Reverse-phase high-performance liquid chromatography (RP-HPLC) is a useful tool for the separation and analysis of purified peptides. In brief, lyophilized samples are applied to a gel filtration column pre-equilibrated with 25 mm Tris HCl buffer (pH 7.8) containing 0.1 M NaCl, then eluted with the same buffer at a flow rate of 0.1 ml/min. The fractions are then collected by an automatic fraction collector and measured at 280 nm by using a microplate reader. The determined samples are desalted on a C₄ column by RP-HPLC, then eluted using a C₁₈ column. Finally, active peaks are collected (Li et al., 2018).

Mass spectrometry can be used to analyze the primary structure of peptide molecules, conduct amino acid sequencing of purified peptide molecules, and perform Edman degradation and amino acid residue recognition, thus helping to obtain a complete amino acid sequence (Mu et al., 2014).

The construction of a cDNA library involves in vitro recombination of cDNA and clone vector DNA, transformation of host cells of clone vector DNA, and procurement of bacterial or phage clones containing recombinant DNA. These sequences represent the entire mRNA population of a certain tissue or cell type at a specific stage of development or differentiation. Given its simple operation and low probability of false positives, this method is widely used to obtain complete nucleotide and encoded amino acid sequences and to clarify their structures to facilitate subsequent synthesis of peptides (Mu et al., 2014; Cao et al., 2018; Song et al., 2019).

Various experiments can be performed to screen peptide molecules for their wound repair activity, such as calculating cell scratch repair rates, cell proliferation rates, and secretory factor levels after peptide application. The wound healing activity of different peptides can also be assessed by direct application on skin wounds (e.g., whole cortex, scald, and diabetic wound models), with an evaluation of wound healing speed and tissue staining (e.g., H&E and Masson staining) (Liu et al., 2019; Song et al., 2019; Zhang et al., 2022).

The AH90 peptide (molecular weight 2.6 kDa) identified from the skin of Odorrana grahami is composed of 24 amino acid residues and shows significant efficacy in accelerating whole skin wound re-epithelialization and granular tissue contraction in mice (Liu et al., 2014b). The Ot-WHP peptide identified from the Chinese frog Odorrana tormota consists of 24 amino acid residues and exhibits 83% similarity with the amino acid sequence of AH90 (He et al., 2019). Ot-WHP also shows significant wound healing effects and a mechanism similar to that of AH90. Both AH90 and ot-WHP can activates the mitogen-activated protein kinase (MAPK) and factor-κB (NF-κB) signaling pathways to induce macrophages to produce chemokines, cytokines, and growth factors and activates the transforming growth factor-β (TGF-β)/Smad signaling pathway to promote cell adhesion and integrin expression to induce keratinocyte migration and fibroblast to myofibroblast transformation, thereby enhancing wound healing. In addition, integrin α5 and α6 expression levels are elevated in keratinocytes after AH90 treatment. Given the cross-talk between integrin and the TGF-β signaling pathway, which also affects TGF-β expression, cell adhesion and migration can be mediated by integrin during skin re-epithelialization, and thus AH90 may promote wound healing speed by up-regulating integrin (Liu et al., 2014b). Ot-WHP can also increase the number of neutrophils and macrophages at the wound site, promote neutrophil phagocytosis, and enhance macrophage, keratinocyte, and fibroblast cross-talk, thus inducing skin wound repair (He et al., 2019). The peptide Bm-TFF2 derived from Bombina maxima skin is not only a platelet agonist, but also demonstrated almost an 80% wound closure rate within 36 h in vitro scratch experiments on IEC-6 cells (Zhang et al., 2010).

Salamanders are the only adult vertebrates capable of regenerating structurally and functionally intact limbs, signifying excellent wound repair capabilities (Tseng and Levin, 2008; Barros et al., 2021). Tylotoin, the first salamander-derived peptide, induces the formation of endothelial cell tubes, promotes α-SMA expression and angiogenesis, accelerates the transformation of fibroblasts into myofibroblasts, and promotes skin wound healing. In cells, tylotoin induces macrophage migration, stimulates TGF-β1 and Interleukin 6 (IL-6) secretion in macrophages, and activates the Smad and MAPK signaling pathways to regenerate skin tissue (Mu et al., 2014). TK-CATH, another salamander-derived bioactive peptide consisting of 55 amino acid residues, shows similar wound healing activity. Notably, TK-CATH induces macrophages to produce cytokines, growth factors, and chemokines by activating the MAPK signaling pathway and promotes the migration and proliferation of keratinocytes, thereby improving the inflammatory process and tissue remodeling during skin wound healing (Luo et al., 2021).

As a unique amphibian in China (Yin et al., 2020), O. andersonii is highly susceptible to skin damage due to the harsh environment in which it lives, thus requiring rapid repair for survival (Bian et al., 2018). As such, O. andersonii skin is an important source of potential wound healing peptides, most of these peptides can promote cell scratch repair in a time and dose dependent manner, and exhibit strong healing ability in mouse skin wound models. OA-GL21 (2.2 kDa, 21 amino acid residues) is a bioactive amphibian peptide with wound healing activity but without a disulfide bond or free cysteine residue (Bian et al., 2018). Studies have shown that OA-GL21 Research shows that OA-GL21 can affect cell migration rather than direct proliferation to help wound repair, and form fewer scars during the repair process. Compared with the positive control drug KangFuXin (KFX), OA-GL21 also shows significant effects on mouse skin wounds, with faster and higher healing rates (Bian et al., 2018). The OM-LV20 peptide (1.9 kDa) contains a pair of intramolecular disulfide bonds. In the skin wound healing process, OM-LV20 treatment has a better proliferate effect on HaCaT cells than fibroblasts (Li et al., 2018). OA-GL12, a peptide with one cysteine, may recruit more macrophages to migrate to the wound site by promoting the expression of TNF and TGF-β1 in RAW264.7 cells. In addition, the pronounced free radical scavenging activity of OA-GL12 may also play a role in wound healing (Song et al., 2019). OA-FF10, which is only 10 amino acid residues in length and contains an intramolecular disulfide bridge (Rana box), was found to have higher sensitivity to HaCaT cells. This characteristic enables OA-FF10 to accelerate the migration and proliferation of HaCaT cells without affecting HSF cells. Moreover, OA-FF10 (1 μM) also displays better activity than KFX (100 mg/L), thus showing the potential as a wound healing molecule (Liu et al., 2019). The CW49 peptide is very effective in promoting chronic wound healing (Liu et al., 2014a). Chronic diabetic wounds often remain in the inflammatory stage for a long time and inhibiting excessive inflammatory responses can hinder disease progress (Zhao et al., 2016). In diabetic wounds, CW49 application results in significant anti-inflammatory effects (inhibiting IL-6 and TNF-α expression) and angiogenesis (stabilizing HIF-1α expression and up-regulating NO production), thereby preventing excessive inflammatory responses (Liu et al., 2014a). As the first identified natural peptide homodimer shown to promote wound repair, OA-GP11d not only promotes the migration of HaCaT cells but also inhibits the release of inflammatory factors by activating the MAPK and NF-κB signaling pathways. In mouse skin burn and injury models, OA-GP11d also exhibits strong repair ability (Fu et al., 2022). The other natural repair-promoting homodimer peptide OA-GL17d (OA-GL17 dimer) has a half-life of 1.86 h (longer than some peptides), this means that it may have more advantages in the effective time than other similar wound-healing peptides. OA-GL17d shows strong repair ability in many animal wound models (e.g., mouse whole skin and scald wound models). This repair effect may occur by reducing miR-663 levels, increasing TGF-β1 levels, activating the TGF-β1/Smad signaling pathway, and ultimately accelerating re-epithelialization and granular tissue formation in skin wounds (Zhang et al., 2022).

Despite its wide distribution in Southeast Asia, little research has been conducted on the Rana limnocharis frog species and its peptides (Sumida et al., 2002). RL-RL10, an active peptide molecule with a molecular weight of only 1.3 kDa, has been shown to improve the proliferation and migration of HaCaT cells in a concentration-dependent manner, and enhance full-thickness wound healing in mice (Wang S. et al., 2021a). RL-QN15, another short peptide derived from skin secretions of R. limnocharis, also shows potent repair effects in various wound models (chronic wounds, skin fibrosis, and oral ulcers). RL-QN15 can induce keratinocyte migration and proliferation, which are crucial for initial wound healing. RL-QN15 can also significantly inhibit the pro-inflammatory factor TNF-α and promote IL-1β to recruit macrophage migration to the wound. During wound healing, excessive inflammation can lead to scar formation, thus inhibiting TNF-α levels may contribute to scar reduction (Kumar and Yin, 2018). In addition, different release levels of TGF-β1 and TGF-α in different periods caused by RL-QN15, can help balance the TGF-β1 and TGF-α ratio during wound repair, which is also one of the reasons for faster wound healing and reduction of scar formation. This above phenomenon may be caused by the activation of the MAPK and Smad signaling pathways in wound skin (Wang Y. et al., 2021b). RL-QN15 also shows improved wound healing ability in combination with nanomaterials, as discussed later in this review.

In the process of wound healing, endogenous infection caused by microorganisms can directly damage wound epithelialization, which is an important factor hindering wound repair (Ki and Rotstein, 2008). Therefore, enhancing the antibacterial effects of skin during wound infection should promote the healing speed of skin wounds.

The cathelicidin-NV peptide from the plateau frog Nanorana ventripunctata belongs to the cathelicidin family and consists of 24 amino acid residues (Wu et al., 2018). The cathelicidin family, which only participates in vertebrate humoral immunity, is an antibacterial and immunostimulatory family and plays an important role in immunoregulation, wound healing, and angiogenesis (Cao et al., 2018). Peptide tylotoin, TK-CATH (both derived from salamanders), and cathelicidin-OA1 (isolated from O. andersonii) also belong to this family (Mu et al., 2014; Liu et al., 2019; Luo et al., 2021). As the first frog species-derived peptide to show wound healing activity (Cao et al., 2018), cathelicidin-OA1 also exhibits antioxidant activity. Because the oxidative stress is detrimental to wound repair, so the healing effects of cathelicidin-OA1 are achieved not only by direct intervention in the wound healing process but also via antioxidant activity (Cao et al., 2018). Local application of cathelicidin-NV in whole skin wounds promotes wound re-epithelialization and accelerates healing. Cathelicidin-NV can also induce fibroblasts to produce collagen and promote keratinocyte proliferation to facilitate granulation tissue formation. Furthermore, cathelicidin-NV treatment can increase the levels of Monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor, -α (TNF-α), vascular endothelial growth factor (VEGF), and TGF-β1 in skin, which is favorable for the formation of blood vessels (Park et al., 2017; Wu et al., 2018).

Peptide B-2Ta, identified from the European frog Pelophylax kl. esculentus, is an effective antibacterial peptide. B-2Ta administration suppresses the inflammatory response and promotes angiogenesis in injured rats. Treatment of infected granulation tissue with B-2Ta inhibits inflammation of injured tissue, promotes angiogenesis and epithelial migration, and accelerates wound healing. Thus, B-2Ta is both an antimicrobial and wound healing peptide (Liu et al., 2017). The antimicrobial peptide fragment Esculenti-1a (1-21) NH2, isolated from the skin of Pelophylax lessonae/ridibundus, also promotes wound healing, especially in chronic skin ulcers. In vitro, Esculenti-1a (1-21) NH2 promotes HaCaT cell and primary epidermal keratinocyte migration to enhance wound epithelialization. In mice, Esculenti-1a (1-21) NH2 releases TGF-β1 and activates the EGFR signaling pathway to promote wound repair (Di Grazia et al., 2015). Temporins A and B, antimicrobial peptides derived from Rana temporaria, also exhibit skin wound healing activity. Notably, temporins A and B can reduce Staphylococcus aureus in HaCaT cells in a dose-dependent manner to induce cell proliferation, thereby promoting keratinocytes to initiate wound closure. These peptides can also induce HaCaT cell migration to the wound by activating the EGFR signaling pathway (Di Grazia et al., 2014). Cathelicidin-DM, an antibacterial peptide derived from Duttaphrynus melanostictus, also has wound healing properties. In mice, cathelicidin-DM markedly accelerates skin infection wound healing, with a stronger effect than that of the aminoglycoside antibiotic gentamicin (Shi et al., 2020).

Modification of peptides can not only improve peptide activity but also reduce production costs. Peptide molecules are generally composed of several to dozens of amino acid residues (Bolhassani, 2019; Sun Z. G. et al., 2021b) and can be transformed by directly modifying the peptide chain skeleton, shortening the length of the peptide chain skeleton, and intercepting the final effective peptide segment.

Tiger17, an 11 amino acid residue peptide synthesized base on peptides tigerinins (from the skin secretions of Fejervarya cancrivora), shows potent skin wound healing activity in mice. This promotion ability may occur by inducing macrophages to re-aggregate to the wound site and promoting keratinocyte and fibroblast migration and proliferation, leading to re-epithelialization and granulation tissue formation, activation of the MAPK signaling pathway, and transfer of TGF-β1 and IL-6 in macrophages. Compared with basic peptide molecules, these peptides tend to have shorter sequences, higher activity, and wider application potential (Tang et al., 2014).

TGF-β is an important growth factor in the body and is essential for wound healing, particular the TGF-β1, 2, and 3 subtypes (Lichtman et al., 2016). TGF-β1 is often released during the acute reaction period of trauma, which helps macrophages and fibroblasts to become chemotactic towards the wound and promotes keratinocyte proliferation (Peplow and Chatterjee, 2013). TGF-β 3 also shows an important influence on cell migration regulation (Le et al., 2012). SMAD is the downstream effector of TGF-β, and TGF-β/SMAD signaling pathway activation can increase skin angiogenesis, promote wound contraction, and inhibit inflammation by inducing fibroblast transformation and integrin expression (Wang Y. et al., 2021b). The MAPK and NF-κB signaling pathways form the intersection of various signaling pathways in wound repair. They are closely related to inflammation and inhibiting their phosphorylation can help reduce inflammatory factor levels and inflammation during wound healing (Fu et al., 2022). Furthermore, studies have identified cross-talk between the TGF-β and MAPK signaling pathways (Liu et al., 2014b).

The ceRNA mechanism has attracted considerable attention and has helped to reveal the interactions between RNAs. Notably, ceRNAs can change the expression of target genes by competing for shared microRNAs (miRNAs) at the post-transcriptional level (Wu et al., 2020). In the human genome, most DNA is transcribed into RNA to become non-coding RNA, which cannot encode proteins. Among them, miRNAs play a variety of important regulatory roles in cells, including repression of target protein translation (Lu and Rothenberg, 2018). In recent years, the ceRNA mechanism has been implicated in many diseases, implying important biological significance. As miRNAs participate in almost all stages of wound healing, they are considered important targets in the intervention of skin wound healing. For example, miR203 is associated with the promotion of keratinocyte proliferation and migration (Liu et al., 2022), and miR19a and 20a are involved in the activation of the NF-κB signaling pathway (Li et al., 2021). However, although certain miRNAs appear to be involved in wound healing, research remains poor in comparison to the huge miRNA family, and exploration of miRNA functions and the ceRNA mechanism is also limited. As mentioned above, miR-663a is the first wound healing miRNA identified using an amphibian-derived peptide (OA-GL17d) as a molecular probe. Inhibition of miR-663a can improve TGF-β levels and activate the TGF-β/SMAD signaling pathway, thereby accelerating wound repair (Zhang et al., 2022). Thus, these findings suggest that ceRNAs are important for a deeper understanding of the mechanisms underpinning wound healing and emphasize the importance of peptides as molecular probes to analyze such mechanisms.