Animals
Male Wistar rats (220-260 g, Laboratory Animal Center, Shandong University) were maintained on a routine diet to acclimate for 1 week before the experiment. The rats were assigned to two groups at random: a ligation (L) group and a normal (N) group. Protocols of the study met the approval of the Ethics in the Care and Use of Laboratory Animals Committee of the School of Stomatology of Shandong University.
Rat experimental periodontitis model
Rats in the L group were placed under general anesthesia and underwent an operation to establish the experimental periodontitis model[16]. A 4‐0 silk suture and an orthodontic ligature wire were placed around the cervical region of the right first lower molars and then ligated firmly. After 6 weeks, all rats in the two groups were euthanized with a lethal dose (150 mg/kg) of sodium thiopental. The gingiva and alveolar bone tissues were collected and fixed in 4% paraformaldehyde for 48 hours. Then, the specimens were dehydrated, cleared and finally embedded in paraffin. Serial sections (5‐μm thick) were obtained for hematoxylin-eosin staining (HE) staining and substance P and HIF-1α immunohistochemical staining.
Cell culture and treatment
Ten Wistar rats (80-100 g) were killed by cervical dislocation. Fresh healthy gingiva was separated and washed three times with phosphate buffered saline (PBS) supplemented with 200 IU/ml penicillin and 200 mg/ml streptomycin (Solarbio, Beijing, China). The gingival tissues were minced by ophthalmic scissors and then digested for one hour at 37 °C with a constant temperature shaker in a solution of collagenase type I (3 mg/mL; Solarbio) and dispase (4 mg/mL; Sigma Aldrich, St Louis, USA). After enzymatic digestion, the filtered single-cell suspension was maintained in α-minimal essential medium (α-MEM; HyClone, Logan, USA) containing 20% fetal bovine serum (FBS; Biological Industries, Kibbutz, Israel), 100 IU/ml penicillin and 100 mg/ml streptomycin at 37°C in an incubator with a 95% O2-5% CO2 atmosphere. After reaching confluence, the cells were detached with 0.25% Trypsin-EDTA solution (Solarbio) and subcultured in α-MEM with 10% FBS. The medium was changed every 48 hours. Cells between the fourth and sixth passages were used for subsequent experiments.
Primary mouse bone marrow-derived macrophages (BMMs) were isolated from the femurs and tibias of ten C57/BL6 male mice (3 weeks old) after cervical dislocation and were cultured in complete α-MEM containing 10% FBS and 30 ng/ml macrophage colony stimulating factor (M-CSF) at 37°C in an incubator with a 95% O2-5% CO2 atmosphere. We added 50 ng/ml RANKL for 4 days to induce BMMs to differentiate into osteoclasts. To observe the effect of substance P on osteoclastogenesis, we added 10 nM substance P (Sigma Aldrich) with or without 1 µg/ml LPS (Invivo Gen, San Diego, USA) to the culture medium (RANKL+10 nM SP group and RANKL+50 nM SP group, RANKL only group as control) and then stained for TRAP.
TRAP staining
The cells were fixed with 4% paraformaldehyde and then stained for TRAP using a commercially available kit (Joy Tech Bio. Co., Hangzhou, China). Osteoclasts were identified as TRAP-positive multinucleated cells containing three or more nuclei.
Immunohistochemical staining
After deparaffinization using xylene and hydration in gradient ethanols, the tissue sections were treated with 3 % H2O2 for 10 min at room temperature to inhibit endogenous peroxidase activities and then incubated with primary antibodies against substance P (diluted 1:200, Abcam, Cambridge, UK) and HIF-1α (diluted 1:200, Abcam) overnight at 4°C. The method was the same as our previously described research. After washing with 0.01 M PBS, the sections were incubated with polymer auxiliary agent for 15 min at 37 °C, washed with 0.01 M PBS 5 min × 3 times, and then incubated with Poly-HRP secondary antibody goat anti-mouse/goat anti-rabbit IgG (ZSBio, Beijing, China) for 15 min at 37 °C. After three washes in 0.01 M PBS for 3 min each, the sections were visualized with 3,3-diaminobenzidine tetrahydrochloride (ZSBio) as recommended by the manufacturer. The negative control used 0.01 M PBS instead of antibodies. The sections were examined and photographed with a light microscope (OLYMPUS CX-71, Japan).
Western blot analysis
RIPA lysis buffer (Solarbio) was used to extract the total proteins. The protein concentrations were measured by using a bicinchoninic acid (BCA) assay kit (Solarbio) according to the manufacturer’s instructions. Equal loading quantities of proteins were separated by 10% SDS-PAGE and electroblotted to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA). The membranes were blocked with 5% nonfat milk dissolved in TBST at room temperature for 1 h and then incubated overnight at 4°C with primary antibodies against HIF-1α (diluted 1:500, Abcam), TNF-α (diluted 1:500, Abcam), OPG (diluted 1:500; Bioss, Beijing, China) and RANKL (diluted 1:500; Bioss). After washing with TBST, the membranes were incubated with secondary horseradish peroxidase (HRP)-linked goat-anti rabbit IgG antibody (diluted 1:10000, CWBiotech, Beijing, China) at room temperature for 1 h. The blots were visualized by using an ECL kit (Millipore).
Statistical analysis
All data are expressed as the mean ± SD. Unpaired Student's t-tests were conducted with GraphPad Prism 5 software. The results for multiple group comparisons were analyzed using one-way analysis of variance (ANOVA) followed by a Newman–Keuls post hoc test. A value of P < 0.05 was considered statistically significant.