Mice and reagents
Male 7–8-week-old BALB/cA mice were purchased from CLEA Japan, Inc. (Tokyo, Japan), maintained under standard conditions in individual cages, and provided with sterile food and water ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of Niigata University (approval no. SA00181). Sivelestat, a NE inhibitor, was purchased from Ono Pharmaceutical Co., Ltd. (Osaka, Japan). It was dissolved in phosphate-buffered saline (PBS) to a concentration of 10 mg/mL.
Construction of recombinant mouse SLPI
Recombinant mouse SLPI was constructed as previously described with some modifications 36. Mouse SLPI DNA (accession number: NP_035544) was synthesized by Eurofins Genomics. The ORF of SLPI was amplified from the synthetic DNA using forward primer 5’-GTCGCATGCCCCTGGACTGTGGAAGGAG-3’ and reverse primer 5’-GTTCTGCAGtcacatcgggggcag-3’. SphI–PstI site of the pQE-30 vector (QIAGEN) in Escherichia coli Rosetta-gami B (DE3) strain (Novagen) was transformed with the plasmid, and transformants were incubated at 30°C for 16 h in the presence of 1 mM isopropyl-β-D-thiogalactopyranoside (FUJIFILM Wako Pure Chemical). Recombinant SLPI expressed in the insoluble fraction was solubilized in a lysis buffer containing 6 M guanidine hydrochloride, 500 mM NaCl, 10 mM imidazole, and 20 mM sodium phosphate (pH 7.8), and recombinant SLPI was purified using Ni-NTA agarose (QIAGEN). Active recombinant SLPI was obtained by dialyzing recombinant SLPI purified under denaturing conditions in a refolding buffer containing 0.5 M urea, 0.4 M L-arginine, 0.5 mM GSSG, 150 mM NaCl, and 50 mM Tris-HCl at 4°C for 16 h and finally replacing the buffer with PBS.
Mouse Tooth Ligated Model
To establish this model, a 5 − 0 silk ligature (Akiyama MEDICAL MFG. Co., Ltd., Tokyo, Japan) was tied around the maxillary second molar to induce periodontitis 37. Thereafter, 50 µg NE inhibitor in 5 µL PBS (ligature + NE inhibitor group), 500 ng recombinant mice SLPI in 5 µL PBS (ligature + SLPI group) or 5 µL PBS was injected into the palatal gingiva of the molar once daily for 3 days for the NE activity assay or 7 days for other analyses. All animal experiments were conducted by a researcher who analyzed the results and was blinded to the actual experiment. The humane endpoint was decided as 20% reduction in body weight from baseline or signs of intense pain.
NE activity assay
NE activity in the palatal gingival tissue of mice has been described previously23. Briefly, three hours after the last injection of mouse SLPI, NE inhibitor, or PBS, the palatal gingival tissues were homogenized in Tris-HCl buffer using a BioMasher (Nippi, Tokyo, Japan). These samples were centrifuged at 300×g for 3 min, and NE activity in the supernatant was determined using N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Merck Millipore, Billerica, MA, USA).
Measurement of periodontal bone loss
Each maxilla was skinned, then the dry skulls were stained with methylene blue (5% in water) for 5 min. Periodontal bone loss in the maxilla was morphometrically assessed using a stereoscopic microscope (Leica Microsystems, Wetzlar, Germany). According to a previously described method 37, the distance from the cement-enamel junction to the alveolar bone crest was measured at six predetermined sites on the ligated second molar and adjacent affected regions. Bone change was calculated by subtracting the sum of the cemento-enamel and alveolar bone crest values from the six corresponding values in the unligated area. Negative values (mm) indicate bone loss relative to baseline (unligated control).
Histologic Analysis
The maxillae prepared from the murine model were fixed in a 4% paraformaldehyde solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 24 h. The specimens were decalcified in decalcifying solution B (Wako Pure Chemical Industries) for 1 week at 4°C. The specimens were then embedded in the O. C. T. compound (Sakura Finetek, Torrance, CA, USA) and frozen in liquid nitrogen. The coronary sections were prepared using a cryostat (Leica Biosystems). The prepared coronal sections (10 µm) were stained with tartrate-resistant acid phosphatase (TRAP) staining (Cosmo Bio Co., Tokyo, Japan).
Immunofluorescence
SLPI expression in frozen coronal sections was assessed by immunofluorescence using an SLPI-specific antibody (Novus Bio, Centennial, USA). After overnight incubation with the primary antibody at 4°C, SLPI was visualized using Alexa Fluor 488 conjugated anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA). Nuclei were identified using Hoechst33342 for 15 min at 37°C. Sections were observed under a confocal laser scanning microscope (Carl Zeiss, Jena, Germany).
Osteoclast differentiation assay using murine bone marrow macrophages
As described previously38, BMMs were collected from the femurs and tibias of mice and age-matched unligated mice were used as controls under sterile conditions. BMMs were cultured in 96-well plates (1.0×105 per well) with recombinant macrophage colony-stimulating factor (M-CSF) (30 ng/mL; R&D Systems, Minneapolis, MN, USA) for 3 h in minimum essential medium alpha (MEMα; Wako Pure Chemical Co., Tokyo, Japan) with 10% fetal bovine serum (FBS) at 37°C in 5% CO2. After removing nonadherent cells, adherent cells were further cultured in MEMα media supplemented with 10% FBS, 100 ng/mL recombinant soluble receptor activator of nuclear factor-kappa B ligated (RANKL; R&D Systems), and 100 ng/mL M-CSF in the presence or absence of recombinant mice SLPI (1 or 10 µg/mL) or NE inhibitor (1 or 10 µg/mL) for 7 days. The medium was replaced with a fresh solution every 3 days during the incubation period. Multinucleation of osteoblasts was confirmed by TRAP staining (to confirm multinucleation). To determine the effect of SLPI on the absorption activity, BMMs were cultured in FACPS/CaP-coated 96-well plates (200 µL/well) (PG Research Co., Ltd., Tokyo, Japan). After 7 days, the supernatant was collected and fluorescence intensity (excitation: 485 nm, emission: 535 nm) was measured using GloMax (Promega Corporation, Madison, WI, USA)24.
Osteoblastogenesis assay
The murine osteoblastic progenitor cell line, MC3T3-E1, was obtained from the RIKEN Bioresource Center (RCB1126). MC3T3-E1 cells were maintained in MEMα media supplemented with 10% FBS and penicillin-streptomycin solution (×100) (Wako Pure Chemical Co.) at 37°C in 5% CO2. To determine the osteogenic differentiation, cells were cultured with 50 µg/mL ascorbic acid (Wako Pure Chemical Co.) and 10 mM β-glycerophosphate (Wako Pure Chemical Co.) in MEMα media supplemented with 10% FBS. The cell culture medium was replaced every three days. ALP activity after 5 days was detected using an ALP staining kit (Cosmo Bio Co., Ltd. Tokyo, Japan) and photographed under a stereoscopic microscope (Leica Microsystems, Wetzlar, Germany) (25×). The ALP-positive area was quantified using ImageJ software version 1.53t (National Institute of Health, Bethesda, MD, USA). Mineralization of bone nodules was detected by staining with Alizarin Red S (Wako Pure Chemical Co.) 24 days after the differentiation of MC3T3-E1 cells. Alizarin Red S-stained calcified deposits were extracted using formic acid and quantified by measuring the optical density at 405 nm using a microplate reader.
Quantitative Real-Time PCR
Total RNA was extracted from mouse maxillary palatal gingiva, BMMs, or MC3T3-E1 cells using TRI reagent (Molecular Research Center, Inc., Cincinnati, OG, USA). RNA was reverse-transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO Co., Ltd., Osaka, Japan). Quantitative PCR with cDNA was performed according to the manufacturer’s protocol using a Step One Plus real-time PCR system (Thermo Fisher Scientific). The data was analyzed using the comparative CT (ΔΔCT) method. TaqMan probes and primers for the expression of housekeeping gene (Gapdh, Mm99999915_g1) along with Il6 (Mm00434228_m1), Il1b (Mm00434228_m1), Nfatc1 (Mm01265944_m1), and Tnfrsf11a (Rank, Mm00437132_m1), Acp5 (Mm00475698_m1), Ctsk (Mm00484039_m1), Runx2 (Mm00501584_m1), Sp7 (Mm00504574_m1), Bglap (Mm03413826_mH) were purchased from Thermo Fisher Scientific.
Statistical Analysis
Data were analyzed by analysis of variance with Dunnett’s or Tukey’s multiple comparison test using GraphPad Prism 8.4.3 (GraphPad Software, Inc., La Jolla, CA, USA).
Data Availability Statement
The authors confirm that the data supporting the findings of this study are available within the article.