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

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.

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 p

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).

The wound healing ability of ARPE-19 was assessed according to the protocol for Incucyte Scratch Wound Assay [ 26 ]. Cells were seeded at 40 × 10 3 /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.

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).

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).

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. 1 E) 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 × 10 3 /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

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. 3 A-C). The Epitalon tetrapeptide restrained the HG-induced gene expression of SNAIL-1 in a concentration-dependent manner (Fig. 3 A), ZEB-1 (Fig. 3 B), and TWIST1 (Fig. 3 C) at all tested concentrations. Furthermore, the mRNA expression of fibrosis-related genes vimentin (VIM) (Fig. 4 A), fibronectin (FN1) (Fig. 4 B), and actin alpha 2 smooth muscle (ACTA2) (Fig. 4 C) 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. 4 D-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)

Below is the link to the electronic supplementary material. Supplementary Material 1