Streptozocin, Masson Staining Kit were purchased from Solarbio Life Science, Beijing, China; Sterile Soybean Oil for Injection (CAS: 8001-22-7) was purchased from Sheng Rui Medicine, Xi’an, China; Hydroxyproline Assay Kit was purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China; HE Staining Kit, Triton X-100, RIPA was purchased from Beyotime Biotechnology, Shanghai, China; α-SMA primary antibody for immunohistochemistry experiment was purchased from Boster Biological Technology, Wuhan, China; secondary antibody for immunohistochemistry experiment was purchased from ZSGB-Bio, Beijing, China; Human umbilical vein endothelial cells (HUVECs) was purchased from Sciencell Research Laboratories (San Diego, CA, USA). Phospho-ERK1/2, β-actin antibody was purchased from Cell Signaling Technology, Danvers, USA; anti-HRP antibody was purchased from LI-COR Biosciences, USA; MTT was purchased from Sigma-Aldrich, USA; AGEs was purchased from Biovision, San Francisco, USA; Matrigel was purchased from BD Biosciences, Franklin Lakes, USA; Calcein AM, PD173074, ZM306416, AG1296 and PD153035 was purchased from Abcam, Cambridge, UK.
Preparation of drugs
KLX with a purity of 99% was provided by department of pharmaceutical chemistry (College of Pharmacy, Harbin Medical University) and dissolved in Sterile Soybean Oil for Injection at the concentration of 30, 100, 300 μg/mL.
In vivo study
KM mice (male, 18-20 g, SPF level) were purchased from Laboratory Animal Center of Second Affiliated Hospital of Harbin Medical University. Mice were randomly divided into two groups according to their original body weight: normal mice and diabetic mice. Diabetic mice were intraperitoneally injected with streptozocin (180 mg/kg) to establish the animal model of Type 1 Diabetes. After 3 days, the fasting glucose was measured. The mice with fasting glucose higher than 11.1 mmol/L can be used as diabetic mice for subsequent experiments. Diabetic mice were randomly divided into 6 groups according to their fasting glucose level: diabetic wound without treatment (DW), diabetic wound treated with blank oil (DW + Blank oil), diabetic wound treated with KLX oil 30 μg/mL (DW + KLX 30 μg/mL), diabetic wound treated with KLX oil 100 μg/mL (DW + KLX 100 μg/mL), diabetic wound treated with KLX oil 300 μg/mL (DW + KLX 300 μg/mL). All mice were anesthetized with 1% sodium pentobarbital (0.1 mL/10g), the back was shaved, and a skin wound with a diameter of 5 mm was made on the back with a skin punch. The NW and DW group were not treated with any drugs, and the rest groups were treated with 10 μL of the corresponding drugs once a day at the wound site for 14 consecutive days. The wound healing of each mouse was recorded by taking pictures at the same focal length every day. Wound area is measured by Image Pro Plus software and the healing rate was calculated by the following formula:
Angiogenesis on the subsurface of the skin
Mice were sacrificed on the 7th day after administration with corresponding drugs. Skin samples with a diameter of 1 cm around the wound were cut and the angiogenesis of the subsurface of the skin was recorded.
Mice were sacrificed on the 7th day after administration with corresponding drugs. Skin samples with a diameter of 1cm around the wound were cut and fixed with 10% paraformaldehyde for 48 h. Then the tissue was embedded with paraffin and sliced into tissue sections with the thickness of 6 μm. Then tissue sections were processed into hematoxylin-eosin (HE) staining using HE Staining Kit. The nuclei was stained blue, and cytoplasm and connective tissue were stained red. Representative images of each group were taken by Leica microsystems (Solms, Germany).
Mice were sacrificed on the 7th day after administration with corresponding drugs. Skin samples with a diameter of 1cm around the wound were cut and fixed with 10% paraformaldehyde for 48 h. Then the tissue was embedded with paraffin and sliced into tissue sections with the thickness of 6 μm. Then tissue sections were processed into Masson staining using Masson Staining Kit. Collagen fibers were stained blue or green. It is worth noting that the inherent collagen fibers were stained dark blue and the new collagen fibers were stained light blue. Representative images were taken by Leica microsystems (Solms, Germany).
Mice were sacrificed on the 7th day after administration with corresponding drugs. Skin samples with a diameter of 1cm around the wound were cut and fixed with 10% paraformaldehyde for 48 hours. Then the tissue was embedded with paraffin and sliced into tissue sections with the thickness of 6 μm. Then tissue sections were fixed on the glass slide pretreated with gelatin and washed twice in PBS for 5 minutes each. Then the tissue sections were treated with 0.1% triton X-100 penetration solution, washed with PBS for 30 minutes, and blocked at room temperature for 2 hours. Then the sections were incubated with α-SMA primary antibody (1: 1000) at 4 ℃ for 24 hours. After incubation, the sections were washed 6 times in PBS for 5 minutes each. Finally, the sections were incubated with secondary antibody for 2 hours. After incubation, the sections were washed 6 times in PBS for 5 minutes each, and sealed with glycerine sealant. Representative images were taken by Leica microsystems (Solms, Germany).
Determination of hydroxyproline content
Mice were sacrificed on the 3th, 7th, 11th day after administration with corresponding drugs. Skin samples with a diameter of 1cm around the wound were cut and the hydroxyproline content was measured. Determination was performed according to the requirements of the Hydroxyproline Assay Kit. The preparation method of test samples is as follows: skin tissue 10 mg was hydrolyzed with hydrolysate 200 μL for 20 minutes in 95 ℃ metal bath. After cooling with running water, 10 μL of indicator solvent was add to each sample. After shaking well, each sample was mixed with 200 μL pH solution A. Then pH solution B was added dropwise to each sample tube and the sample solution was mixed thoroughly until the liquid becomes yellow green. Then the sample solution was diluted with 2 mL of deionized water. Transfer the diluted sample solution to a new tube and add an appropriate amount of activated carbon. After thorough mixing, the mixture was centrifuged at 3500 rpm for 10 minutes and 200 μL of supernatant was taken as the test sample. Then add the detection reagents 1, 2 and 3 in the assay kit to the sample tubes respectively (deionized water served as a blank Control and 5 μg/mL hydroxyproline solution served as a standard control) and heat in a 60℃ water bath for 15 minutes. After cooling, the mixture was centrifuged at 3500 rpm for 10 minutes and the supernatant was taken and its absorbance at the wavelength of 550 nm was measured. The content of hydroxyproline was calculated according to the following equation:
Determination of cell proliferation
Cell proliferation was determined by MTT assay according to previous method. Cultured HUVECs were seeded into 96-well cell culture plates at a density of 1×104/well and divided into six groups: Control, AGEs, AGEs + DMSO, AGEs + KLX 1 μM, AGEs + KLX 10 μM and AGEs + KLX 100 μM. Cells were pretreated with AGEs 100 μg/mL for 24 hours to establish a diabetic wound cell model and treated with the corresponding drugs for 30 minutes. In another experiment, cells were pretreated with PD173074 (FGFR inhibitor), PD153035 (EGFR inhibitor), ZM306416 (VEGFR inhibitor) and AG1296 (PDGFR inhibitor) for 12 hours to inhibit the activity of above receptors before incubating with KLX. Then the culture medium was discarded and MTT solution was added to each well in the dark. After incubating for 4 hours at 37℃, the MTT-containing medium was discarded and 150 μL of DMSO was added to each well. After shaking for 1 minute, the absorbance of each sample was detected at 490 nm with a microplate spectrophotometer (Bio-Tek, USA).
Determination of cell migration
Cell migration was determined by scratch assay. Cultured HUVEC was seeded into a 6-well cell culture plates at a density of 1×106/well and divided into six groups: Control, AGEs, AGEs + DMSO, AGEs + KLX 0.1 μM, AGEs + KLX 1 μM, AGEs + KLX 10 μM. A cell model of wound was prepared by drawing a straight line with a uniform width using the tip of a sterile pipette at the bottom of the culture plate. Then the cells were washed 3 times with PBS to remove cell debris. Except for the control group, cells in the other group were treated with AGEs 100 μg/mL and corresponding drugs. In another experiment, cells were pretreated with PD173074 (FGFR inhibitor) for 12 hours to inhibit the activity of FGFR before incubating with KLX. Cell migration in each well was recorded by taking photos at 0 hours, 12 hours and 24 hours after administration. The wound healing was calculated by Image-Pro-Plus Software.
Determination of tubular structure formation
The entire experiment should be performed at 4°C. The equipment and reagents required for the experiment should be cooled in advance. Matrigel was placed at 4℃ overnight to dissolve and diluted with FBS free PRIM-1640 medium in proportion of 1:1. Add 100 μL of diluted Matrigel to the center well of a precooled laser confocal dishes, and transfer the dishes to the incubator for 30 minutes to allow Matrigel to solidify. At the same time, HUVECs cell suspension was prepared and mixed with corresponding drugs, and then uniformly seeded into laser confocal cultural dishes. Please note that air bubbles should be avoided throughout the process. Then the dished were incubated at 37°C and 5% CO2 for 4 hours. The culture medium was discarded and the cells were stained with Calcein AM dye for 30 minutes. Then the cells were gently washed 3 times with PBS. The formation of the tubular structure was observed and recorded under Leica microsystems (Solms, Germany). Mesh number, node number and total tube length of the tubular structure was calculated by Image-Pro-Plus software.
Western blot analysis
Western blot analysis was performed according to the previous method . Protein extraction experiments should be performed at 4°C. Cells or tissues were lysed with ice-cold protein lysates (RIPA : phosphatase inhibitor : protease inhibitor = 100:10:1) and the lysed mixture was collected into a tube. The lysed mixture was sonicated and centrifuged at 13500 rpm and 4°C for 15 minutes. The supernatant was collected and the protein concentration was measured. The protein samples were heated in a metal bath at 95°C for 10 minutes. Then the samples were cooled and subjected to gel electrophoresis. Protein samples were added to the gel wells for separation. After complete separation, protein was transferred to an NC membrane and blocked with 5% skim milk for 2 hours to block nonspecific bands. Then the NC membrane was washed with PBST on a shaker for 30 minutes and incubated with phospho-ERK1/2 and β-actin primary antibody (1:1000) at 4 ℃ overnight. Then the NC membrane was washed with PBST on a shaker for 30 minutes and incubated with the anti-HRP antibody (1:10000) for 50 minutes in the dark at room temperature. Then the NC membrane was washed with TBST on a shaker in the dark for 30 minutes. The expression of the target protein was detected by an infrared fluorescence scanning system, and the optical density integral value of the protein bands was analyzed by Odyssey1.3 software.
The discovery Studio 3.5 docking program was adopted here . The preparation of protein structure (PDB code 3POZ) included adding hydrogen atoms, removing water molecules, and assigning Charm forcefield . Goldscore was selected as the score function, and the other parameters were set as default. For each docking study, a total of 10 docking poses were retained. The root-mean square deviation (RMSD) between docking poses were calculated.
Data are expressed as mean ± SEM. Data of each group was performed T-Test or Paired T-Test for statistical analysis. p < 0.05 was considered statistically significant.