Amphibian-derived peptide homodimer OA-GL17d promotes skin wound regeneration through the miR-663a/TGF-β1/Smad axis

Background:Amphibian-derived peptides exhibit considerable potential in the discovery and development of new therapeutic interventions for clinically challenging chronic skin wounds.MicroRNAs(miRNAs)are also considered promising targets for the development of effective therapies against skin wounds.However,further research in this field is anticipated.This study aims to identify and provide a new peptide drug candidate,as well as to explore the underlying miRNA mechanisms and possible miRNA drug target for skin wound healing.Methods:A combination of Edman degradation,mass spectrometry and cDNA cloning were adopted to determine the amino acid sequence of a peptide thatwas fractionated from the secretion of Odorrana andersonii frog skin using gel-filtration and reversed-phase high-performance liquid chromatography.The toxicity of the peptide was evaluated by Calcein-AM/propidium iodide(PI)double staining against human keratinocytes(HaCaT cells),hemolytic activity against mice blood cells and acute toxicity against mice.The stability of the peptide in plasma was also evaluated.The prohealing potency of the peptide was determined by MTS,scratch healing and a Transwell experiment against HaCaT cells,full-thickness injury wounds and scald wounds in the dorsal skin of mice.miRNA transcriptome sequencing analysis,enzyme-linked immunosorbent assay,real-time polymerase chain reaction and western blotting were performed to explore the molecular mechanisms.Results:A novel peptide homodimer(named OA-GL17d)that contains a disulfide bond between the 16th cysteine residue of the peptide monomer and the sequence‘GLFKWHPRCGEEQSMWT’was identified.Analysis showed that OA-GL17d exhibited no hemolytic activity or acute toxicity,but effectively promoted keratinocyte proliferation and migration and strongly stimulated the repair of full-thickness injury wounds and scald wounds in the dorsal skin of mice.Mechanistically,OA-GL17d decreased the level of miR-663a to increase the level of transforming growth factor-β1(TGF-β1)and activate the subsequent TGF-β1/Smad signaling pathway,thereby resulting in accelerated skin wound re-epithelialization and granular tissue formation.Conclusions:Our results suggest that OA-GL17d is a new peptide drug candidate for skin wound repair.This study emphasizes the importance of exogenous peptides as molecular probes for exploring competing endogenous RNA mechanisms and indicates that miR-663a may be an effective target for promoting skin repair.