Chronic nonhealing wounds remain a considerable challenge in clinical treatment due to excessive inflammation and impeded reepithelialization and angiogenesis. Therefore, the discovery of novel prohealing agents for chronic skin wounds are urgent and important. Amphibian-derived prohealing peptides, especially immunomodulatory peptides, provide a promising strategy for the treatment of chronic skin trauma. However, the mechanism of immunomodulatory peptides accelerating the skin wound healing remains poorly understood. The prohealing ability of peptide Andersonin-W1 (AW1) was assessed by cell scratch, cell proliferation, transwell, and tube formation. Next, full-thickness, deep second-degree burns and diabetic full-thickness skin wounds in mice were performed to detect the therapeutic effects of AW1. Moreover, the tissue regeneration and expression of inflammatory cytokines were evaluated by hematoxylin and eosin (H&E), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry staining. Molecular docking, colocalization, and western blotting were used to explore the mechanism of AW1 in promoting wound healing. We provide solid evidence to display excellent prohealing effects of AW1, identified as a short antimicrobial peptide in our previous report. At relative low concentration of nM, AW1 promoted the proliferation, migration, and scratch repair of keratinocyte, macrophage proliferation, and tube formation of HUVEC. AW1 also facilitated reepithelialization, granulation regeneration, and angiogenesis, thus significantly boosting the healing of full-thickness, deep second-degree burns and diabetic skin wounds in mice. Mechanistically, in macrophages, AW1 directly bound to Toll-like receptor 4 (TLR4) in the extracellular region and regulated the downstream nuclear factor‐κB (NF-κB) signaling pathway to facilitate the inflammatory factor secretion and suppress excessive inflammation induced by lipopolysaccharide (LPS). Moreover, AW1 regulated macrophage polarization to promote the transition from the inflammatory to the proliferative phase and then facilitated reepithelialization, granulation regeneration, and angiogenesis, thus exhibiting excellent therapeutic effects on diabetic skin wounds. AW1 modulates inflammation and the wound healing process by the TLR4/NF-κB molecular axis, thus facilitating reepithelialization, granulation regeneration, and angiogenesis. These findings not only provided a promising multifunctional prohealing drug candidate for chronic nonhealing skin wounds but also highlighted the unique roles of “small” peptides in the elucidation of “big” human disease mechanisms.

中文翻译：

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生物活性肽Andersonin-W1与TLR4的直接结合可加速糖尿病皮肤伤口的愈合

由于过度炎症和阻碍上皮化和血管生成，慢性不愈合伤口在临床治疗中仍然是一个相当大的挑战。因此，发现新型慢性皮肤伤口促愈合剂迫在眉睫且重要。两栖动物衍生的促愈合肽，特别是免疫调节肽，为治疗慢性皮肤创伤提供了一种有前景的策略。然而，免疫调节肽加速皮肤伤口愈合的机制仍然知之甚少。通过细胞划痕、细胞增殖、Transwell 和管形成来评估肽 Andersonin-W1 (AW1) 的促愈合能力。接下来，对小鼠进行全层深二度烧伤和糖尿病全层皮肤创面检测AW1的治疗效果。此外，通过苏木精和伊红（H＆E）、酶联免疫吸附测定（ELISA）和免疫组织化学染色评估组织再生和炎性细胞因子的表达。采用分子对接、共定位和蛋白质印迹等方法探讨AW1促进伤口愈合的机制。我们提供了确凿的证据来证明 AW1 具有出色的促愈合作用，AW1 在我们之前的报告中被确定为一种短抗菌肽。在相对较低浓度的nM下，AW1促进角质形成细胞的增殖、迁移和划痕修复，巨噬细胞增殖和HUVEC的管形成。 AW1还促进上皮再形成、肉芽再生和血管生成，从而显着促进小鼠全层深二度烧伤和糖尿病皮肤伤口的愈合。从机制上讲，在巨噬细胞中，AW1直接与胞外区的Toll样受体4（TLR4）结合，并调节下游核因子-κB（NF-κB）信号通路，促进炎症因子分泌，抑制脂多糖诱导的过度炎症。脂多糖）。此外，AW1调节巨噬细胞极化，促进炎症期向增殖期转变，进而促进上皮化、肉芽再生和血管生成，从而对糖尿病皮肤创面表现出优异的治疗效果。 AW1 通过 TLR4/NF-κB 分子轴调节炎症和伤口愈合过程，从而促进上皮再形成、肉芽再生和血管生成。这些发现不仅为慢性不愈合皮肤伤口提供了一种有前景的多功能促愈合候选药物，而且强调了“小”肽在阐明“大”人类疾病机制中的独特作用。