Mice
The experiments were performed in 7-week-old specific pathogen free C57BL/6 female mice weighing ~18 g, with intact dentition and no caries or periodontal disease. Mice were obtained from the Animal Center of Jinzhou Medical University. Animals were kept in individual cages with ad libitum access to food and water and fed adaptively for 1 week before establishment of the periodontitis model. The experimental procedures and animal treatment protocols were approved by the Ethics Committee of Jinzhou Medical University.
Mouse periodontitis model
Porphyromonas gingivalis W83 was cultivated on brain heart infusion (BHI) blood agar medium (Solarbio, Beijing, China) (containing 5% sheep’s blood (Solarbio), 1% hemin (Sigma-Aldrich, MO, USA), and 0.1% vitamin K1 (Huayang, Jiangsu, China)) for 5–7 days. Colonies were scraped off the plates and re-suspended in BHI liquid culture medium. P. gingivalis W83 was collected after 24 h. The bacterial density was adjusted to 1 × 109/mL in phosphate buffer containing 2% sodium carboxy methylcellulose (CMC) (Macklin, Shanghai, China).
Fifteen mice were randomly divided into three groups: Sham, P.g+ATRA, and P.g+CMC (n=5 mice/group). Periodontitis model was induced in mice by oral infection with 109 P. gingivalis W83, suspended in 100 μl sterile phosphate-buffered saline (PBS) containing 2% CMC, twice a day for 7 days. Sham mice were administered 100 μl of PBS containing 2% CMC by a micropipette for 7 days. Mice in the P.g+ATRA group were intra-gastrically administered 200 μL of ATRA per mouse dissolved in 1% CMC solution at 0.25 mg/μl every other day from day 0 to day 42, and mice in the P.g+CMC and Sham group were intra-gastrically administered 200 μl of 1%CMC solution per mouse. All mice were killed by asphyxiation with CO2 on day 42 and subjected to subsequent analyses described below.
Bone loss measurement
The right maxilla was dissected out and the soft tissue was removed by soaking in 3% hydrogen peroxide for 6 h, followed by boiling for 5 min to remove the residual soft tissue; the tissues were then stained with 0.1% methylene blue. The distance from the cement enamel junction (CEJ) of the maxillary first molar to the alveolar bone crest (ABC) of the third molar (CEJ-ABC distance) was measured under a stereomicroscope at 32× magnification. The mesial as well as the buccal and palatal molar surfaces were measured in each tooth, and the average values of 12 sites were determined. The total CEJ-ABC distance of the 12 sites in each group was calculated. The bone resorption rate of the experimental group (%) was calculated as = (total CEJ-ABC distance of the experimental group − total CEJ-ABC distance of the Sham group) / total CEJ-ABC distance of the Sham group.
Histological examination
Staining with hematoxylin and eosin (H&E) was performed to visualize the pathological damage of periodontal tissue. The molar segment of the left maxilla was washed with phosphate buffer, fixed with 4% paraformaldehyde for 48 h, and decalcified in 10% neutral ethylenediamine tetra acetic acid (EDTA; Solarbio). After dehydration in an alcohol series and paraffin embedding, the samples were cut into proximal and distal sections (5 μm thick). The sections were then stained with H&E and examined under a dissecting microscope at 100× magnification.
Flow cytometric analysis
Isolation of gingival mononuclear cells
The isolated periodontal tissue was washed with PBS and then added to 1 mL of PBS containing 2% FCS, 2 mg/mL collagenase II (Invitrogen, CA, USA), 1 mg/mL DNase I (Solarbio), and 1 mg/mL hyaluronidase (Sigma-Aldrich) in centrifuge tubes. Next, 20 μL of 0.5 M EDTA was added, and the tubes were incubated horizontally in a water bath preheated at 37°C with shaking at 200 rpm for 60 min. After centrifugation at 400 xg, 4°C for 8 min, the supernatant was discarded and the pellet was passed through a 200 mesh sieve. The filtered cell suspension was then centrifuged at 320 xg, 4°C for 8 min, and the supernatant was discarded. The pellet was resuspended in 200 μL of 2% fetal bovine serum (FBS) in PBS (FBS-PBS) and the cell numbers were counted.
Extraction of peritoneal lavage fluid
Under aseptic conditions, the abdominal cavity of mice was opened and injected with 5 mL of precooled 2% FBS-PBS gently over a period of 2–3 min. After holding for an additional 5 min, the peritoneal lavage fluid was absorbed and lavaged again. The fluid was collected into 15-mL centrifuge tubes and the tubes were centrifuged at 300 × g, 4°C for 8 min. The supernatant was discarded; the pellet was resuspended in 200 μL of 2% FBS-PBS and the cell numbers were counted.
Preparation of single cell suspensions of spleen cells
The spleens of mice were removed aseptically according to routine procedures, rinsed with 1 mL of PBS, ground on a 200-mesh stainless steel screen, and filtered through a 30-μm nylon mesh to remove agglomerated cells. The cell suspension was centrifuged at 300xg, 4°C for 5 min, and then the supernatant was aspirated. Next, 1 mL red blood cell lysate was added to the precipitated cell mass. RPMl1640 medium containing 10% FBS and 1% penicillin-streptomycin was then added to terminate the reaction. After centrifugation at 300 xg, 4°C for 5 min, the supernatant was discarded. The pellet was then washed twice with PBS and the supernatant was discarded. Cell viability was determined by trypan blue staining, and the cells were counted.
To identify macrophages and macrophage subpopulations, cells were stained with FITC-labeled monoclonal anti-mouse F4/80 and APC-labeled monoclonal anti-mouse CD206 antibodies (BioLegend, San Diego, CA, USA). The cells were washed twice with PBS containing 1% FBS, resuspended in staining buffer, and examined by flow cytometry (BD FACSAria II, NY, USA). F4/80+CD206+ cells (M2 macrophages) were analyzed using Flow Jo10.4 software.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
For qRT-PCR, total RNA was extracted from gingival tissues, peritoneal cells, and splenocytes using TRIzol reagent (Vazyme Biotechnology Co. Ltd, Nanjing, China) in accordance with the manufacturer’s protocol. The quality and quantity of RNA were measured using a spectrophotometer. The absorbance was measured at wavelengths of 260 nm and 280 nm and the ratios of the optical densities (ODs) at 260/280 nm for the samples were between 2.0 and 2.2. Total RNA was reverse transcribed into cDNA for amplification using reverse transcription (RT) reaction (PrimeScript RT system; Vazyme Biotechnology Co. Ltd.) according to the manufacturer’s instructions. PCR was performed in an ABIPRISM cycler with the SYBR Green PCR Kit and specific primers for IL-10, Arg-1, TGF-b1, and b-actin mRNAs. PCR was performed in a total volume of 20 μL consisting of 10 μL of 2×ChamQuniversal SYBR qPCR Master Mix, 0.4 μL each of forward and reverse primers, 2 μL of cDNA, and ddH2O.
The primer sequences are as follows:
mouse IL-10, forward, 5′-CCTCGTTTGTACCTCTCTCCG-3′;
mouse IL-10, reverse, 5′-AGGACACCATAGCAAAGGGC-3′;
mouse TGF-b1, forward, 5′-AGCTGCGCTTGCAGAGATTA-3′;
mouse TGF-b1, reverse, 5′-AGCCCTGTATTCCGTCTCCT-3′;
mouse Arg-1, forward, 5′-CAGCACTGAGGAAAGCTGGT-3′;
mouse Arg-1, reverse, 5′-ACAGACCGTGGGTTCTTCAC-3′;
mouse b-actin, forward, 5′-GGCTGTATTCCCCTCCATCG-3′;
mouse b-actin, reverse, 5′-CCAGTTGGTAACAATGCCATGT-3′.
The 2−ΔΔCt method was used to determine the relative expression level of each gene. The target gene mRNA expression was normalized relative to b-actin mRNA, and the adjusted gene expression of mice in the Sham group was used as a reference (fold change = 1).
Statistical analysis
The data are expressed as the mean ± standard deviation (SD) and were analyzed using GraphPad Prism 22.0 software. The results were analyzed by one-way analysis of variance (ANOVA) and Student’s t test. In all analyses, P < 0.05 was taken to indicate statistical significance.