Peptide synthesis

AW1 peptide (ATNIPFKVHFRCKAAFC), fluorescein isothiocyanate (FITC)-labeled AW1, and scrambled peptide (ATVHNAAFIPFKFRCKC) were commercially synthesized by Wuhan Bioyeargene Biotechnology Co., Ltd. (Wuhan, China) with a purity greater than 95%. The purity of AW1 was detected by high-performance liquid chromatography (HPLC) according to a previous study [32].

Cell culture

The mouse leukemic monocyte/macrophage cell line (RAW264.7, KCB200603YJ), human immortalized keratinocyte cell line (HaCaT, KCB200442YJ), and human umbilical vein endothelial cells (HUVECs, KCB2010233YJ) were obtained from the Chinese Academy of Sciences Kunming Cell Bank (Yunnan, China). HaCaT were seeded in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12, BI, Israel) supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone, USA) and antibiotics (100 units/mL penicillin and 100 units/mL streptomycin). HUVECs were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM, BI, Israel) with 10% FBS and 1% penicillin–streptomycin. The RAW264.7 cells were cultured in DMEM (BI, Israel) supplemented with inactivated 10% (v/v) FBS (Hyclone, USA) and antibiotics (100 units/mL penicillin and 100 units/mL streptomycin). All cells were cultured at 37 °C under a 5% CO2 atmosphere.

Cell scratch assay

A cell scratch assay was performed according to a previous study [26] to explore the effects of AW1 and scrambled peptide on keratinocyte scratch repair. HaCaT (2 × 105) were seeded in the 24-well plate and grown to confluency. Then, cell scratch was created by a 200-μL pipette tip and treated with vehicle [phosphate-buffered saline (PBS)], positive control [recombinant human basic fibroblast growth factor (rh-bFGF)], AW1 (1, 10, and 100 nM), and scrambled peptide (100 nM) for 24 h. The status of cell scratch was recorded by inverted microscope (Motic, AE2000, China) and calculated by Image J. All data were from three independent experiments performed in triplicate.

Cell viability

The proproliferative activities of different concentrations of AW1 (1, 10, and 100 nM) and scrambled peptide (100 nM) on keratinocytes and macrophages were detected according to previous research [33]. Briefly, HaCaT and RAW264.7 (2.5 × 103) were seeded in the 96-well plates. Then the cells were treated with PBS, AW1 (1, 10, and 100 nM), and scrambled peptide (100 nM) for 24 h, and MTS Cell Proliferation Assay Kit (Promega, USA) was used to detect cell viability. All data were from three independent experiments performed in triplicate.

Cell migration

Transwell assay was performed to explore the effects of AW1 and scrambled peptide against keratinocyte migration following a previous study [34]. Keratinocytes (2 × 105 cells/well) in the serum-free medium were seeded into the upper chamber of 24-well plates (Corning, USA) with 8-μm pore filters and treated with vehicle (PBS), different concentrations of AW1 (1, 10, and 100 nM), and scrambled peptide (100 nM). A total of 500 μL DMEM/F12  with 10% FBS was added to the lower chamber. After incubation for 24 h, the keratinocytes on the upper surface of the filter membranes were removed, and the migrated cells were stained with 0.1% crystal violet for 15 min. The migrated cells were recorded by inverted microscope. Finally, the stained cells were eluted by 33% glacial acetic acid, and absorbance was detected at 570 nm to indirectly reflect the number of migrated cells. Each experiment was performed three times independently in triplicate.

Tube formation assay

Tube formation assay was performed by Matrigel basement membrane matrix (356234, Biosciences, San Jose, USA) according to a previous study [35]. Briefly, after thawing overnight at 4 °C, 50 μL Matrigel was added to 96-well plates and incubated at 37 °C to solidify. HUVECs (2 × 104) were cultured in the 96-well plates and treated with vehicle (PBS) and different concentrations of AW1 (1, 10, and 100 nM) for 6 h. The tube formation was recorded by an inverted microscope and the tube length was randomly measured in five fields by ImageJ software. Each experiment was repeated three times independently.

Experimental animals

Male Kunming mice and male C57BL/6 mice (20–24 g, 6–8 weeks) were purchased from Kunming Medical University (Kunming, China). All mice were fed individually in PVC cages under a 12-h light/12-h dark cycle. Before the experiment, the mice were adaptively cultivated for 1 week.

Full-thickness skin wounds in Kunming mice

The therapeutic effects of AW1 (1, 10, and 100 nM) and scrambled peptide (100 nM) on full-thickness skin wounds in mice were determined according to a previous study [26]. After anesthetizing, the back hair of the mice was removed, and two equally large symmetrical wounds (10 mm) were created on the dorsal of each mouse using a punch. The mice were randomly divided into six groups, with five mice in each group. The wounds were topically treated with vehicle (PBS), recombinant human basic fibroblast growth factor (rh-bFGF, Beijing Bersee Science and Technology Co. Ltd., China, 100 ng/mL), different concentrations of AW1 (1, 10, and 100 nM), or scrambled peptide (100 nM) twice a day (20 μL per administration). Wound status was recorded every other day postsurgery until day 8, and wound area was quantified using ImageJ and analyzed using GraphPad Prism. Skin wound samples were acquired at days 4 and 8 postsurgery. Each experiment was performed three times independently.

H&E staining

To explore the regeneration of skin wounds after treatment, H&E staining was performed as per a previous study [29]. Wound regeneration was recorded using a light microscope at the same magnification (×40). To evaluate the thickness of the neoepidermis and new granulation tissue in skin wounds of mice, five values were randomly measured in one field using ImageJ, and mean values were calculated and then analyzed by GraphPad Prism [3].

Immunohistochemistry

To demonstrate the proliferation of epidermal cells and detect the inflammatory response, immunohistochemistry stain was performed according to a previous study [26]. The sliced tissues were incubated with primary antibodies, including rabbit anti-mouse interleukin-1β (IL-1β, Affinity; AF5103, China; 1:100), interleukin-6 (IL-6, Servicebio; GB11117, China; 1:600), tumor necrosis factor α (TNF-α, Servicebio; GB11188, China; 1:500), and Ki67 (Servicebio; GB11499, China; 1:500) at 4 °C for 12 h. Secondary antibodies (Servicebio; GB23303, China; 1:400) were used for incubation at 37 °C for 1 h, and diaminobenzidine staining (Biosharp, China) was performed. The positive staining of IL-1β, IL-6, TNF-α, and Ki67 was recorded by light microscopy (Primo Star, Zeiss, Germany) and measured by ImageJ software.

Deep second-degree burns in mice

Deep second-degree burns were established in mice following a previous study [33]. Briefly, two deep second-degree burns were created on the dorsal of each mouse. Wounds were topically treated with vehicle (PBS), rh-bFGF (100 ng/mL), or different concentrations of AW1 (1, 10, and 100 nM) twice a day (20 μL per administration). Skin wound condition was documented on days 0, 4, 8, and 14 postoperation. Skin samples from the central area of the wounds were obtained on days 3, 4, 8, and 14 and used for histological analysis. Each experiment was repeated three times independently.

Full-thickness skin wounds in diabetic mice

A type 2 diabetes C57BL/6 mouse model was established according to previous research [36]. Fasting blood glucose levels of mice were detected on days 3, 7, 14, 22, and 30 after the injection of streptozotocin (Solarbio, China). The mice with fasting blood glucose levels higher than 16.7 mmol/L were considered as type 2 diabetic. The therapeutic effects of AW1 (1, 10, and 100 nM) on diabetic mouse full-thickness skin wounds were determined according to a previous study [26]. In short, two symmetrical full-thickness wounds (10 mm) were created on the dorsal of type 2 diabetic mice. The diabetic mice with full-thickness skin wounds were randomly divided into five groups: diabetic control (PBS), rh-bFGF (100 ng/mL), and AW1 groups (1, 10, and 100 nM). Wild-type mice with full-thickness skin wounds were used as normal control (PBS). Wounds were topically treated with PBS, rh-bFGF, or different concentrations of AW1 twice a day. Wound condition was recorded on days 0, 4, 8, 12, and 18. Wound tissues were acquired on postoperative days 3, 8, 12, and 18. Each experiment was repeated three times independently.

Enzyme-linked immunosorbent assay (ELISA)

To explore the effects of AW1 (1, 10, and 100 nM) on inflammatory factor expression in macrophages and chronic wounds, ELISA was performed following previous research [26, 34]. Briefly, macrophages (2 × 106 cells/well) were seeded in 6-well plates and randomly treated with PBS (vehicle), lipopolysaccharide (LPS, 1 μg/mL), or different concentrations of AW1 for 24 h. The supernatants were collected, and the expression levels of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, were detected on the basis of instructions provided with the ELISA kits (NeoBioscience, Shanghai, China). The expression of TNF-α, IL-6, and IL-1β in chronic skin wound tissues (days 3, 8, and 12) were detected by ELISA kits (NeoBioscience, Shanghai, China). Each experiment was performed three times independently.

Immunofluorescence

The wound tissues were blocked using 5% (w/w) goat serum containing 0.3% TritonX-100 for 1 h, then incubated with primary antibodies, including rat anti-mouse F4/80 (Abcam, ab6640, USA; 1:100), inducible nitric oxide synthase (iNOS, Abcam, ab178945, USA; 1:300), arginase (ARG)-1 (Affinity, DF6657, China; 1:200), CD31 (Affinity AF0077, China; 1:200), vascular endothelial growth factor (VEGF, Abcam, ab233693, USA; 1:500), and α-smooth muscle actin (α-SMA, Cell Signaling Technology, no. 19245, USA; 1:200), at 4 °C for 12 h, respectively. The wound tissues were then incubated with secondary antibodies, including goat anti-rabbit IgG Alexa Fluor® 488 (Abcam, ab150077, USA; 1:200) and goat anti-rat IgG Alexa Fluor® 647 (Abcam, ab150159, USA; 1:200), for 1 h at 37 °C, then stained with 4′,6-diamidino-2-phenylindole (DAPI). A confocal laser scanning microscope (Zeiss LSM800, Germany) was used for recording, with quantification of fluorescence intensity and statistical analysis performed using ImageJ and GraphPad Prism.

Molecular docking

Molecular docking between AW1 and TLR4 was performed according to a previous study [37].

Colocalization of TLR4 and AW1

Macrophages (2 × 103 cells/well) were seeded in 12-well plates containing circular microscope cover glass (14 mm, Nest). After attachment, macrophages were treated with FITC-labeled AW1 (100 nM) for 1 h, and fluorescent staining was performed according to previous study [38]. Briefly, macrophages were incubated with TLR4 primary antibody (Affinity, China, 1:300) for 12 h at 4 °C, then incubated with secondary antibody (goat anti-rabbit IgG Fluor 594-conjugated, Affinity, China, 1:300) for 1 h at 37 °C. After DAPI staining and sealing, a confocal laser scanning microscope (Zeiss LSM800, Germany) was used for observation.

Western blotting

Mouse macrophages (2 × 106) were seeded in 6-well plates and treated with PBS, AW1 (1, 10, and 100 nM), LPS (1 μg/mL), or a TLR4 inhibitor (1 μg/mL) for 24 h. Macrophages and mouse skin tissue with deep second-degree burns (days 3 and 8) were lysed using cell lysate (RIPA:PMSF:phosphatase inhibitor of 100:1:1). The protein supernatants were collected after centrifugation (14,000g, 20 min, 4 °C) and quantified using the Bradford method (BCA protein analysis kit, Meilun, Dalian, China). Western blotting was performed to detect the effects of AW1 on the nuclear factor‐κB (NF-κB) signaling pathway and TLR4/NF-κB molecular axis according to previous studies [3, 39]. Primary antibodies, including GAPDH, Iκb, P-iκb, P65, and P-P65 (Affinity; China, 1:1 000), were used for western blotting according to the provided instructions. Each experiment was performed five times independently.