Salamanders are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs ( 7 ). Wounding is believed to initiate limb regeneration. One of the earliest steps for wound healing is reepithelialization of the wounded surface. Wound healing proceeds incredibly quickly in salamanders. Compared with the process of reepithelialization in mammalian wounds, which normally takes 2‐3 d, the same process in salamanders takes <10 h. Little is known about skin wound healing in amphibians. Some genes, including prx1, Tbx5, Fgf8, Fgf10 , and Msx1 , have been found to take part in amphibian skin wound healing and limb regeneration ( 8 ), but no effector substance that exerts a skin wound‐healing function has been identified from amphibians. The current work aims to identify bioactive substance with wound‐healing‐promoting activity from salamander skin.

Adult skin consists of two tissue layers: a keratinized stratified epidermis and an underlying thick layer of collagen‐rich dermal connective tissue providing support and nourishment ( 1 ‐ 2 ). The skin serves as a protective barrier against the outside world, and any break in it must be rapidly and efficiently mended. Wound healing is essential for organisms to survive. Cutaneous wound healing is divided into 3 continuous and overlapping processes: inflammation, proliferation, and remodeling phase ( 1 , 3 ‐ 5 ). In the inflammatory phase, inflammatory cells, composed mainly of neutrophils and macrophages, infiltrate to the wound site and trigger the process of wound healing ( 2 ). In the proliferative phase, the major events are reepithelialization and granulation tissue formation, both of which require cell proliferation and migration of keratinocytes, fibroblasts, and endothelial cells ( 3 ). In the contraction and remodeling phase, myofibroblasts differentiated from fibroblasts play a key role in wound contraction and control synthesis and degradation of ECM proteins. This complex process is executed and regulated by an equally complex signaling network involving numerous growth factors, cytokines, and chemokines, such as transforming growth factor β (TGF‐β), interleukin 1 (IL‐1), and interleukin 6 (IL‐6) ( 6 ).

MATERIALS AND METHODS

Tylototriton verrucosus sample Adult T. verrucosus (either sex, 20±5 g) were collected from the Yunnan province of China. Animals were anesthetized using 2.5% vaporized inhaled isofluorane, and the dorsal skin was cut off after cleansing with distilled water. The skin was homogenized by tissue homogenizer with 0.1 M phosphate‐buffered saline (PBS; pH 6.0) containing 1% (v/v) protease inhibitor cocktail (P8340‐5; Sigma, St. Louis, MO, USA). The skin homogenate solutions were quickly centrifuged (10,000 g for 10 min), and the supernatants were lyophilized. All experiments were approved by Kunming Institute of Zoology, Chinese Academy of Sciences.

Peptide purification An aliquot (1 g) of lyophilized homogenate supernatant of skin was dissolved in 10 ml PBS and centrifuged at 5000 g for 10 min. The supernatant was applied to a Sephadex G‐50 (Superfine, 2.6 cm diameter, 100 cm length; Amersham Biosciences, Castle Hill, NSW, Australia) gel filtration column equilibrated with 0.1 M PBS for preliminary separation. Elution was performed with the same buffer, collecting fractions of 3.0 ml. The eluted fractions were monitored at 280 nm and subjected to cell proliferation assays. The fraction containing cell proliferation activity was further purified by a C 18 reversed‐phase high‐performance liquid chromatography (RP‐HPLC) column (5 μM particle size, 110 Å pore size, 250 mm length, 4.6 mm diameter; Gemini C 18 , Phenomenex, Torrance, CA, USA). The elution was performed using a linear gradient of 0‐80% acetonitrile containing 0.1% (v/v) trifluoroacetic acid in 0.1% (v/v) trifluoroacetic acid/water over 60 min as illustrated in Supplemental Fig. S1A. UV‐absorbing peaks were collected, lyophilized, and assayed for cell proliferation activity.

Primary structure analysis Purified peptides were subjected to amino acid sequencing by automated Edman degradation analysis on a pulsed liquid‐phase Shimadzu protein sequencer (PPSQ‐31A; Shimadzu, Kyoto, Japan) according to the manufacturer's instructions. Purified peptide (0.5 μl) in 0.1% (v/v) trifluoroacetic acid/water was spotted onto a matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) plate with 0.5 μl α‐cyano‐4‐hydroxycinnamic acid matrix (10 mg/ml in 60% acetonitrile) and analyzed by an UltraFlex I mass spectrometer (Bruker Daltonics, Billerica, MA, USA) in positive ion mode.

Construction and screening of a cDNA library Total RNA was extracted from the skin of T. verrucosus using the RNeasy Protect Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The Smart cDNA Library Construction Kit (Clontech, Mountain View, CA, USA) was used to synthesize cDNA. The synthesized cDNA was used as template for PCR to screen the cDNAs encoding the peptide (tylotoin). Two pairs of oligonucleotide primers [S1: 5'‐AC(A/C/G/T)C(G/T)(C/T)TT(A/G)TT(A/G)‐TT(C/T)TG(A/C/G/T)C‐3', according to the sequence determined by Edman degradation, in the antisense direction, and primer II A: 5'‐AAGCAGTGGTATCAACGCAGAGT‐3; S2: 5'‐ATGGAGCTATGCCTCATACTCAC‐3' and primer II A] were used in PCR reactions. The PCR conditions were 2 min at 95°C and 30 cycles of 10 s at 92°C, 30 s at 50°C, and 40 s at 72°C, followed by 10 min extension at 72°C. The PCR products were cloned into pGEM‐T Easy vector (Promega, Madison, WI, USA). DNA sequencing was performed on an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA).

Synthetic peptide Tylotoin (KCVRQNNKRVCK) was synthesized by GL Biochem Ltd. (Shanghai, China) and analyzed by HPLC and mass spectrometry to confirm its purity >98%.

Cell proliferation assay The proliferation of immortalized human HaCaT keratinocyte cells, human skin fibroblasts (HSFs), and human umbilical vein endothelial cells (HUVECs) was measured using a colorimetric assay. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/ml)‐streptomycin (100 mg/ml) at 37°C in a humidified 5% CO 2 atmosphere. HaCaT cells, HSFs, and HUVECs (2×104 cells/well, 180 μl) were plated into 96‐well plates. After adhering to the plate, cells were incubated with vehicle (DMEM) or tylotoin at different concentrations (2, 5, 10, 20 μg/ml) for 24 h. Then 20 μl of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) solution (R&D Systems Inc. Minneapolis, MN, USA) was added to each well for a further 4 h incubation at 37°C. After the cells were washed 3 times with PBS (pH 7.4), the insoluble formazan product was dissolved by incubation with 150 μl DMSO. The absorbance of each well was measured on an enzyme‐linked immunosorbent assay (ELISA) microplate reader at 570 nm. The optical density reflects the level of cell metabolic activity. Each experiment was performed in quintuplicate.

Regulation of cytokine production. Raw 264.7 murine macrophage cells (1×106) were seeded and adhered to a 96‐well culture plate. The cells were treated with tylotoin (2, 5, 10, and 20 μg/ml) or vehicle for 16 h, then supernatants were collected for EGF, IL‐1, IL‐6, TGF‐β1, and tumor necrosis factor α (TNF‐α) analysis using ELISA kits (Dakawe, Beijing, China). ELISA was performed according to the manufacturer's instructions.

Endothelial cell tube formation assay HUVECs were cultured in M200 medium (Invitrogen) supplemented with 20% FBS and 1× low serum growth supplement (LSGS; Invitrogen) at 37°C in a humidified 5% CO 2 atmosphere. Matrigel matrix (50 μl) was added to each well of a 96‐well plate and incubated at 37°C for 30 min to allow for gel formation. After detaching by trypsinization, HUVECs were washed and resuspended in serum‐free medium containing 1× LSGS. The cells (1×106) were seeded into the 96‐well plate pretreated with Matrigel matrix. Cells were incubated with vehicle (M200), tylotoin, or stylotoin at 20 μg/ml concentration for 18 h. Tubes were photographed using a microscope (Olympus, Tokyo, Japan) and quantified using the OpenLab program (Improvision, Coventry, UK).

Western blot analysis Raw 264.7 murine macrophage cells (1×106/well) were plated into a 6‐well culture plate and transferred to serum‐free DMEM for a 16 h incubation. After incubation with tylotoin (2, 5, 10, and 20 μg/ml) or vehicle, 1 h incubation for mitogen‐activated protein kinase (MAPK) pathway and 18 h incubation for Smad pathway, the cells were collected by centrifugation (1000 g for 5 min) and washed twice with ice‐cold PBS. The washed cell pellets were lysed in 250 μl RIPA lysis buffer (50 mM Tris‐HCl, pH 7.4; 1% Nonidet P‐40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM phenylmethylsulfonyl fluoride; 1 μg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM sodium orthovanadate; and 1 mM NaF) and incubated for 30 min on ice. The concentration of protein was determined by the Bradford protein assay. Next, 30 μg of cellular proteins was separated on a 12% SDS‐PAGE gel and electroblotted onto a polyvinylidene difluoride membrane. Primary antibodies against β‐actin (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and extracellular regulated protein kinase 1/2 (Erk1/2), SAPK/c‐Jun NH2‐terminal kinase (JNK), p38 MAPK, Smad2, Smad3, and Smad7 (1:2000; Cell Signaling Technology, Beverly, MA, USA) were used in Western blot analysis.

Wound healing scratched assay HaCaT cells (1×106) were seeded into a 6‐well plate and grown to monolayer confluency. After 24 h of serum starvation (DMEM supplemented with 1% FBS), the cell monolayer was subjected to a mechanical scratch wound using a sterile pipette tip. After washing twice with PBS to remove floating cells, cells were then cultured for additional periods (from 0 to 48 h) in a serum‐free basal medium in the continued presence of vehicle, tylotoin, or stylotoin (20 μg/ml). Images of the wounded cell monolayers were obtained using a microscope (Olympus) at 0, 24, and 48 h after scratch wounding. Cell migration activity was expressed as the percentage of the gap relative to the total area of the cell‐free region immediately after scratch wounding, named the repair rate of scarification, using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA). For each plate, 6 randomly selected images were acquired. All experiments were carried out in quintuplicate.

Full‐thickness wounds and quantification of healing Male Kunming mice (age 6‐8 wk) were anesthetized using 1% pentobarbital sodium (0.1 ml/20 g body weight). Dorsal hairs were removed by an electric clipper, and the dorsal skin was cleansed with Betadine. Two full‐thickness wounds were created in the skin on the back of each mouse using a 9‐mm‐diameter biopsy punch. After wounding, mice were caged individually until termination of the experiment. Mice were treated with vehicle, tylotoin (20 μl, 20 μg/ml) or EGF (20 μl, 100 μg/ml) applied directly to the wound site twice daily. Wound healing was macroscopically monitored by taking digital photographs at the indicated time points. The wound areas were calculated from the photographs using PhotoShop (Adobe Photoshop Element 2.0; Adobe Systems, San Jose, CA, USA; n=10/group). The experimental protocols were approved by the Animal Care and Use Committee at Kunming Institute of Zoology, Chinese Academy of Sciences.

Histology and immunohistochemistry The biopsy specimens involving the central part of the wounds (different days after wounding) were obtained from mice for light microscopy. Skin specimens were fixed in 10% formalin, dehydrated through a graded series of ethanol, cleared in xylene, and embedded in paraffin wax. Thick sections (5 μM) were prepared and stained with hematoxylin and eosin (H&E) for histological analysis. IPLab imaging software (BD Biosciences, Bedford, MA, USA) was used to measure changes in the wound. Width of the wound and distance of the neoepithelium were measured on H&E‐stained sections, and percentage reepithelialization was calculated as (distance covered by neoepithelium)/(distance between wound bed) × 100 (n=6/group). All slices were used to evaluate epidermal regeneration and granulation, by using a semiquantitative score system (9). In this system, a 3‐point scale was used to evaluate dermal and epidermal regeneration (1, little regeneration; 2, moderate regeneration; 3, complete regeneration) and 4‐point scales were used to evaluate granulation tissue formation (1, thin granulation layer; 2, moderate granulation layer; 3, thick granulation layer; 4, very thick granulation layer). For immunohistochemistry, 3 μM sections were reacted with anti‐F4/80 primary antibody (1:200 dilution, ab111101; Abcam, Cambridge, MA, USA), anti‐TGF‐β1 primary antibody (1:500, ab92486; Abcam), and anti‐α SMA primary antibody (1:1000 dilution, ab32575; Abcam) after blocking endogenous peroxidase and nonspecific binding. Then, they were incubated with biotinylated goat anti‐rabbit IgG (1:200 dilution, ab150077; Abcam) for 1 h at room temperature. As a control, sections were treated with the same dilution buffer without the primary antibodies. Infiltration of macrophages was evaluated by counting the cells immunostained with anti‐F4/80 antibody (n=6/group).

Skin tissue ELISA The biopsy specimens involving the central part of the wounds (d 0, 1, 2, 3, 4, 6, 8, and 10) were obtained from mice for tissue ELISA. Skin specimens were homogenized in 1 ml 0.1 M PBS/g tissue using a glass homogenizer. The homogenates were transferred to 1.5 ml Eppendorf tubes and centrifuged at 13,000 g for 30 min at 4°C, and the supernatant was stored at ‐80°C until analyzed. TGF‐β1 protein levels were determined using ELISA kits (Dakawe).