The Antioxidant Tetrapeptide Epitalon Enhances Delayed Wound Healing in an in Vitro Model of Diabetic Retinopathy

Abstract

Diabetic retinopathy (DR) is the most common complication of diabetes mellitus and a leading cause of vision loss. Short peptides, such as di-, tri-, and tetrapeptides, have various beneficial activities, including antioxidant, antimicrobial, and anti-inflammatory effects. This study aims to test the hypothesis that the antioxidant effect of the synthetic tetrapeptide AEDG (Ala-Glu-Asp-Gly, Epitalon) improves the delayed healing process associated with hyperglycemia in DR, using a high glucose (HG)-injured human retinal pigment epithelial cell line (ARPE-19). We found that HG exposure delayed wound healing in ARPE-19 cells and increased intracellular levels of reactive oxygen species (ROS), while decreasing antioxidant gene expression. HG also induced epithelial-mesenchymal transition (EMT) and upregulated fibrosis-related genes, suggesting that HG-induced EMT contributes to subretinal fibrosis, the end-stage of eye diseases, including proliferative DR. The antioxidant Epitalon restored impaired wound healing in HG-injured ARPE-19 cells by inhibiting hyperglycemia-induced EMT and fibrosis. These findings support using the antioxidant agent Epitalon as a promising therapeutic strategy for DR to improve retinal wound healing compromised by hyperglycemia. More mechanistic investigations are needed to confirm Epitalon’s benefits and safety. Developing ophthalmic forms of Epitalon may enhance its delivery directly to the retina, potentially improving its therapeutic efficacy.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12015-025-10911-x.

Introduction

Retinal pigment epithelial (RPE) cells, which lie between the choroid and neurosensory retina, constitute the outer blood-retinal barrier and play a crucial role in the pathological processes that lead to vision loss. When this barrier is disrupted, RPE cell activation occurs, leading to the proliferation, epithelial-mesenchymal transition (EMT), and secretion of extracellular matrix molecules, thus promoting vitreoretinal disorders, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and proliferative diabetic retinopathy (PDR) [1–3].

Diabetic retinopathy (DR) is a complication of diabetes that can lead to blindness in adults. If left untreated, it can progress from a mild, non-proliferative stage to moderate and severe stages, eventually leading to PDR, in which the formation of fibrotic membranes promotes retinal detachment and vision loss [4, 5]. The available treatments for PDR currently include anti-Vascular Endothelial Growth Factor - (VEGF) agents and invasive laser therapies [6].

In diabetic patients, as occurs in the lower extremities, the eye may suffer from progressive damage to the retinal microvasculature due to hyperglycemia and other factors linked to diabetes, resulting in optic neuropathy and partial or complete blindness [7]. A connection between diabetic corneal ulcers and diabetic foot ulcers (DFUs) has been proposed: they share many aspects of impaired wound healing resulting from neurovascular, sensory, and immunologic compromise [7].

Aberrant redox signaling is widely recognized as a contributor to the development of diabetic complications, including DR and several pathological mechanisms contribute to the accumulation of reactive oxygen species (ROS) in diabetic wounds [8]. Hyperglycemia may directly cause oxidative stress in all retinal cells or contribute to oxygen deficits in the inner retinal layers due to the retina’s high metabolic activity, making it prone to oxidative stress. This is reflected by the activity of protective antioxidant pathways such as mitochondrial uncoupling and the Nuclear factor E2-related factor 2 (NRF2), which are weakened or bypassed in diabetes, allowing oxidative damage to accumulate [9]. Persistent oxidative stress can result in irreversible cell damage and dysfunction, contributing to the clinical manifestations of DR, including neurodegeneration, vascular leakage, and vessel degeneration [9]. Therapeutics targeting oxidative stress hold great promise for the treatment of DR but have not yet been translated into clinical practice.

Different studies have shown the effects of oligopeptides (having up to 10 amino acids) on cell senescence and aging. Short peptides (di-, tri-, and tetrapeptides) can affect the transmission of biological information, modulation of transcription, and restoration of genetically conditioned alterations occurring with age [10]. Additionally, based on the number of amino acid residues in their structure, peptides can support many key processes in the body due to their antioxidant, antimicrobial, antibacterial, anti-inflammatory, antitumor, and immunoregulatory activities [11]. Therefore, elucidating the mechanisms of action of these peptides is of great interest to researchers working in molecular biology, pharmacology, and medicine. Some bioregulator peptides were developed at St. Petersburg Institute of Bioregulation and Gerontology over 40 years ago under the supervision of Professor Vladimir Khavinson [12]. Among them, the synthetic tetrapeptide AEDG (Ala-Glu-Asp-Gly, Epitalon) has been obtained from a natural peptide called epithalamin, produced in the pineal gland [13]. Epitalon was patented about twenty-five years ago [14]. During this time, in vitro, in vivo, and in silico studies on this tetrapeptide have been performed, supporting the geroprotective and neuroendocrine nature of this peptide as result of its antioxidant, neuro-protective, and antimutagenic effects [13]. It has been demonstrated that Epitalon has a direct influence on melatonin synthesis [15], alters the mRNA levels of interleukin-2 [16], modulates the mitogenic activity of murine thymocytes [17], and enhances the activity of various enzymes, including Acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) [18] and telomerase [19]. However, the mechanism of action of this peptide remains to be further elucidated.

Short peptides are signaling molecules that can potentially bind to DNA and histone proteins, serving as regulatory factors [10]. The ability of Epitalon to permeate the cell membrane and enter the nucleus has been demonstrated [20], thus suggesting that its activity may be associated with cytoplasmic and nuclear components. Epitalon can bind certain DNA sequences, particularly with CAG sequences, which are susceptible to DNA cytosine methylation [20]. In human gingival mesenchymal stem cells, Epitalon regulates gene expression and protein synthesis: it preferentially binds to histones H1/6 and H1/3 at sites that interact with DNA, thus suggesting that binding to histones may be an additional mechanism for enhancing the expression of genes that regulate neuronal differentiation and protein synthesis [10]. Altogether, these data suggest a role for epigenetics in Epitalon bioregulatory activities.

Previous studies have shown that Epitalon increases bioelectric activity and enhances the functional activity of the retina by preserving its morphological structure. Besides, the tetrapeptide had a stimulatory effect on the expression of differentiation markers of retinal neurons and pigment epithelium, thus suggesting the retinoprotective effect of Epitalon [21, 22]. In this study, we developed an in vitro model of DR by exposing the human retinal pigment epithelial cell line (ARPE-19) to high glucose (HG) injury to test the hypothesis that the antioxidant effect of the Epitalon tetrapeptide improves the delayed healing process associated with hyperglycemia in DR.

Materials and Methods

Cell Culture

The human retinal pigment epithelium cell line, ARPE-19 (ATCC, Manassas, USA), was cultured in Dulbecco’s modified Eagle’s medium Nutrient Mixture F-12 (1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12; (DMEM/F12, Gibco, Gaithersburg, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Gaithersburg, USA) and 1% penicillin/streptomycin (Invitrogen, Life Technologies, USA) in a humidified incubator with 5% CO₂  at 37 °C. For this study, cells were divided into two experimental settings: the high glucose (HG) setting, mimicking diabetes in vitro, and the standard glucose (SG) setting [23]. Cells in the HG condition were cultured in the medium containing 35 mM of D-glucose (Sigma-Aldrich), and cells in the SG condition were cultured in DMEM/F12 (this represents control, CTR, condition). Cells were also treated with 35 mM of mannitol to exclude the possible effect of osmolarity [24].

Epitalon Preparation

Prof Khavinson (Saint Petersburg, Russia) kindly provided the tested peptide Epitalon, which was dissolved in water at a stock concentration of 100 µg/mL, then further diluted to prepare the three concentrations for testing (20, 40, and 60 ng/mL).

Assessment of Cell Viability

ARPE-19 cell viability was evaluated by performing the 3-(4,5-dimethylthiazol-2-yl)−5-(3-carboxymethoxyphenyl)−2-(4-sulfophenyl)−2 H-tetrazolium (MTS) assay as previously described [25]. Following the manufacturer’s protocol, we carried out the CellTiter 96^® AQueous One Solution Cell Proliferation Assay (Promega Italia s.r.l., Milan, Italy). Briefly, ARPE-19 cells were seeded into 96-well plates (5 × 10³/well), and incubated overnight. After cell adhesion, the medium was replaced, and cells were incubated with SG (CTR) or HG 35 mM for 24, 48, and 72 h. Absorbance at 490 nm was measured using a Synergy H1 BioTek (Agilent) spectrophotometer after a 3-hour dark incubation of cells with the reagent. DMEM/F12 was used as a control for background absorbance.

IncuCyte^® Scratch Wound Cell Migration Assay

The wound healing ability of ARPE-19 was assessed according to the protocol for Incucyte Scratch Wound Assay [26]. Cells were seeded at 40 × 10³/well density onto a 96-well ImageLock tissue culture plate to reach a maximum density of 100% and incubated overnight in standard growth conditions. The following day, scratch wounds were created simultaneously in all wells using the WoundMaker™ according to the manufacturer’s protocol. After removing detached cells by PBS washing, wounded cells were treated with 100 µL of medium containing one of the following experimental conditions: SG (CTR), HG, HG supplemented with Epitalon at different concentrations (20, 40, or 60 ng/mL) or mannitol. The plate was placed in the IncuCyte live-cell analysis system, and migration was monitored using a 10X phase contrast objective. Scans were taken every 3 h, with data collected up to 48 h. Results were reported by correlating relative wound density with wound closure time. Relative wound density was analyzed using the Incucyte^® Scratch Wound Analysis Software Module, representing a measure (%) of the wound region’s density relative to the cell region’s density.

Measurement of ROS Production

ARPE-19 Cells (5,000/well) were seeded in white opaque 96-well plates (Corning, Sigma-Aldrich) and incubated overnight. ROS production was then assessed by using the ROS-Glo™ H₂O₂ Assay kit (Promega, Madison, MI, United States), following the manufacturer’s protocol. After adhesion, cells were treated to establish the following experimental conditions: SG (CTR), HG, and HG supplemented with 20, 40, or 60 ng/mL of Epitalon or mannitol. The treatments were performed for 72 h, and for the final 6 h of treatment, H₂O₂ substrate solution was added to the wells, and 2 h before the end of the treatment, 100 µL of ROS-Glo detection solution was added to each well. After incubation at room temperature for 20 min, the luminescence was measured with a GloMax^® Multi Detection System luminometer (Agilent, Santa Clara, United States). Seventy-two hours of treatment with menadione (20 µM) was carried out as a positive control. DMEM/F12 was used as a control for background luminescence. ROS production was expressed as luminescence intensity, and the percentage of control cells (CTR, SG condition) was reported.

Assessment of Gene Expression by qPCR

Epitalon (20, 40, and 60 ng/ml) was added to ARPE-19 cells (500,000 cells/well) exposed to high concentrations of glucose (HG, 35 mM) for 24–72 h. The assessment of gene expression by qPCR was performed as previously described [27, 28]. Briefly, total RNA from cells was isolated using a Pure link RNA Mini kit (Life Technologies, USA), according to the manufacturer’s protocols. Then, total RNA (2 µg) was reversely transcribed into cDNA using Iscript cDNA Synthesis Kit (Bio-Rad, Milan, Italy), following the manufacturer’s instructions. Then, we used 100 ng of cDNA for the reaction mixture. Gene expression analysis in ARPE-19 cells was conducted by amplifying the following genes: SOD2 (protein name: superoxide dismutase 2, mitochondrial), CAT (protein name: catalase), HMOX1 (protein name: heme oxygenase 1), SNAL1 (protein name: Snail Family Transcriptional Repressor 1), ZEB1 (protein name: Zinc finger E-box-binding homeobox 1), TWIST1 (protein name: Twist Family BHLH Transcription Factor 1), VIM (protein name: Vimentin), FN1 (protein name: Fibronectin 1), ACTA2 (protein name: actin alpha 2, smooth muscle) and GAPDH (protein name: glyceraldehyde-3-phosphate dehydrogenase). The amplification was performed by using TaqMan gene expression assays (Hs00167309, Hs00156308, Hs01110250, Hs00195591, Hs01566408, Hs04989912, Hs00958111, Hs04189111, Hs00426835, and Hs99999905) according to the manufacturer’s instructions. The levels of mRNA of target genes were normalized to GAPDH using a QuantStudio 7 Real-Time PCR system (Thermo Fisher, MA, USA).

Assessment of alpha-SMA Protein Expression by Western Blot

Epitalon (20, 40, and 60 ng/ml) was added to ARPE-19 cells (500,000 cells/well) exposed to high concentrations of glucose (HG, 35 mM) for 48 h. Protein expression of alpha-SMA was assessed by Western blot [29–31]. Briefly, cells were lysed in PBS containing 1% Triton and 1 mM phenylmethylsulfonyl fluoride (PMSF), along with a complete set of EDTA-free protease inhibitors (Thermo Scientific, Rockford, IL, USA). Protein samples were separated by 4–12% SDS-PAGE, transferred onto PVDF membranes (GE Healthcare, Milan, Italy), and incubated with antibodies targeting and Alpha-Smooth Muscle Actin (catalog n° A2547, Sigma-Aldrich). Membranes were developed using ECL Western Blotting Detection Reagents and analyzed with Alliance 1D software (UVITEC, Cambridge, UK). The bands’ optical density (OD) was quantified and normalized to β-actin OD values.

DNA Extraction and Global DNA Methylation Assessment

ARPE-19 Cells (500,000/well) were treated to establish the following experimental conditions: SG, HG, and HG supplemented with Epitalon 20, 40, or 60 ng/mL. The treatments were performed for 24 h. Total DNA was extracted with Genomic DNA Isolation Kit (Norgen Biotek Corp., Thorold, Ontario, Canada), according to the manufacturer’s protocol. The quantification of extracted DNA was performed using the Qubit DNA assay kit (Life Technologies, ThermoFisher Scientific, MA, USA). Finally, the MethylFlash Global DNA Methylation (5mC) ELISA Easy Kit (colorimetric) was used according to the manufacturer’s instructions, to assess global levels of 5-methylcytosine (5mC).

Statistical Analyses

Statistical analyses of cell experiments were conducted by using the GraphPad Prism (GraphPad Software, version 9, San Diego, CA). Differences between the groups were analyzed using the Student’s t-test, and P ≤ 0.05 were considered statistically significant. Data are reported as means ± the standard error of the mean (SEM), representing at least three independent experiments.

Results

Epitalon Treatment Restored the Delayed Wound Healing in HG-Injured Cells

Individuals with diabetes mellitus (DM) have abnormal wound-healing pathways in the eye [7]. Thus, in HG-injured ARPE-19 cells, we assessed the impact of Khavinson Peptides Epitalon (20, 40, and 60 ng/mL) on the wound-healing process, evaluated in a tissue culture plate using real-time imaging (IncuCyte S3 Live-Cell Analysis System) [26]. Cells were plated to confluency and scratched; then, wound closure was monitored at 3-hour intervals for the duration of the experiment (48 h). The wound closure was quantified by measuring the relative density of the wound in the scratched area compared to the confluent area and expressed as relative wound density (%). As reported in Fig. 1(A-D), HG exposure slowed wound closure compared to control cells (CTR, SG condition), and this effect was prevented by Epitalon treatment. No effect was found by mannitol treatment (data not shown). The quantitative analysis of the area under the curve (AUC) (Fig. 1E) confirmed that Epitalon treatment restored the delayed healing of HG-injured cells at all tested concentrations.

Fig. 1.

Effect of Epitalon tetrapeptide on in vitro wound healing of ARPE-19 cells exposed to high glucose (HG) concentration. ARPE-19 cells were seeded at 40 × 10³/well density onto a 96-well ImageLock tissue culture plate to reach a maximum density of 100% and incubated overnight in standard growth conditions. The following day, scratch wounds were created simultaneously in all wells. After removing detached cells by washing with PBS, wounded cells were treated with 100 µL of medium containing one of the following experimental conditions: SG (CTR), HG, or HG supplemented with Epitalon at different concentrations: 20 ng/mL (A), 40 ng/mL (B), or 60 ng/mL (C). The plate was placed in the IncuCyte live-cell analysis system, and migration was monitored using a 10X phase contrast objective. Scans were taken every 3 h, with data collected up to 48 h. Results were reported by correlating relative wound density with wound closure time. Relative wound density was analyzed using the Incucyte^® Scratch Wound Analysis Software Module, representing a measure (%) of the wound region’s density relative to the cell region’s density. The test was performed using at least three independent experiments for each condition. Images show differences in wound closure at 0 h, 12 h and 24 h of incubation. Scale bar is 400 μm (D). Area under the curve (AUC) values for each experimental condition (E). Data are reported as mean ± SEM (n = 3–4). §p < 0.05 vs. CTR, *p < 0.05 and **p < 0.01 vs. HG 35 mM, respectively

Epitalon Treatment Reduced HG-induced Oxidative Stress

Redox signaling imbalance has significant implications for the pathogenesis of delayed healing in diabetes. In diabetic patients, elevated ROS levels result from their increased production and weakened antioxidant defenses, particularly in wounds. Hyperglycemia triggers various pathological mechanisms that lead to excessive ROS, including H₂O₂, in diabetic wounds [8]. In our in vitro model of DR, exposure of ARPE-19 cells to HG for 72 h significantly increased H₂O₂ generation compared to control (SG condition) (p < 0.01), similar to the pro-oxidant stimulus menadione at 20 µM. Epitalon 40 and 60 ng/mL diminished HG-induced H₂O₂ generation in ARPE-19 cells (p < 0.05 and p < 0.01 respectively) (Fig. 2A). No effect was found by mannitol treatment (data not shown). Moreover, in HG-stimulated cells, downregulation of the antioxidant genes superoxide dismutase 2 (SOD2) (Fig. 2B), catalase (CAT) (Fig. 2C), and heme oxygenase 1 (HMOX1) (Fig. 2D) was observed compared to control (SG condition), which was restrained by Epitalon at 20, 40 and 60 ng/mL.

Fig. 2.

Concentration-dependent effect of Epitalon tetrapeptide on the intracellular generation of reactive oxygen species (ROS) and on the expression of antioxidant genes in ARPE-19 cells exposed to high glucose (HG) concentration. Epitalon tetrapeptide at different concentrations (20, 40, and 60 ng/ml) or the oxidative stress inducer Menadione (20µM) as positive control were added to ARPE-19 cells (5,000 cells/well) exposed to high concentrations of glucose (HG, 35 mM) for 72 h. ROS-Glo™ H₂O₂ assay was performed to assess ROS generation. ROS production was expressed as luminescence intensity and the percentage of control cells (CTR, SG condition) was reported (A). Epitalon tetrapeptide at 20, 40, or 60 ng/ml was added to ARPE-19 cells (500,000 cells/well) exposed to high concentrations of glucose (HG, 35 mM) for 72 h. mRNA expression of SOD2 (protein name: superoxide dismutase 2) (B), CAT (protein name: catalase) (C), and HMOX1 (protein name: Heme Oxygenase 1) (D) were analyzed using qPCR. Relative fold change in gene expression was calculated using cells under SG condition (CTR, fold change = 1) and normalized to GAPDH as a housekeeping gene. Data are reported as mean ± SEM of at least three independent experiments. §p < 0.05 vs. CTR, *p < 0.05 and **p < 0.01 vs. HG 35 mM, respectively (A). #p < 0.01 vs. CTR, *p < 0.05 and **p < 0.01 vs. HG 35 mM, respectively (B); §p < 0.05 vs. CTR, *p < 0.05 and **p < 0.01 vs. HG 35 mM, respectively (C); §p < 0.05 vs. CTR, *p < 0.05 and **p < 0.01 vs. HG 35 mM, respectively (D)

Epitalon Treatment Restrains HG-induced Expression of EMT and fibrosis-related Genes

EMT of RPE cells is a crucial process in the development of fibrosis associated with vitreoretinal diseases, including PDR [32]. The most characterized transcription factors involved in the regulation of EMT include snail family transcriptional repressor (SNAIL)−1, zinc finger E-box binding homeobox 1 (ZEB1), and twist family bHLH transcription factor 1 (TWIST1) [33, 34].

In ARPE-19 cells, we found that, compared to SG, HG significantly increased the mRNA expression of these genes 24 h after exposure (Fig. 3A-C). The Epitalon tetrapeptide restrained the HG-induced gene expression of SNAIL-1 in a concentration-dependent manner (Fig. 3A), ZEB-1 (Fig. 3B), and TWIST1 (Fig. 3C) at all tested concentrations. Furthermore, the mRNA expression of fibrosis-related genes vimentin (VIM) (Fig. 4A), fibronectin (FN1) (Fig. 4B), and actin alpha 2 smooth muscle (ACTA2) (Fig. 4C) was significantly upregulated by HG exposure and inhibited by Epitalon tetrapeptide treatment. Protein expression analysis by Western blot confirmed that alpha-smooth muscle (a-SMA) was significantly induced 48 h after HG exposure. Epitalon treatment restrained this effect in a concentration-dependent manner (Fig. 4D-E and Supplementary Fig. 3).

Fig. 3.

Concentration-dependent effect of Epitalon tetrapeptide on the expression of EMT genes in ARPE-19 cells exposed to high glucose (HG) concentration. Epitalon tetrapeptide at different concentrations (20, 40, or 60 ng/mL) were added to ARPE-19 cells (500,000 cells/well) exposed to HG, 35 mM for 24 h. mRNA expression of SNAl1 (protein name: Snail Family Transcriptional Repressor 1) (A), ZEB1 (protein name: Zinc finger E-box-binding homeobox 1) (B), TWIST1 (protein name: Twist Family BHLH Transcription Factor 1) (C) was analyzed using qPCR. Relative fold change in gene expression was calculated using cells under SG condition (CTR, fold change = 1) and normalized to GAPDH as a housekeeping gene. Data are reported as mean ± SEM of at least three independent experiments. §p < 0.05 vs. CTR, and **p < 0.01 vs. HG 35 mM, respectively (A); §p < 0.05 vs. CTR, *p < 0.05 and **p < 0.01 vs. HG 35 mM, respectively (B); §p < 0.05 vs. CTR and **p < 0.01 vs. HG 35 mM, respectively (C)

Fig. 4.

Concentration-dependent effect of Epitalon tetrapeptide on the expression of fibrosis-related genes and protein expression of Alpha-Smooth Muscle Actin (alpha-SMA) in ARPE-19 cells exposed to high glucose (HG) concentration. Epitalon tetrapeptide at different concentrations (20, 40, and 60 ng/mL) were added to ARPE-19 cells (500,000 cells/well) exposed to HG, 35 mM for 24 h (A-C). mRNA expression of VIM (protein name: Vimentin) (A), FN1 (protein name: fibronectin 1) (B), and ACTA2 (protein name: actin alpha 2, smooth muscle) (C) was analyzed using qPCR. Relative fold change in gene expression was calculated using cells under SG condition (CTR, fold change = 1) and normalized to GAPDH as a housekeeping gene (A-C). Epitalon tetrapeptide at 20, 40, or 60 ng/mL was added to ARPE-19 cells (500,000 cells/well) exposed to HG, 35 mM for 48 h (D-E). Protein expression of alpha-SMA was assessed by Western blot (D). Densitometric analysis of the expression levels of alpha-SMA (OD value/β-actin OD) (E). Data are reported as mean ± SEM (n = 3–6). §p < 0.05 vs. CTR, *p < 0.05 and **p < 0.01 vs. HG 35 mM vs. HG 35 mM, respectively (A-E)

Discussion

DR is a leading global cause of blindness [35], primarily due to its complications, such as PDR and diabetic macular edema (DME) [36]. These conditions arise from microvascular damage and vascular hyperpermeability, driven by inflammatory and pro-angiogenic factors in the eye [37]. Proven treatments, including retinal photocoagulation (PRP), VEGF inhibitors, and corticosteroids, have demonstrated efficacy in both clinical trials and real-world applications [37]. However, while numerous studies indicate that pharmacologic treatment may serve as a first-line option for PDR, it is currently limited to delaying the necessity for PRP [37]. PRP remains the standard treatment for PDR, but it may lead to permanent peripheral visual field loss, reduced night vision, or worsen existing DME, increasing its occurrence [37]. Consequently, current therapies are reactive rather than preventive, highlighting the critical need for disease-modifying treatments that target underlying pathophysiology. Additionally, non-invasive options to alleviate patient burden and enhance compliance, such as eye drops or systemic medications, are essential. In this study, we aimed to assess the protective effects of the tetrapeptide Epitalon in an in vitro model of DR using an HG-injured ARPE-19 cell line.

Epitalon is a short peptide acting as a bioregulator of proliferation and differentiation processes in various types of cells and tissues and showing geroprotective effects in animal models [10]. Results of experimental and clinical investigations demonstrated the effects of the synthetic peptide on the course of retinitis pigmentosa, a degenerative process of the retina resulting from disturbed metabolism of specific proteins in the pigmented epithelium and other retinal layers [38]. In this context, the use of Epitalon in patients with dystrophic retinal lesions showed positive clinical effects (i.e. visual acuity increase, extension of visual field borders, and normalization of electrophysiological indices) in more than 90% of cases. None of the Epitalon-treated patients showed any exacerbation of their clinical condition [38].

In diabetic patients, the progressive retinal damage associated with hyperglycemia and other diabetes-related factors is linked to the abnormal wound healing pathways in the eye [7]. Using the real-time wound healing/scratch assay, we found that HG exposure (for 48 h) caused a delayed wound healing process in ARPE-19 cells. Oxidative stress plays a significant role in regulating normal wound healing by aiding hemostasis, inflammation, wound closure, and the development and maturation of the extracellular matrix (ECM) [8]. Several lines of evidence support the pivotal role of the oxidized cellular state in retinal cell damage [39, 40]. In diabetes, hyperglycemia is associated with dysfunction of RPE, which is crucial for retinal health [41]; therefore, a significant link between DR and oxidative stress is described [41]. Additionally, because of its high oxygen consumption, rich supply of polyunsaturated fatty acids, and exposure to visible light, the retina is particularly susceptible to oxidative stress caused by hyperglycemia [42].

Increased ROS levels in human and murine primary RPE [43] and in ARPE-19 cells [44] cultured under HG conditions have been reported. Several mechanisms contributing to increased oxidative stress in the diabetic milieu have been proposed, including glucose auto-oxidation, weakened antioxidant defense systems, metabolic abnormalities, and mitochondrial damage [45–47]. Thus, we hypothesized that in our in vitro model of DR, normal redox signaling was disrupted by hyperglycemic conditions. In ARPE-19 cells the addition of glucose increased intracellular ROS levels and lowers the expression of the antioxidant genes SOD2, CAT, and HMOX1.

Cell culture studies investigating diabetes and its complications typically use glucose 5.5 mM concentration, representing healthy humans’ physiological blood glucose level [23]. However, for ARPE-19 cells, ATCC recommends a mixture of Dulbecco’s Modified Eagle Medium and Ham’s F12 Medium with 17.5 mM glucose. Accordingly, several in vitro studies aimed at assessing the efficacy of drugs on glucose-related cell damage in ARPE-19 reported that using euglycemic media (5.5 mM) as a control condition may affect normal cell growth [48]. Thus, in our in vitro model of DR, ARPE-19 cells were cultured in DMEM/F12 nutrient mixture having the recommended standard glucose concentration of 17.5 mM, as control condition. Then, to mimic the diabetic condition in vitro, we exogenously added glucose to the control medium, to obtain a final concentration of 35 mM.

As shown in Supplementary Fig. 1, ARPE-19 cell viability was not significantly affected after exposure to 35 mM glucose for 24, 48, and 72 h. These results are consistent with previous studies, which reported no significant changes in cell growth or morphology in ARPE-19 cells exposed to a final concentration of 30 mM glucose [23]. Furthermore, this HG concentration was associated with a sustained increase in ROS generation (up to 96 h), suggesting it effectively induces oxidative stress, partly due to the simultaneous disruption of the antioxidant defense system [23].

Previous studies reported the antioxidant properties of Epitalon by increasing SOD activity and reducing the levels of lipid peroxidation (LPO) products, diene conjugates, and ROS [49–51].

In HG-injured ARPE-19, we found that Epitalon 40 and 60 ng/mL reduced redox signaling imbalance by constraining H₂O₂ generation (Fig. 2A). Furthermore, the decrease expression of the antioxidant genes SOD2 (Fig. 2B), CAT (Fig. 2C), and HMOX1 (Fig. 2D) induced by HG exposure was also attenuated. The antioxidant activity of Epitalon was linked to the recovery of delayed healing in HG-injured cells across all tested concentrations (Fig. 1).

EMT is a multi-step process in which epithelial cells lose their characteristic traits and gain mesenchymal features, including the loss of cell junctions and apicobasal polarity [33]. It occurs in three biological contexts with different functional consequences: type 1 EMT is involved in embryonic development and growth [52], type 2 EMT is involved in wound healing, tissue regeneration, and fibrosis [52], and Type 3 EMT, also known as oncogenic EMT, contributes to cancer metastasis [52–55]. EMT is observed in the process of renal interstitial, pulmonary and liver fibrosis [56–59], as well as in specific ocular tissue [32]. In RPE cells, it plays a key role in proliferative retinal diseases, such as age-related macular degeneration, by contributing to subretinal fibrosis [32]. In diabetes, HG levels can trigger the expression of mesenchymal markers in RPE cells [60].

In our model of DR, we found that HG exposure induces the expression of key EMT genes, specifically SNAIL-1 (Fig. 3A), ZEB-1 (Fig. 3B), and TWIST1 (Fig. 3C). This effect was associated with the upregulation of fibrosis-related genes VIM (Fig. 4A), FN1 (Fig. 4B) and ACTA2 (Fig. 4C), thereby supporting the hypothesis that HG-induced EMT contributes to subretinal fibrosis, which represents the end-stage of various eye diseases, including PDR [5].

Clinical studies have demonstrated that fibrotic membranes in PDR exhibit uncontrolled growth, contributing to retinal detachment from RPE cells [5]. However, the underlying mechanisms remain unclear. Daley and colleagues revealed that hyperglycemia triggers Akt2 activation (phosphorylation) in RPE cells, which in turn increases the expression of EMT markers and fibrosis-related proteins via ERK signaling [6]. Furthermore, it has been proposed that hyperglycemia elevates superoxide production, leading to the accelerated formation of advanced glycation end-products (AGEs). AGEs play a significant role in the pathogenesis of ocular diseases, with studies showing that AGE mimetic administration induces RPE dysfunction [61]. The disruption of the outer blood-retinal barrier observed in DR patients and diabetic mouse models [62] can activate RPE cells, triggering the EMT process. In our research, we found that the antioxidant Epitalon restored impaired wound healing in HG-injured ARPE-19 cells by inhibiting hyperglycemia-induced EMT and fibrosis (Figs. 3 and 4). Whether this protective effect involves interference with HG-induced Akt2 activation or AGE formation requires further investigation.

Altered DNA methylation patterns, specifically levels of methylation (5 mC) and hydroxymethylation (5 hmC), are commonly associated with type 2 diabetes (T2D) and general oxidative stress [63]. In DR, oxidative stress also influences DNA methylation [64], Aberrant methylation of key genes involved in the inflammatory response in the retina has been observed. Among them, NOD-like receptor (NLRP3) plays a role in inflammatory body formation and activation, as well as in the regulation of transforming growth factor beta-1 (TGFβ1), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor ligand superfamily member 2 (TNFSF2), all of which contribute to vascular complications in diabetes [65]. Methylation levels of NLRP3, TGFβ1, MCP-1, and TNFSF2 are significantly lower in DR patients compared to healthy individuals, suggesting that hypomethylation of these genes contributes to DR progression [66].

In our DR model, Epitalon treatment prevented HG-induced hypomethylation in ARPE-19 cells, as assessed by global 5 mC levels (Supplementary Fig. 2). This supports the hypothesis that Epitalon’s protective effects in DR may involve modulating epigenetic changes caused by hyperglycemia. Further investigation is needed to explore how Epitalon’s impact on global methylation patterns could influence gene expression related to oxidative stress, EMT, and fibrosis in DR.

The findings of this study are derived from in vitro experiments and, while valuable, have inherent limitations in their translatability to human outcomes. First, although HG exposure is widely recognized in the literature as a common in vitro model to mimic diabetes-induced cellular damage, it does not fully replicate the complexity of the DR. Diabetes is characterized by chronic systemic hyperglycemia, which activates several secondary metabolic pathways—such as the polyol pathway, accumulation of AGE products, and protein kinase C (PKC) activation—that contribute to retinal and systemic vascular damage [67]. Exposing retinal cells to elevated glucose levels in vitro does not account for the diverse circulating mediators and systemic factors involved in the diabetic condition. Second, although ARPE-19 cells are a common model for studying retinal function [68, 69], their standard culture medium already contains elevated glucose levels higher than physiological norms. Thus, to mimic hyperglycemia, additional glucose is added to further increase glucose concentrations [23].

In conclusion, our data suggest that the treatment with the antioxidant peptide Epitalon enhanced impaired wound healing processes in HG-injured ARPE-19 cells by inhibiting hyperglycemia-induced EMT and fibrosis, thus supporting the therapeutic benefits of this interesting peptide for DR management. However, the suitability of current in vitro models, including those based on ARPE-19 cells, for accurately recapitulating RPE physiology in the context of diabetes warrants further optimization.

In the future, the development of ophthalmic delivery systems for Epitalon could facilitate its targeted administration to the retina, potentially enhancing its therapeutic efficacy. However, further efforts are needed to standardize in vitro protocols for evaluating Epitalon’s biological activity and to clarify its precise molecular mechanisms, which remain only partially understood. In this context, the application of advanced in vitro models, such as three-dimensional (3D) cell cultures and organ-on-chip systems, deserves particular attention. These models offer a more physiologically relevant environment by better replicating the structural and functional complexity of human tissues under diabetic conditions. By enabling more predictive and mechanistic investigations, they hold significant promise for advancing the preclinical development of Epitalon as a multifunctional therapeutic candidate for the management of diabetes and its systemic complications.

Electronic Supplementary Material

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Author Contributions

M.G. and M.D: acquisition, analysis, interpretation of data, writing—original draft; C. M. and A.G.R.: acquisition, analysis, interpretation of data; M.S., I.A., A.T: interpretation of data, revising manuscript critically for important intellectual content; V. K.: conceptualization, design; S. V.: conceptualization, design, revising manuscript critically for important intellectual content; A. B.: conceptualization, design, interpretation, writing—original draft, revising manuscript critically for important intellectual content; P.B.: conceptualization, design, interpretation, funding, writing—original draft, revising manuscript critically for important intellectual content. All authors were involved in reviewing and approving the final version of the manuscript.

Funding

This research was funded by the European Union—Next Generation EU—under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 -M4 C2, Investment 1.5-Call for Tender No. 3277 of 30 December 2021, Italian Ministry of University, Award No. ECS00000041.

Project Title: “Innovation, digitalization, and sustainability for the diffused economy in Central Italy”, Concession Degree No. 1057 of 23 June 2022, adopted by the Italian Ministry of University. CUP: D73 C22000840006; Ministero dell’Università e della Ricerca (MUR) (University Scientific Research Funds).

Data Availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics Approval

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Consent To Participate

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Consent for Publication

Not Applicable.

Competing Interests

The authors declare no competing interests.

Clinical Trial Number

Not applicable.