As the largest organ of the human body, the skin serves as an important barrier that separates the internal and external environments of the body (Wang et al., 2021; Hwang et al., 2023). It possesses various physiological functions such as sweating, heat dissipation, and sensation of stimuli (Kim et al., 2019). In addition to protective structures such as the epidermis, dermis, and subcutaneous tissues, the human skin consists of crucial accessory organs such as blood vessels, sweat glands, and nerves (Raziyeva et al., 2021; Natesan and Kim, 2023). Dysfunction caused by acute and chronic skin damage can lead to imbalances in the internal and external body environments, thereby resulting in serious consequences such as infections and electrolyte imbalances (Mullin et al., 2023). More importantly, vascular and nerve damage leads to delayed transport of various active substances and signal transduction, which seriously hinders wound healing and imposes substantial pressure on both patients and the healthcare system (Carmeliet, 2003; Zhao et al., 2023). Currently, clinical treatments for wound repair primarily involve surgical procedures combined with drug therapy (Eriksson et al., 2022; Zhang et al., 2024). However, existing drugs, such as antimicrobial dressings, cytokines, small molecules from plants, and small-molecule drugs such as heat shock proteins, have various drawbacks, including low activity, poor stability, difficult storage, and high costs (Las Heras et al., 2020). Therefore, there is an urgent need for novel drugs capable of accelerating wound healing.

Amphibian skin is capable of rapid repair and secretes diverse biologically active molecules, including antimicrobial peptides, antioxidant peptides, neuropeptides, and cytokines (Zhou et al., 2024). For example, Sugiura et al. identified axolotl MARCKS-like protein, an extracellularly released factor that induces the initial cell cycle response during axolotl appendage regeneration (Sugiura et al., 2016). Zhulyn et al. discovered unique expansions in the mTOR protein sequence among urodele amphibians and demonstrated that rapid activation of protein synthesis is a unique feature of the injury response that is critical for limb regeneration in the axolotl (Zhulyn et al., 2023). Cao et al. identified a novel cathelicidin (cathelicidin-OA1) present in the skin of Odorrana andersonii that exhibited antioxidant activity and accelerated wound healing in human keratinocytes (HaCaTs) and skin fibroblasts (HSF) in a time- and dose-dependent manner (Cao et al., 2018). In addition, their unique skin and active molecules effectively protect them from ultraviolet radiation, microorganisms, and physical injury in various hazardous environments (Cao et al., 2018). Based on these properties, it is speculated that active substances in amphibian skin will exert positive effects on wound healing in humans. To examine this hypothesis, Menger et al. cloned and classified an amphibian epidermal lipoxygenase and observed a positive influence on human cell migration (Menger et al., 2011). Fu et al. also identified the first naturally occurring peptide homodimer, the OA-GP11 dimer (OA-GP11d), from Odorrana andersonii, which effectively promoted the repair of full-thickness and burn wounds in mice (Fu et al., 2022). Although these studies have suggested the active molecule from amphibian skin as a potential new pro-regenerative drug candidate, relevant active molecules as biotherapeutics and their application in wound healing have rarely been reported (Guo et al., 2022).

In the current study, a biologically active macromolecule (JH015Y) was extracted and isolated from amphibians, and a stable peptide structure was identified. Subsequently, the therapeutic efficacy of JH015Y protein in different wounds, both in vitro & in vivo, was demonstrated. Collectively, the results revealed that the identified natural extract has tremendous potential for wound repair in animals. This study expands our understanding of the biologically active peptides derived from amphibians and holds promise for the development of new wound-healing biotherapeutics.

Materials

This study utilized thefollowing listed materials:HaCaTcells (maintained in our laboratory); HSF cells (maintained in our laboratory); JH015Y protein; recombinant murine epidermal growth factor (EGF) (PeproTech, 5 Cedarbrook Drive, Cranbury, NJ, USA); DMEM culture medium; fetal bovine serum (Gibco, Carlsbad, CA, USA); penicillin/streptomycin stock solution (Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, 236 Baidi Road, Tianjin, China); CCK-8 assay kit (Servicebio, Wuhan, China); Clean bench (BSC-1100IIA, Beijing Donglian Haer Instrument Manufacturing Co., Ltd., Beijing, China); CO2 cell culture incubator (Shanghai Boxun Medical Biological Instrument Co., Ltd, Shanghai, China); microplate reader (Jiangnan Corporation, Ningbo, China); Depilatory cream (Veet, London, UK); sterile dressing (6×7 cm, Zhejiang Ou Jie Technology Co., Ltd., Deqing, China); glutaraldehyde (JiZhiSheng Biology, Shanghai, China); OCT embedding agent (Tissue-TEK, Sakura, Torrance, CA, USA); Masson’s trichrome staining kit (Nanjing Kaiji Biotechnology Development Co., Ltd., Nanjing, China); cryostat (CM1950, Leica, Wetzlar, Germany); YLS-5Q desktop constant temperature and pressure scald instrument (Tianjin Norei Xinda Technology Co., Ltd., Tianjin, China); blood glucose meter (Sinocare, Changsha, China); Sprague Dawley rats (male, 120-160 g, four-week-old), C57BLKS/J (db/db) mice (male, 40-50 g, six-week-old), provided by Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China; BCA protein quantification kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China); electrophoresis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China); protein molecular weight standards (Shanghai Yaen Biotechnology Co., Ltd., Shanghai, China); sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gel rapid preparation kit (Dalian Meilun Biotechnology Co., Ltd., Dalian, China); protein loading buffer; chemiluminescence imager (BioRad, Hercules, CA, USA); vertical protein electrophoresis apparatus (Hangzhou Nuoyang Biotechnology Co., Ltd., Hangzhou, China); Maldi TOF mass spectrometer (Bruker, Saarbrucken, Germany); ultra-high resolution mass spectrometer (Thermo Fisher, Waltham, MA, USA).

Cells proliferation assay (Lee et al., 2024)

Briefly, HaCaT and HSF cells (2.5×10⁴ cells/mL) were seeded into 96-well plates and cultured overnight to reach ~80% confluency. To each well, the JH015Y protein was applied to attain final concentrations of 5, 25, and 125 μg/mL; the positive control (EGF) was used at a final concentration of 10 ng/mL (Peng et al., 2016). Acetic acid (0.1 M) was used as the solvent to prepare the JH015Y protein solution and solvent control group (blank group). Three replicates were performed for each concentration.

Animal model and surgical procedures (Lee et al., 2023)

In vivo, wound healing was examined using six-week-old (170-200 g) Sprague-Dawley rats and C57BLKS/J (db/db), supplied by Shanghai SLAC Laboratory Animal Co., Ltd. All experimental procedures were in accordance with the Zhejiang University guidelines for the welfare of experimental animals (Peng et al., 2012, 2020, 2013a). The healing properties of the JH015Y protein were examined by establishing the following models: (1) Acute wound model: A 15 mm×15 mm skin excision of created in rats by excising the skin within the confines of the square down to the level of subcutaneous panniculus carnosus (Peng et al., 2013b); (2) Burn model: Burns were induced using a scalding instrument under the following conditions: 0.5 kg injury pressure, 4 m2 injury area, 80°C injury temperature, and 8 s injury time; (3) Diabetic ulcer model: Full-thickness skin excision wound (1 cm×1 cm) in C57BLKS/J (db/db) mice. Rats/mice with wounds were randomly divided into 5 groups (n=9 animals/group) as follows: (1) Blank control group without treatment; (2) EGF treatment group; (3) Low-dose protein treatment group; (4) Medium-dose protein treatment group; (5) High-dose protein treatment group. To prevent interference between rats/mice, each rat/2-3 mice were individually housed in a cage. Animals were anesthetized, their back hair was shaved, and their skin was cleaned using povidone-iodine solution, followed by wiping with sterile water. Every two days, a phosphate-buffered saline solution, different doses of JH015Y protein solution, and EGF solution were locally administered to the wound site once. The high-dose JH015Y protein group received 2.5 mg/dose, the medium-dose JH015Y protein group received 0.5 mg/dose, and the low-dose JH015Y protein group received 0.1 mg/dose; EGF was administered at a dose of 200 ng.

Histopathological assessment (Boo et al., 2023)

Healed skin samples, including the epidermis, dermis, and hypodermis, were fixed in 4% buffered paraformaldehyde, dehydrated, and embedded in paraffin for sectioning. The sections (5 μm thick) were stained using Masson’s trichrome staining kits in accordance with the manufacturer’s protocols. The stained skin sections were subsequently examined and photographed using a Leica image analysis system (Zhang et al., 2016).

Physicochemical properties of the JH015Y protein

After preparing, separating, and concentrating the gel, samples were electrophoresed at 90V for 30 min. Subsequently, staining with Coomassie Brilliant Blue was performed for at least 10 min, followed by several destaining steps until a clear background was observed. Following SDS-PAGE, spectral analysis of the JH015Y protein was conducted using FlexControl software (Chicago, IL, USA) to determine its molecular weight. Additionally, amino acid sequencing of the JH015Y protein was performed using mass spectrometry to determine its amino acid sequence characteristics (Li et al., 2020).

Statistical analysis

Data analyses were performed using GraphPad Prism 8.0.1 (GraphPad Software, Inc., La Jolla, CA, USA). Measurement data are expressed as the mean ± standard deviation SD, and the count data are expressed as %. Comparisons between the two groups were performed using the independent samples t-test. *P<0.05 as the difference was significant, **p<0.01 as the difference was highly significant.

Identification of JH015Y protein

As shown in Fig. 1A and 1C, JH015Y comprises a variety of proteins with molecular weights ranging between 15 and 70 kDa. Among them, components with molecular weights of 26 ± 3, 17 ± 3, and 50 ± 5 kDa exhibited the highest protein concentrations. According to the results of the amino acid analysis, the amino acid sequence of the JH015Y protein showed low similarity with samples in existing protein databases, indicating that JH015Y may be a newly discovered protein. In addition, as shown in Fig. 1B, the mass spectrometry results identified a protein carrying a single charge with a mass-to-charge ratio of approximately 2.5, indicating a high concentration; the result corresponded to a band with a molecular weight of 26 ± 3 kDa detected in the SDS-PAGE results.

Figure 1. Identification of JH015Y protein. (A) Protein electrophoresis map. (B) Mass spectrum. (C) Analysis of JH015Y protein and its matching degree with protein database data. HIVEP3: HIVEP Zinc Finger 3; A member of the human immunodeficiency virus type 1 enhancer binding protein family. NAC-A/B: domains of NAC transcription factors unique to plants. LIM: A highly conserved double zinc finger domains in evolution.

In vitro cell proliferative capacity of JH015Y protein

The biological behaviors of keratinocytes and fibroblasts play a crucial role in the skin wound healing process. Therefore, we examined the cell proliferative capacity of JH015Y protein in HaCaT and HSF model cells. As shown in Fig. 2, groups treated with different concentrations of JH015Y protein significantly promoted the proliferation of HaCaT and HSF cells compared with the blank group while showing a similar effect to the EGF-positive group. In addition, the proliferative effect of JH015Y protein was concentration-dependent between 5-125 μg/mL. With increasing JH015Y protein concentration, the proliferation rate gradually increased from 112% in the control group to 147%, which was comparable with that observed in the EGF group (10 ng/mL). Therefore, the JH015Y protein may promote keratinocyte and fibroblast proliferation and wound healing in acute and chronic skin injuries.

Figure 2. Promoting of JH015Y protein on cell proliferation in vitro. (A) Influence of JH015Y protein in the proliferation of HaCaT cells. (B) Influence of JH015Y protein in the proliferation of HSF cells. ***p<0.001, **p<0.01, *p<0.05. n=5. EGF: Epidermal Growth Factor. HSF: Human Skin Fibroblasts. HaCaT: Human Keratinocytes Cells.

In vitro cell migration and cytokine secretion properties of JH015Y protein

In addition to cell proliferation, cell migration and related cytokine secretion are important for the skin wound healing process. Therefore, we examined the impact of the JH015Y protein on cell migration and cytokine secretion in HaCaT and HSF model cells. As shown in Fig. 3, the high- and low-concentration JH015Y protein group (125 μg/mL and 5 μg/mL, respectively) significantly enhanced migration of HaCaT and HSF cells when compared with the blank group and exhibited a similar effect to the EGF group (10 ng/mL). In addition, both the high- and low-concentration JH015Y protein groups promoted cytokine secretion. Compared with the blank group, the high-concentration JH015Y protein group increased the secretion of vascular endothelial growth factor (VEGF) and EGF in HSF cells by 3.4 and 2.9 times, while the low-concentration group increased secretion by 2.5 times; these increments were similar to the 3.7 and 4.1 times demonstrated by the EGF group. Likewise, the high-concentration JH015Y protein group enhanced the secretion of VEGF and EGF in HaCaT cells by 3.5 and 5.5 times, respectively, while the low-concentration group enhanced secretion by 2.3 and 2.5 times; these increased levels closely resembled the 5.3 and 5.9 times demonstrated by the EGF group. Therefore, these findings suggested that the JH015Y protein may promote keratinocyte and fibroblast proliferation and wound healing in acute and chronic skin injuries.

Figure 3. The promoting effect of JH015Y protein on in vitro cell migration. (A) The effect of JH015Y protein on the migration of HaCaT cells. (B) The effect of JH015Y protein on HSF cell migration. (C) The effect of JH015Y protein on cytokine secretion of HaCaT. (D) The effect of JH015Y protein on cytokine secretion in HSF. ***p<0.001, **p<0.01, *p<0.05. n=5. HSF: Human Skin Fibroblasts. EGF: Epidermal Growth Factor. VEGF: Vascular Endothelial Growth Factor.

In vivo therapeutic effects of JH015Y protein on acute wound models

To investigate the therapeutic effect of JH015Y on wound healing in vivo, we established a classic model of acute wounds, the full-thickness skin defect rat model. From day 4 onward (Fig. 4A, 4B), wounds treated with 0.5 and 2.5 mg/dose JH015Y protein exhibited significantly higher healing rates, 1.26 and 1.27 times, than those of the blank group, respectively. In addition, the therapeutic effects of 0.5 and 2.5 mg/dose of JH015Y protein were similar to those of EGF (200 ng/dose). The healed skin was histologically analyzed using Masson staining. As shown in Fig. 4C, from day 4 onward, a small amount of collagen deposition was already present in the healed skin of the high-dose group (2.5 mg/dose) and the EGF group (200 ng/dose). The skin wounds of all groups appeared to be healed by day 18. Notably, the high-dose JH015Y protein (2.5 mg/dose) and EGF (200 ng/dose) groups exhibited greater collagen deposition and thicker and more mature fibers. Conversely, the healed skin of the blank and low-dose JH015Y protein (0.1 mg/dose) groups exhibited finer and less mature collagen fibers. These results suggested that JH015Y protein could support the rapid healing of severe full-thickness skin defects, and the healing process appears to be similar to the therapeutic effects of traditional growth factor drugs.

Figure 4. Therapeutic effect of JH015Y protein on acute wounds in vivo. (A) The wound healing rate of rats in different groups and days. (B) Photos of rat wounds from different groups on different days. (C) Masson staining results of skin samples from different groups of rats on different days (scale: 100 μm). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. n=8. EGF: Epidermal Growth Factor.

In vivo therapeutic effects of JH015Y protein on burn wounds

Compared with acute wounds that require prompt care, burn wounds typically take longer to heal owing to extensive skin loss, destructive soft tissue damage, and scarring. Accordingly, we also investigated the therapeutic effects of JH015Y on burn wounds in vivo. As shown in Fig. 5A, 5B, in the burn model, the medium-dose (0.5 mg/dose) and high-dose (2.5 mg/dose) JH015Y protein groups demonstrated accelerated burn wound healing from day 4 and a trend of delayed scar formation, with wound healing rates 1.27 and 1.28 times that of the blank group, respectively. In addition, according to the results of Masson’s staining (Fig. 5C), all groups showed wound healing by day 21. In the medium/high-dose JH015Y protein and EGF groups, healed skin exhibited abundant collagen deposition and mature fibers. In contrast, the blank and the low-dose JH015Y protein (0.1 mg/dose) groups exhibited fewer collagen fibers in the healed skin. In this study, the medium-dose JH015Y protein group (0.5 mg/dose) exhibited therapeutic effects comparable to EGF (200 ng/dose) on rat skin burns. Importantly, the therapeutic effects of the high-dose JH015Y protein group (2.5 mg/dose) were superior to those of the EGF group (200 ng/dose). These findings indicated that JH015Y protein could exert good therapeutic effects on rat skin burns.

Figure 5. Therapeutic effect of JH015Y protein on burn wounds in vivo. (A) Photos of rat wounds from different groups on different days. (B) The healing rate of scalds in rats of different groups and days. (C) Masson staining results of skin samples from different groups of rats on different days (scale: 100 μm). ***p<0.001, **p<0.01, *p<0.05. n=8. EGF: Epidermal Growth Factor.

In vivo therapeutic effects of JH015Y protein on diabetic ulcers

Diabetic ulcers are typical chronic refractory wounds caused by a series of neurovascular lesions and inflammatory reactions owing to the infiltration of a high-glucose environment. In the ulcer model, as shown in Fig. 6A, 6B, compared with the blank group, the medium/high-dose JH015Y protein groups demonstrated a trend of accelerated skin healing from day 7, with wound healing rates 1.14 and 1.3 times that of the blank group, respectively. Furthermore, the JH015Y protein exhibited similar therapeutic effects to EGF (200 ng/dose) on ulcer wounds, indicating that the JH015Y protein exerts good therapeutic effects on diabetic ulcers.

Figure 6. Therapeutic effect of JH015Y protein on diabetic ulcer in vivo. (A) Photos of mouse wounds from different groups on different days. (B) Healing rate of ulcer wounds in mice of different groups and days. (C) Masson staining results of skin samples from different groups of rats on different days (scale: 100 μm). **p<0.01, *p<0.05. n=8. EGF: Epidermal Growth Factor.

According to the results of Masson staining histological analysis (Fig. 6C), although the blank and low-dose JH015Y protein groups still exhibited signs of localized inflammatory cell infiltration and bleeding in the healed skin on day 19, these phenomena were not detected in the medium/high-dose JH015Y protein and EGF (200 ng/dose) groups. These results suggested that the JH015Y protein could reduce the inflammatory response in the diabetic ulcer wound-healing process and create a favorable physiological environment for collagen deposition and chronic wound healing.

Biological safety

The survival rate and body weight of experimental animals are important indices for evaluating the therapeutic effects of drugs. As shown in Fig. 7, the body weights of the rats and mice in the different groups increased gradually and stabilized eventually, with no deaths observed. This indicated that the tested concentrations of JH015Y protein exhibited low biological toxicity in the three different acute and chronic wound models.

Figure 7. The survival rate and body weight of animals. (A) Trauma model. (B) Burn model. (C) Diabetes ulcer model. n=8. EGF: Epidermal Growth Factor.

In amphibians, the reprogramming of cell lineages (Plikus et al., 2017) and novel regeneration-organizing cells (Aztekin et al., 2019) have been widely explored, and their advantages are expected to be applied in mammalian wound healing. However, no mammalian cell displays extensive adult regenerative properties like urodele amphibians because of the cross-species bottleneck, which typically results in scarring and non-functional repair (Lopez et al., 2014). Fortunately, obtaining protein and other extracts from amphibians has advantages such as easy preparation and clear composition, and studies have demonstrated the important mechanism of amphibian proteins as biotherapeutics in wound healing. For example, Mandato et al. reported that microtubules are drawn to wound borders via actomyosin-based contraction during wound healing (Bement et al., 1999; Mandato and Bement, 2003). The clinical applications of a variety of amphibian proteins such as novel peptides, thrombin (Tanaka et al., 1999) and plakoglobin (Kofron et al., 2002) appear promising in mechanical injury and diabetic ulcer healing.

In this study, the efficacy of JH015Y as a biotherapeutic agent for acute and chronic wound healing was comprehensively investigated. JH015Y exerted a concentration-dependent proliferative effect on both HaCaT and HSF cells. The optimal efficacy was comparable to that of EGF. Second, in various wound healing experiments, from days 4 to 14, the skin healing effect of the JH015Y protein gradually approached that of EGF and was substantially better than that of the blank (control) group. Given the robust skin-healing ability of JH015Y, we believe that it holds great potential in various skin trauma fields. Homologous pro-healing proteins are also known to demonstrate this property. For example, Li et al. (Andersonin-W1 (Li et al., 2024), reported the potential of OA-RD17 (Li et al., 2023)), an amphibian-derived pro-healing peptide. The authors found that this peptide could facilitate re-epithelialization, granulation regeneration, and angiogenesis, thus substantially boosting the healing of full-thickness deep second-degree burns and diabetic skin wounds in mice. Additionally, Liu et al. (2014) reported the potential wound-healing properties of a peptide (CW49) that promoted wound healing of a full-thickness dermal wound in both normal and diabetic animals. The JH015Y protein derived from amphibians in complex wild environments has shown wound-healing effects (Fig. 8), underscoring the importance of exploring the vast untapped treasures in the animal and plant kingdoms. Studies of these amphibian-derived proteins as biotherapeutics provide a missing link in our understanding of vertebrate regenerative potential.

Figure 8. The therapeutic effects of the JH015Y protein in different wounds.

The study was supported by the National Natural Science Foundation and National Key Research and Development Program of China (Grant no. 82374043, U23A20505, 2022YFC3501904), Zhejiang Province Commonweal Projects (Grant no. LGF22H280001), China-ASEAN International Innovative Center for Health Industry of Traditional Chinese Medicine (Gui Ke AD20297142).

The authors declare no conflict of interest.

LH P: Conceptualization, methodology. HR C: writing-original draft. NZ: methodology. YD L: writing-original draft, writing-reviewing.