Xinwang Yang

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Peptide agents are regarded as hopeful candidates to solve life-threatening resistance of pathogenic microorganisms to classic antibiotics due to their unique action mechanisms. Peptidomic and genomic investigation of natural antimicrobial peptides (AMPs) from amphibian skin secretions can provide a large amount of structure-functional information to design peptide antibiotics with therapeutic potential. In the present study, we identified a large number of AMPs from the skins of nine kinds of Chinese odorous frogs. Eighty AMPs were purified from three different odorous frogs and confirmed by peptidomic analysis. Our results indicated that post-translational modification of AMPs rarely happened in odorous frogs. cDNAs encoding precursors of 728 AMPs, including all the precursors of the confirmed 80 native peptides, were cloned from the constructed AMP cDNA libraries of nine Chinese odorous frogs. On the basis of the sequence similarity of deduced mature peptides, these 728 AMPs were grouped into 97 different families in which 71 novel families were identified. Out of these 728 AMPs, 662 AMPs were novel and 28 AMPs were reported previously in other frog species. Our results revealed that identical AMPs were widely distributed in odorous frogs; 49 presently identified AMPs could find their identical molecules in different amphibian species. Purified peptides showed strong antimicrobial activities against 4 tested microbe strains. Twenty-three deduced peptides were synthesized and their bioactivities, including antimicrobial, antioxidant, hemolytic, immunomodulatory and insulin-releasing activities, were evaluated. Our findings demonstrate the extreme diversity of AMPs in amphibian skins and provide plenty of templates to develop novel peptide antibiotics.

Current treatment for chronic infectious wounds is limited due to severe drug resistance in certain bacteria. Therefore, the development of new composite hydrogels with nonantibiotic antibacterial and pro-wound repair is important. Here, we present a photothermal antibacterial composite hydrogel fabricated with a coating of Fe2+ cross-linked carboxymethyl chitosan (FeCMCS) following the incorporation of melanin nanoparticles (MNPs) and the CyRL-QN15 peptide. Various physical and photothermal properties of the hydrogel were characterized. Cell proliferation, migration, cycle, and free-radical scavenging activity were assessed, and the antimicrobial properties of the hydrogel were probed by photothermal therapy. The effects of the hydrogel were validated in a model of methicillin-resistant Staphylococcus aureus (MRSA) infection with full-thickness injury. This effect was further confirmed by changes in cytokines associated with inflammation, re-epithelialization, and angiogenesis on the seventh day after wound formation. The MNPs demonstrated robust photothermal conversion capabilities. The composite hydrogel (MNPs/CyRL-QN15/FeCMCS) promoted keratinocyte and fibroblast proliferation and migration while exhibiting high antibacterial efficacy, effectively killing more than 95% of Gram-positive and Gram-negative bacteria. In vivo study using an MRSA-infected full-thickness injury model demonstrated good therapeutic efficacy of the hydrogel in promoting regeneration and remodeling of chronically infected wounds by alleviating inflammatory response and accelerating re-epithelialization and collagen deposition. The MNPs/CyRL-QN15/FeCMCS hydrogel showed excellent antibacterial and prohealing effects on infected wounds, indicating potential as a promising candidate for wound healing promotion.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Cathelicidins play pivotal roles in host defense. The discovery of novel cathelicidins is important research; however, despite the identification of many cathelicidins in vertebrates, few have been reported in amphibians. Here we identified a novel cathelicidin (named cathelicidin-OA1) from the skin of an amphibian species, Odorrana andersonii. Produced by posttranslational processing of a 198-residue prepropeptide, cathelicidin-OA1 presented an amino acid sequence of 'IGRDPTWSHLAASCLKCIFDDLPKTHN' and a molecular mass of 3038.5 Da. Functional analysis showed that, unlike other cathelicidins, cathelicidin-OA1 demonstrated no direct microbe-killing, acute toxicity and hemolytic activity, but did exhibit antioxidant activity. Importantly, cathelicidin-OA1 accelerated wound healing against human keratinocytes (HaCaT) and skin fibroblasts (HSF) in both time- and dose-dependent manners. Notably, cathelicidin-OA1 also showed wound-healing promotion in a mouse model with full-thickness skin wounds, accelerating re-epithelialization and granulation tissue formation by enhancing the recruitment of macrophages to the wound site, inducing HaCaT cell proliferation and HSF cell migration. This is the first cathelicidin identified from an amphibian that shows potent wound-healing activity. These results will help in the development of new types of wound-healing agents and in our understanding of the biological functions of cathelicidins.