S.V., Leronni, D., Mihalik, A.C., He, Y., Cecon, E., Wehbi, V.L., Kim, J., Heath, B.E., Baranova, O.V., Wang, X., Gable, M.J., Kretz, E.S., Di Benedetto, G., Lezon, T.R., Ferrando, L.M., Larkin, T.M., Sullivan, M., Yablonska, S., Wang, J., Minnigh, M.B., Guillaumet, G., Suzenet, F., Richardson, R.M., Poloyac, S.M., Stolz, D.B., Jockers, R., Witt-Enderby, P.A., Carlisle, D.L., Vilardaga, J.P. and Friedlander, R.M. (2017): Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release. Proc. Natl. Acad. Sci. USA, 114, E7997-E8006.
• Tan, D.X., Hardeland, R., Manchester, L.C., Korkmaz, A., Ma, S., Rosales-Corral, S. and Reiter, R.J. (2012): Functional roles of melatonin in plants, and perspectives in nutritional and agricultural science. J. Exp. Bot., 63, 577-597.
• Tosini, G., Baba, K., Hwang, C.K. and Iuvone, P.M. (2012): Melatonin: an underappreciated player in retinal physiology and pathophysiology. Exp. Eye Res., 103, 82-89.
• Tosini, G., Iuvone, M. and Boatright, J.H. (2013): Is the melatonin receptor type 1 involved in the pathogenesis of glaucoma? J. Glaucoma, 22 (Suppl 5), S49-S50.
• Tsai, F.J., Li, T.M., Ko, C.H., Cheng, C.F., Ho, T.J., Liu, X., Tsang, H., Lin, T.H., Liao, C.C., Li, J.P., Huang, S.M., Lin, J.C., Lin, C.C., Liang, W.M. and Lin, Y.J. (2017): Effects of Chinese herbal medicines on the occurrence of diabetic retinopathy in type 2 diabetes patients and protection of ARPE-19 retina cells by inhibiting oxidative stress. Oncotarget, 8, 63528-63550.
• Uz, T., Arslan, A.D., Kurtuncu, M., Imbesi, M., Akhisaroglu, M., Dwivedi, Y., Pandey, G.N. and Manev, H. (2005): The regional and cellular expression profile of the melatonin receptor MT1 in the central dopaminergic system. Brain Res. Mol. Brain Res., 136, 45-53.
• Wiechmann, A.F. and Sherry, D.M. (2013): Role of melatonin and its receptors in the vertebrate retina. Int. Rev. Cell Mol. Biol., 300, 211-242.
• Xie, X., Feng, J., Kang, Z., Zhang, S., Zhang, L., Zhang, Y., Li, X. and Tang, Y. (2017): Taxifolin protects RPE cells against oxidative stress-induced apoptosis. Mol. Vis., 23, 520-528.
• Yu, L., Sun, Y., Cheng, L., Jin, Z., Yang, Y., Zhai, M., Pei, H., Wang, X., Zhang, H., Meng, Q., Zhang, Y., Yu, S. and Duan, W. (2014): Melatonin receptor-mediated protection against myocardial ischemia/reperfusion injury: role of SIRT1. J. Pineal Res., 57, 228-238.