Synthetic peptides, strains, cell lines and animals

Synpeptide Co., Ltd. (Shanghai, China) was commissioned to synthesize peptides hLFT-68 (IAENRADAV), hLFT-309 (GSPSGQKDLLF), and bLGB-111 (LDTDYKKY) by solid-state method to confirm their purity purities >95%.

E. coli K12 and S. aureus were all purchased from the China Center of Industrial Culture Collection (CICC), and preserved in glycerol at − 80 °C. Before the experiment, the two strains were inoculated into liquid Luria-Bertani (LB) broth medium (Sangon Biotech (Shanghai) Co., Ltd., China) and tryptic soy broth (TSB) medium (Shanghai Acmec Biochemical Co., Ltd., China), respectively, and incubated at 37 °C for 12 h to revive, with the process repeated 3 times.

Human skin fibroblasts (HSF), human immortal keratinocytes (HaCaT), and RAW 264.7 cell lines were all purchased from Procell Life Science&Technology Co., Ltd. (Wuhan, China). The cell lines have been checked free of mycoplasma contamination by fluorescent staining. The cell lines were cultured in Dulbecco’s modified eagle medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 units/mL of penicillin, and 100 units/mL of streptomycin (Gibco, Grand Island, NY, USA) in a humidified 5% CO₂ atmosphere at 37 °C, respectively. All experiments used subconfluent cells (about 80% confluence) grown in a DMEM medium.

Eight-week-old, specific pathogen-free adult male C57BL/6 mice were ordered from Liaoning Changsheng Biotech Co., Ltd. (Liaoning, China) and kept at Dalian Medical University Laboratory Animal Center, Liaoning, China. Experiments were approved and then performed in compliance with the guidelines of the Dalian Institute of Chemical Physics Science Ethics Committee (ethics approval number DICPEC2326). All mice were kept under clean conditions on a 12-hour light/12-hour dark cycle at 22 °C with 75% humidity. Mice were provided with sterilized standard mouse food and water.

Antimicrobial activity assay

The antimicrobial activity of the peptides was evaluated against E. coli and S. aureus. The minimal inhibit concentrations (MIC, expressed as mg/mL) of hLFT-68, hLFT-309, and hLGB-111 were determined by the broth microdilution method as described in the literature^26,27. The measurement concentration ranges of hLFT-68, hLFT-309, and hLGB-111 were 102.4, 51.2, 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 mg/mL. Vancomycin (VAN) was used as the positive control. The MICs were determined as the lowest concentrations of peptide that inhibited visible bacterial growth. The absorbance at 600 nm of each well was measured using a microplate reader (BioTek, VT, USA), and the inhibition rate was calculated by the absorbance. The half maximal inhibitory concentration (IC₅₀, expressed as mg/mL) of hLFT-68, hLFT-309, and hLGB-111 were calculated by inhibition rates and Graphpad Prism version 9.5 software (Graph Software, San Diego, CA, USA)²⁸.

Inhibition of lipopolysaccharide (LPS)-induced inflammation in vitro

RAW 264.7 cells were used for constructing an LPS-induced inflammatory cell model. Cells were cultured in 96-well plates at a density of 5 × 10⁴ cells/wall overnight. Till cell adherence, the hLFT-68 and hLFT-309 were added per wall in the treatment group to different final concentrations. After 2 h of treatment, LPS (MedChemExpress, NJ, USA) with a final concentration of 1 µg/mL was added to both the treatment and control groups, and the incubation was continued for 24 h²⁹. The non-LPS group was set up as a control for model validation. Neither LPS nor other treatments were added to the cells in this group. Then the supernatants were collected and went through centrifugation at 12,000 rpm for 30 min at 4°C. Supernatants were stored at − 80 °C until usage. Tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β released in the cell-free supernatants from non-stimulated or stimulated culture macrophage cells were measured with enzyme-linked immunosorbent test (ELISA) kits from R&D Systems (Ruixinbio, Quanzhou, China). The ELISA was performed according to the manufacturer’s instructions.

Cell proliferation assay

HSF and HaCaT cells were used for the cell proliferation assay by Cell Counting Kit-8 (CCK-8, Seven Biotech, Beijing, China). Cells were added to 96-well plates at a density of 1.5 × 10⁴ cells/wall for 6 h. Then, hLFT-68, hLFT-309, and hLGB-111, respectively, were added per wall to achieve different final peptide concentrations for 24 h. Additionally, human epidermal growth factor (EGF, MedChemExpress, NJ, USA) at final concentrations of 10 µg/mL was added as the positive control. After that, 10 µL of CCK8 solution was added per well, and cells were incubated for a further 1–4 hours. Absorbance at 450 nm was then quantified with a microplate reader. According to the formula below, the cell proliferation rate was calculated:

where A₀ represents the zero adjusted hole, A_x reflects the absorbance of cells treated with the MAPs, and A_control represents the absorbance of cells treated with none of the MAPs. The values of half maximal effective concentration (EC₅₀, expressed as µg/mL) of hLFT-68, hLFT-309, and hLGB-111 were calculated by cell proliferation rates and Graphpad Prism version 9.5 software³⁰.

Infected full-thickness wound model

Male C57BL/6 mice were used for constructing an infected full-thickness wound model. Mice were injected intraperitoneally with 1% sodium pentobarbital for anesthesia before surgery. After the removal of the dorsal hair, two symmetrical circular full-thickness skin wounds (0.8 cm in diameter) were created on the depilated back skin. Then 50 µL of S. aureus at a concentration of 10⁷ colony-forming units (CFU)/mL was inoculated into each wound³¹. At the end of each experiment, mice were sacrificed by CO₂ inhalation, which was confirmed by ceasing breath and heartbeat. This model method was used in 2.6. & 2.7. animal experiments.

In vivo study of antimicrobial efficacy

After modeling, mice were randomly assigned to 4 groups (10 mice/group): control, hLFT-68, hLFT-309, and VAN (positive control). The following interventions were performed on both dorsal wounds of mice twice a day: treated with 50µL phosphate buffered solution (PBS, Gibco, Grand Island, NY, USA), 50µL 0.5 mg/mL hLFT-68 group, 50µL 0.5 mg/mL hLFT-309 group, and 50µL 0.15 mg/mL VAN (Bioseth, Zhenjiang, China) as the positive control (N = 3) in different groups (PBS as solvent). The intervention was maintained for 4 days. On the third day following surgery, secretions were collected from the wound site of each group. This was achieved by harvesting and grinding 0.5 g of skin tissue from the wound area. The tissue was harvested using a skin biopsy punch (0.8 cm in diameter), and the excess tissue surrounding the wound was removed until the remaining tissue weighed 0.5 g. The collected secretions were diluted with 5 mL of PBS³². 100 µL of the secretion solution was added to the surface of the TSB solid medium for uniform coating and incubated at 37 °C. Photographs of the colonies were taken after 18 h, and the colonies were counted by the Image-Pro Plus 6.0 program.

Quantification of in vivo wound healing

The infected full-thickness wound models shown previously were constructed and group interventions were the same except that the EGF group treated 50 µL of 5000 IU/mL rhEGF (recombinant Human EGF, Kanghesu, Shanghai Haohai Biological Technology Co., Ltd., China) as the positive control (N = 10). The intervention was maintained for 12 days. The wound area was photographed with a digital camera. The wound repair rate was calculated from the photographs using an image analysis program (Image J, National Institutes of Health; Bethesda, MD, USA) by tracing the wound margin and calculating the pixel area. The measurements were performed in triplicate, and the mean values of consecutive tracings were computed and expressed as a percentage of closure from the original wound. According to the formula below, the wound repair rate was calculated:

where S_n and S₁ are, respectively, the wound area in mice on days n and 1.

At the same time, the inflammation score was recorded every two days. Inflammation score modified and simplified from the Bates-Jensen Wound Assessment Tool³³: (1) Wound edge: 1 = blurred, unable to distinguish the wound edge; 2 = able to clearly distinguish the wound edge; 3 = clearly distinguishable contour, with the base of the wound lower than the wound edge. (2) Exudate type: 1 = no exudate; 2 = thin light red or pink color exudate; 3 = red or pink color exudate. (3) Exudate volume: 1 = no exudate, wound tissue dry; 2 = wound tissue slightly moist, soaking 0–25% of the dressing; 3 = wound tissue moist, soaking more than 25% of the dressing. (4) Wound color: 1 = normal or pink; 2 = light red; 3 = dark red or purple.

Histological analysis, immunohistochemistry (IHC), and ELISA

Starting on day 0 after surgery, the entire layer of tissue on both sides of the mice’s backs was clipped 0.5 cm around the center of the wound at 4-day intervals, which contains the wound and its surrounding normal skin tissue. Skin tissue samples were harvested and fixed with a 4% paraformaldehyde solution (Servicebio, Wuhan, China) for the preparation of histological slides (N = 3). The slides were subjected to Masson’s trichrome staining, as well as Sirius red staining. In addition, the wound tissue slides were also stained by IHC of CD31. All stains and antibodies were purchased from the Servicebio company. Images of stained samples were captured with an optical microscope (Leica, Wetzlar, Germany) or a polarized light microscopy (Leica, Wetzlar, Germany). Collagen volume fraction, collagen types I and III ratio, and mean integrated optical density (IOD) of CD31 were analyzed and calculated from image information of Masson’s trichrome staining, sirius red staining, and IHC, respectively, by the Image-Pro Plus 6.0 program (Media Cybernetics, MD, USA).

The biopsy specimens involving the central part of the wounds on days 4, 8, and 12 were obtained from mice for tissue ELISA. RIPA lysis buffer (Solarbio, Beijing, China) was prepared and supplemented with 1% PMSF (TargetMol, MA, USA) and 1% phosphatase inhibitors (TargetMol, MA, USA). Using a glass homogenizer, skin specimens were homogenized at a ratio of 0.1 g of ground tissue per 1 mL of lysis buffer. The homogenates were transferred to 1.5 mL tubes and centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was stored at 80 °C until analyzed. Prior to testing, the supernatant protein concentration was quantified by a bicinchoninic acid (BCA) assay, using BCA protein assay kits (Seven Biotech, Beijing, China). The supernatant protein concentration was then normalized to a standard value. TNF-α, IL-6, and IL-1β protein levels were determined using ELISA kits (Ruixinbio, Quanzhou, China).

Statistical analysis

At least three independent experiments were performed. Results are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed by Student’s t-test and Mann-Whitney U test using IBM SPSS Statistics 25 (IBM SPSS, Armonk, NY, USA). P < 0.05 was considered statistically significant. Results are shown as mean ± SEM. Graphs were generated using GraphPad Prism version 9.5.