Establishment of mice diabetic wound model
The animal studies were approved the Animal Experiment Ethics Committee of Nantong University. The healthy male C57/BL6 mice weighing approximately 26 ± 2 g at 8–10 weeks of age were provided by the Laboratory Animal Center of Nantong University. Mice were adaptively fed for 7 days and allowed to free access for water and food at room temperature of 24.0 ± 2.0 ℃. Subsequently, diabetic models were established by intraperitoneal injection of 1% streptozotocin (STZ, pH4.2-4.5 in citrate and sodium citrate buffer solution, 60 mg/kg) for five consecutive days following mice abrosia for 16 hours. Blood glucose value of the mice was determined for three consecutive days to evaluate the success of the diabetes model, which was higher than 16.7 mmol/L.
The mice were anesthetized by the intraperitoneal injection of 3% sodium pentobarbital (30 mg/kg). Then, the fur around the surgical site on the back was shaved and the skin was disinfected with iodophor., A perforator was used to form two symmetrical round wounds with a diameter of 8 mm on the back, and each mouse was placed in a separate cage. A total of 18 mice were randomly divided into two groups, with one group was treated with 0.2 mg/ml COS and the other with sodium chloride as the control. The drug was administered once a week by subcutaneous injection, and the samples were collected at 7, 14 and 21 days post injury. All the mice were euthanized by cervical dislocation to prevent suffering.
Assessment of wound size
In order to determine the size of the wounds, the wounds at different times following COS treatment were photographed with the reference of a scale ruler. Then, Adobe Illustrator software was used to outline the boundary of the wounds, and the percentage of wound healing was calculated. according to following formula:
Wound closure rate = (initial wound area in cm2- wound area at stage in cm2) / (initial wound area in cm2) × 100%
Tissue immunofluorescence
The tissues around the back wounds were collected at different time stages, fixed and embedded in paraffin. Sections were cut into 5 µm thick, deparaffinated and rehydrated according to the standard procedure. Thereafter, they were blocked with 0.01 M PBS containing 3% BSA, 0.1% Triton X-100 and 10% normal goat serum for 1 h at 37 ℃, and incubated overnight at 4 ℃ with primary antibodies: rabbit anti-collagen III antibody (Servicebio), rabbit anti-α-SMA antibody (Servicebio), rabbit anti-CD45 antibody (Servicebio), or rabbit anti-CD31 antibody (Servicebio). After incubation with the second antibody at room temperature for 2 hours, the sections were stained with Meyer’s hematoxylin and 3,3-diaminobenzidine (DAB; Sigma, St. Louis, Missouri, United States) for 1–2 min (Sorabio, Beijing, P.R.C.) and blocked with neutral gum (Invitrogen, San Diego, CA, United States). A microscope (Leica DMR 3000, Leica Microsystem, Bensheim, Germany) was used to observe positive signals.
Cell culture and treatment
Fibroblast NIH/3T3 cell line was purchased from Shanghai Fuheng Biotechnology Co., Ltd. Cells were cultured at 37°C, 5% CO2, and 20% O2 with DMEM (dulbecco's modified eagle medium) containing 10% calf serum (CS) and 1% (Penicillin/Streptomycin) P/S. Fibroblasts were treated with 0–2 mg/ml COS for 24 or 48 hours with or without presence of 10mM LY294002 (inhibitor of PI3K/AKT).
Cell Counting Kit-8 assay
Cell viability was evaluated by Cell Counting Kit-8 assay according to the protocol. Fibroblasts were seed in 96-well plates for 24 hours (200 µL, 1 × 104 cells / well). Then, they were treated with COS for 24 hours. A total of 10 µL of CCK8 were added into each well and incubated the cells for 2 hours. Finally, the absorbance at 450 nm was measured on the micro-plate reader (ThermoFisher, USA). The experiment was repeated for three times.
Cell proliferation assay
Fibroblasts were cultured in 96-well plate. After 24 hours, cells were treated with COS for 36 hours, then they were subjected to determination of cell proliferation by using Cell-Light™ EdU Apollo567 In Vitro Kit (Ribobio). Based on the manufacturer’s protocol, EdU solution was added into the cell supernatant and incubated for 2 hours. Subsequently, the cells were fixed and washed, and the nuclei were stained. The images were photographed by the fluorescence microscope. All experiments have three replicates.
Cell cycle analysis
Fibroblasts were seeded in 6-well plates (3 mL/well, 1 × 106 cells/mL). Then, they were treated with COS at the concentration of 0, 0.5 mg/ml and 1 mg/ml, respectively. The cells were resuspended and collected in 50 µl PBS, followed by a fixation with ice-cold 75% alcohol and reservation overnight at -20℃. After removing alcohol, the cells were stained with a propidium iodide (PI) solution (1 mg/ ml RNase A and 0.05 mg/ml PI; Sigma) for 30 min. Cell cycle was determined by a flow cytometer (Attune NxT, Invitrogen),and analyzed by AttuneTM NxT software (v2.7.0).
Wound healing assay
To determine the effect of COS on fibroblast migration,, the fibroblasts (3 mL/well, 1 × 106 cells/ mL) were seeded into 6-well plates and incubated at 5% CO2 at 37°C. After a confluent monolayer was formed, a sterile pipette tip was used to scratch the cells. The exfoliated cells or cell debris were washed with PBS. The scratch areas were observed and recorded at 0, 24 and 48 hours under the optical microscope. ImageJ software was used to analyze COS-mediated cell migration using the formula below:
Migration area = (initial wound area - wound area at each time point)/(initial wound area) × 100%
Transwell assay
Fibroblasts were suspended in DMEM and transferred to the top chambers of each transwell with 8 µm pores (Corning Incorporated). Each well was added with 300 µL cell medium (4 × 104 cells/well), followed by addition of 700 µL medium containing 10% FBS and COS into the lower chamber to stimulate cell migration. The cells were allowed for migration at 37°C for 48 hours, washed with PBS, and fixed by 4% paraformaldehyde for 30 minutes. Cells adhering to the bottom surface of each membrane were stained with 0.5% crystal violet for 20 minutes, imaged, and counted using a DMR inverted microscope (Leica Microsystems). Assays were performed three times using triplicate wells. The images were analyzed and quantified by ImageJ software.
Western blot assay
Total protein was extracted from cells treated with COS using extraction buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1 mM PMSF. Samples were vortexed for 30 min, followed by a centrifugation at 12000 rpm for 15 min. The supernatants were collected and protein concentrations were determined by the BCA method. Proteins of 20 µg of each sample were heated at 95°C for 5 min, and then separated by 10% SDS-PAGE gel, followed by transferring onto a polyvinylidene difluoride (PVDF) membrane. The membranes were washed with TBST buffer solution (50 mm Tris-HCl, 100 mm NaCl and 0.1% Tween-20, pH 7.6), and blocked with 5% skimmed dry milk for 2 hours. Finally, the membranes were incubated with primary antibody overnight at 4°C. The primary antibodies were used as follows: mouse anti-PNCA antibody (Proteintech); rabbit anti-PI3K antibody (Proteintech); rabbit anti-P-PI3K antibody (Cell Signaling); rabbit anti-AKT antibody (Proteintech) or rabbit anti-P-AKT antibody (Cell Signaling). The membranes were further washed with TBST for three times at room temperature, and then incubated with a secondary antibody for 2 hours. Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA) was used to visualize the protein bands. Imaging system was used to measure the optical density of each band on the membrane. The β-actin was used as an internal control..
Disc Diffusion Assay
The antimicrobial activity of COS against Staphylococcus aureus (provided by Clinical center of the Affiliated Hospital of Nantong University) was examined by Kirby-Bauer disk diffusion susceptibility test. Single colony of bacteria was selected and incubated overnight in Mueller-Hinton broth (MHB) at 250 rpm at 37°C. The bacterial suspension was seed into a Mueller-Hinton Agar (MHA) plates with addition of COS. After incubating of the plate at 37°C for 24 hours, the zone of inhibition (ZOI) around the discs was determined. The formula for calculating the antibacterial rate was refered as follows:
Antibacterial rate = (bacteriostatic circle diameter - initial bacteriostatic circle diameter) / bacteriostatic circle diameter × 100%
Statistical analysis
The results are expressed in mean ± SEM. Statistical analysis was performed using GraphPad Prism version 8.4.3 for Windows (GraphPad Software LLC, La Jolla, CA, USA) by unpaired t-test and one-way ANOVA test. The p value < 0.05 was accepted as statistically significant.