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 that was 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.

中文翻译：

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两栖类肽同源二聚体 OA-GL17d 通过 miR-663a/TGF-β1/Smad 轴促进皮肤伤口再生

背景 两栖动物衍生的肽在发现和开发针对临床具有挑战性的慢性皮肤伤口的新治疗干预措施方面表现出相当大的潜力。MicroRNAs (miRNAs) 也被认为是开发针对皮肤伤口的有效疗法的有希望的靶标。然而，该领域的进一步研究值得期待。本研究旨在鉴定和提供一种新的多肽候选药物，以及探索潜在的 miRNA 机制和可能的 miRNA 药物靶点用于皮肤伤口愈合。方法 采用Edman降解法、质谱法和cDNA克隆法相结合的方法，采用凝胶过滤和反相高效液相色谱法对从安氏蛙皮分泌物中分离得到的肽段进行氨基酸序列测定。该肽的毒性通过 Calcein-AM/碘化丙啶 (PI) 双染色对人角质形成细胞 (HaCaT 细胞)、对小鼠血细胞的溶血活性和对小鼠的急性毒性进行评估。还评估了肽在血浆中的稳定性。该肽的促愈合效力通过 MTS、划痕愈合和针对 HaCaT 细胞的 Transwell 实验、小鼠背侧皮肤的全层损伤伤口和烫伤伤口来确定。进行miRNA转录组测序分析、酶联免疫吸附测定、实时聚合酶链反应和蛋白质印迹以探索分子机制。结果鉴定了一种新的肽同源二聚体（命名为OA-GL17d），它在肽单体的第16个半胱氨酸残基和序列“GLFKWWHPRCGEEQSMWT”之间含有二硫键。分析表明，OA-GL17d无溶血活性或急性毒性，但有效促进角质形成细胞增殖和迁移，强烈刺激小鼠背侧皮肤全层损伤创面和烫伤创面的修复。机制上，OA-GL17d 降低 miR-663a 的水平以增加转化生长因子-β1 (TGF-β1) 的水平并激活随后的 TGF-β1/Smad 信号通路，从而导致加速皮肤伤口再上皮化和颗粒组织形成。结论 我们的研究结果表明 OA-GL17d 是一种用于皮肤伤口修复的新肽候选药物。本研究强调了外源性肽作为分子探针探索竞争性内源性 RNA 机制的重要性，并表明 miR-663a 可能是促进皮肤修复的有效靶标。