2.1 Establishment of an HGF-overexpressing mouse model
All animal experiments were performed according to the Ethical Principles for Animal Experimentation of Guangdong province and were approved by the Ethics Committee for Animal Research (no. 2016-067) of Guangzhou Medical University. Wild-type C57BL/6 (WT) obtained from the Guangdong Experimental Animal Center (Guangzhou Guangdong) and HGF-overexpressing transgenic C57BL/6 (HGF-Tg) mice were used in this study. HGF is high expressed in oral mucosa of HGF-Tg mice(Fig 1). In total, 29 WT and 29 HGF-Tg mice aged 8–10 weeks (22.8 g±5.2 g, 8–10 weeks) were used in this experiment. Mice were kept under 12-hour/12-hour light/dark cycles, with free access to food and water, in the Central Animal Laboratory of Guangzhou Medical University.
2.2 Oral mucosa traumatic ulcer protocol
Oral mucosa traumatic ulcers were generated using the method described by Cavalcante in 2011 . Briefly, the mice were anesthetized with 0.1–0.2 mlof pentobarbital sodium (0.1%) via intraperitoneal injection, and the oral mucosa was sterilized using 0.12% chlorhexidine gluconate. The ulceration was performed on the left oral mucosa using a number 15 scalpel blade; a 5-mm marker was used for standardization. The protocol was performed by the same investigator (Guanying Chen) for standardization. We choose 5th day as the sacrifice day because the mice lose weight during the 5 first days .
2.3 Blood and tissue sample collection
The mice were weighed on day zero and on the day of sacrifice (the 5th day), Weight loss=(final weight-initial weight). The mice were sacrificed by ether anesthesia and neck dislocation. Blood samples were collected and centrifuged for 10 min at 200 x g, after which the separated serum was collected and stored at -80 °C. The blood cells were collected for flow cytometry analysis. The mucosa containing the ulcers were collected and fixed in 10% formalin. After being embedded in paraffin, the tissue was sectioned into 2-µm slices that were then mounted on slides and stained with hematoxylin and eosin (HE) for inflammation score evaluation (from 0 to 4 according to published evaluation criteria) .Weighing, blood collection and sample collection were performed separately on the 5th day.
2.4 Serum cytokine analysis
Serum cytokines were tested in duplicate alongside a standard with the Mouse Cytokine Array Q5 (RayBio, QAM-CYT-5) according to the manufacturer’s instructions. The fluorescent signal intensity was measured using an InnoScan 300 Microarray Scanner (Parc d'Activités Activestre, 31390 Carbonne, France) at a wavelength of 532 nm and a resolution of 10 µm. All results were analyzed using Q-Analyzer software for QAM-cyt-5. Healthy wild-type mice (n=3), WT mice with ulcers (n=5) and HGF-Tg mice with ulcers (n=8) were included in this analysis.
2.5 Flow cytometry analysis
Immune cells from WT (n=29) and HGF-Tg mice (n=29) blood were isolated after red blood cell lysis. Cells were washed and resuspended in PBS (1×106/ml), and cells were stained for 30 min at 4 ℃ with the following antibodies: CD45-APC-Cy7 (BD, Lot: 7096638)，CD4-PE (BD, Lot: 7138675)，CD8-FITC (Invitrogen, Lot: 4329219)，CD11b-Percy5.5 (BD, Lot: 7066558)，Ly6G-FITC (BD, Lot: 7052879)，and F4/80-PE-Cy7 (Invitrogen, Lot: 4323732). The cells were sorted by flow cytometry (BD FACSAria III), and the data were analyzed using FlowJo software (BD FACSDiva software).
2.6 Immunohistochemical staining for NF-кB, CD45, and Ly6G
Slides bearing 4-µm tissue slices were deparaffinized and rehydrated and then processed according to a standard immunohistochemical staining manual. Briefly, endogenous peroxidase was blocked with 3% H2O2, and then the samples were incubated for 1 hour at room temperature with primary antibodies against CD45 (Lot: 7096638, BD, USA, dilution 1:100), NF-κB (bs-0465R, Bioss, USA, dilution 1:50) and Ly6G (bs-2576R, Bioss, USA, dilution 1:100), after which they were incubated with HRP secondary antibodies and substrate. The percentages of NF-κB-positive cells in epithelium were scored as follows: 0: 0% positive cells; 1: 1–33% positive cells; 2: 34–66% positive cells; and 3: 67–100% positive cells . The score of Ly6G-positive cells among inflammation was calculated according to Li with a minor change : the average positive cell ratios were photographed and counted in 3–5 fields (400X) and then scored as follows: 0: 0% positive cells; 1: 1–33% positive cells; 2: 34–66% positive cells; and 3: 67–100% positive cells. CD45 was qualified by the average positive cell number that was counted in 3–5 fields (400X).
2.7 Statistical analysis
SPSS 19.0 (IBM, USA) was used to calculate statistical significance between groups. The unpaired t-test and ANOVA were used to compare the differences between groups. Quantitative data are expressed as the mean ± standard error (mean±SE). GraphPad Prism 6 software (La Jolla, CA, USA) was used to plot the comparison of means, and p≤0.05 was considered a statistically significant difference.