α-modified minimal essential medium (α-MEM) was purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA) and fetal bovine serum (FBS) from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). Recombinant mouse receptor activator of nuclear factor kappa-B ligand (RANKL) was purchased from PeproTech EC Ltd. (London, UK). Anti-IL-23 antibody (cat. no. wl01655) was purchased from Wanlei Bio (Shenyang, China). The anti-β-actin antibody (cat. no. A1978), anti-GAPDH (cat. no. G9545) antibody, NF-κB inhibitor (BAY11-7082) and an inhibitor of phosphoinositide 3-kinase (LY294002) were purchased from Sigma-Aldrich; Merck Millipore. Anti-NF-κB p65 (C-20; cat. no. sc-372) and the anti-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1; 7A6) antibody (cat. no. sc7294) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against inhibitor of κB α (IκBα; cat. no. 9242s), phospho-IκB (cat. no. 9240s), and c-Fos (cat. no. 4384s) were obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA). Other materials used were of the highest grade commercially available.

A total of 22 adult patients with a diagnosis of apical periodontitis and indication for tooth extraction and 22 periodontal healthy subjects requiring tooth extraction for orthodontic reasons were recruited from the Surgery Clinic, School of Stomatology, China Medical University (Shenyang, China). Exclusion criteria included a history of systemic disorders, including diabetes and osteoporosis, and patients who had received antibiotic, anti-inflammatory or hormonal drugs within 3 months prior to the present study. Ethical approval was received from the ethical committee of China Medical University and written informed consent was provided by all participants. Samples of apical lesions and healthy PDLs were stored at −80°C for RNA extraction or fixed in 10% buffered formalin for immunohistochemical analysis.

Total RNA was extracted using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNA synthesis from 1,000 ng of RNA was performed using a reverse transcription kit (Takara Bio, Inc., Otsu, Japan). qPCR analysis was performed using a 7300 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Premix Ex Taq™ (Takara Bio, Inc.). The qPCR thermocycling parameters were as follows: 95°C for 10 min and 40 cycles of 95°C for 15 sec and 60°C for 1 min. RT-qPCR analysis of each gene was performed in triplicate for at least 3 independent experiments. The sequences of the primers were as follows: Forward, 5′-CCGCTTCAAAATCCTTCGCA-3′ and reverse, 5′-TGCTGCCTTTAGGGACTCAG-3′ for human IL-23; forward, 5′-GGCACCCAGCACAATGAAG-3′ and reverse, 5′-GCCGATCCACACGGAGTACT-3′ for human β-actin; and forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′ for human GAPDH. Target gene expression levels were calculated using the 2^−∆∆Cq method (23). β-actin was used as control for the analysis of histological sections, whereas GAPDH was used as a control for cytology analyses.

Biopsies of apical lesions and healthy apical PDLs were fixed in 10% buffered formalin and embedded in paraffin. Sections (6-µm thick) were deparaffinized, dehydrated and blocked with normal horse serum, followed by incubation with an rabbit anti-human polyclonal antibody against IL-23 (dilution, 1:200) overnight at 4°C. Following washing in phosphate-buffered saline (PBS), the sections were incubated with a secondary biotinylated anti-rabbit IgG antibody (cat. no. KIT9710; dilution, 1:1,000) and avidin-biotin-peroxidase complex (Fuzhou Maixin Biotech Co., Ltd., Fuzhou, China). Subsequently, the reaction was developed using a 3,3′-diaminobenzidine kit (Fuzhou Maixin Biotech Co., Ltd.).

P. endodontalis (ATCC^® 35406™) was obtained from the Central Laboratory of Capital Medical University (Beijing, China) and cultured anaerobically at 37°C. Bacteria were collected by centrifugation at 1,000 × g for 15 min at 4°C. LPS was extracted using the hot phenol-water method as previously described (8). The bioactivity of purified P. endodontalis LPS was measured using the Limulus Amoebocyte Lysate Endotoxin assay kit (GenScript USA Inc., Piscataway, NJ, USA). A concentration of 10 µg/ml P. endodontalis LPS was selected for use in the present study, based on our previous study (8).

RAW264.7 murine monocyte/macrophage cells were obtained from RIKEN BioResource Center (Tsukuba, Japan). RAW264.7 cells and SH-9 human PDL cells (24) were cultured in α-MEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO₂. SH-9 human PDL cells were incubated with P. endodontalis LPS (10 µg/ml) for 0, 12, 24, and 36 h. SH-9 human PDL cells were pre-incubated with 10 µM NF-κB inhibitor (BAY11-7082) and 50 µM phosphoinositide 3-kinase (LY294002) for 30 min prior to the addition of P. endodontalis LPS to the culture media. The conditioned medium from SH-9 cells was obtained by centrifugation at 10,000 × g for 10 min at 4°C to remove cell debris, and filtered through a 0.45 mm pore membrane filter (Advantec Toyo Kaisha, Ltd., Tokyo, Japan). Media was stored at −80°C until use. For induction of osteoclastogenesis, RAW264.7 cells were cultured in in α-MEM (20% FBS) with the same volume of conditioned medium from SH-9 cells, in the presence of 10 ng/ml RANKL.

The NF-κB luciferase reporter vector (cat. no. 219078) was obtained from Agilent Technologies, Inc. (Santa Clara, CA, USA). Cells were transfected with NF-κB reporter vector using Lipofectamine^® LTX reagent (Invitrogen; Thermo Fisher Scientific, Inc.). GL3-basic vector (Promega Corporation, Madison, WI, USA) served as a negative control. Cells were treated with P. endodontalis LPS for 1 h. The efficiency of transfection was standardized by co-transfection with pTK-Renilla (Promega Corporation). Total cell lysates were prepared using the Dual-Glo^® Luciferase assay system (Promega Corporation) and subsequently assessed for luciferase activity.

Cells were washed twice with PBS and scraped into lysate buffer [1 mM dithiothreitol, 1 mM phenylmethylsulfonyl, 1 µg/ml leupeptin, 2 µg/ml aprotinin and 5 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. Proteins (15 µg) were loaded onto 10% SDS-PAGE gels, electrophoresed and transferred to polyvinylidene difluoride membranes (Immobilon-P; Merck Millipore). The membranes were blocked with PBS-Tween containing 5% non-fat skim milk for 2 h. The membranes were subsequently incubated with rabbit anti-human monoclonal antibodies against IκB, p-IκB, c-Fos, NFATc1, β-actin and GAPDH (dilution, 1:1,000), followed by incubation with the secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody (cat. no. 7074; dilution, 1:5,000; Cell Signaling Technology, Inc.). Proteins were visualized with an Enhanced Chemiluminescence detection kit (GE Healthcare Life Sciences, Uppsala, Sweden) according to the manufacturer's protocol.

Cells were fixed with 10% buffered formalin for 10 min, permeabilized with methanol and blocked with 4% bovine serum albumin (BSA; Wako Pure Chemical Industries, Ltd., Osaka, Japan). The cells were subsequently incubated with a rabbit anti-human monoclonal antibody against NF-κB (dilution, 1:500) at 4°C overnight. Following incubation with anti-rabbit Alexa Fluor 488-conjugated goat IgG antibody (cat. no. A24922; diluted 1:500 in 4% BSA; Invitrogen; Thermo Fisher Scientific, Inc.) for 40 min and 10 µg/ml Hoechst 33342 for 20 min for nuclear staining, cells were mounted with fluorescent mounting medium (Dako North America, Inc., Carpinteria, CA, USA).

For transient silencing of IL-23, SH-9 cells were transfected with siRNA targeting IL-23 (siIL-23; Invitrogen; Thermo Fisher Scientific, Inc.) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Non-specific siRNA (siCont; catalog no. 12935113; Invitrogen; Thermo Fisher Scientific, Inc.) served as a negative control. The target site of siIL-23 was: 5′-AATCTGCTGAGTCTCCCAGTGGTGA-3′.

RAW264.7 cells cultured with conditioned medium from P. endodontalis LPS-treated SH-9 cells were fixed in acetone-citrate-formaldehyde for 15 min at room temperature. TRAP staining was performed with 0.01% naphthol AS-MX phosphate (Sigma-Aldrich; Merck Millipore) and 0.005% fast red violet LB salt (Sigma-Aldrich; Merck Millipore) in the presence of 50 mM sodium tartrate and 90 mM sodium acetate (pH 5.0) for 15 min at 37°C and rinsed twice with distilled water.

All statistical analyses were performed using the SPSS 17.0 software program (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard error. Statistical significance was determined using Student's t-test and one-way analysis of variance with the Bonferroni post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Expression of IL-23 mRNA was evaluated in periapical lesions and healthy PDLs by RT-PCR. The relative mRNA expression levels of IL-23 were significantly greater in periapical lesions compared with healthy control PDLs (P=0.004; Fig. 1A). Immunohistochemical analysis revealed that the samples from periapical lesions demonstrated increased IL-23 staining compared with healthy PDL tissue (Fig. 1B).

SH-9 human PDL cells were treated with P. endodontalis LPS for 12, 24 or 36 h. As presented in Fig. 2A, treatment with P. endodontalis LPS significantly increased the mRNA expression levels of IL-23 in SH-9 cells at 24 and 36 h (P=0.004 and P<0.001, respectively). To examine the signaling pathway involved in this increase, SH-9 cells were pretreated with 10 µM BAY11-7082 (an inhibitor of NF-κB) and 50 µM LY294002 (an inhibitor of phosphoinositide 3-kinase) for 30 min, and then treated with P. endodontalis LPS for 24 h. BAY11-7082 treatment decreased the mRNA expression levels of IL-23 in the P. endodontalis LPS-treated SH-9 cells (P=0.006), whereas LY294002 treatment demonstrated no significant effect (Fig. 2B). The nuclear translocation of NF-κB was analyzed by immunostaining. In untreated cells, NF-κB localized to the cytoplasm (Fig. 2C, a-c). Nuclear translocation of NF-κB was observed in cells treated with P endodontalis LPS (Fig. 2C, d-f). The activation of the NF-κB signaling pathway was further confirmed by western blot analysis (Fig. 2D). P. endodontalis LPS induced IκB phosphorylation. In accordance with these results, a luciferase assay indicated that P. endodontalis LPS increased NF-κB transcriptional activity in SH-9 cells, which was suppressed by pretreatment with BAY11-7082 (P<0.001; Fig. 2E).

RAW264.7 cells were treated with conditioned medium from P. endodontalis LPS-treated SH-9 cells in the presence of 10 ng/ml RANKL. RAW264.7 cells treated with unconditioned medium containing the same concentrations of P. endodontalis LPS and RANKL served as a control. Western blot analysis and TRAP staining were performed to determine the magnitude of osteoclastogenesis. Treatment with conditioned medium significantly increased the formation of TRAP-positive multinuclear giant cells, compared with control medium-treated cells (P=0.003; Fig. 3A and B), and this was accompanied by the upregulation of the osteoclast marker genes, NFATc1 and c-Fos (Fig. 3C).

To examine the role of IL-23 expression in SH-9 cells in P. endodontalis LPS-induced osteoclastogenesis, gene silencing was performed by siRNA transfection. Transfection of siIL-23 significantly reduced IL-23 expression in SH-9 cells compared with transfection with siCont (P=0.009; Fig. 4A). RAW264.7 cells were treated with the conditioned medium from P. endodontalis LPS-treated siCont or siIL-23 cells in the presence of 10 ng/ml RANKL. Conditioned medium from P. endodontalis LPS-treated siIL-23 cells exhibited a reduced ability to induce osteoclastogensis compared with conditioned medium from siCont cells, as determined by TRAP staining (P=0.006; Fig. 4B and C). In addition, the protein expression of NFATc1 and c-Fos was reduced in RAW264.7 cells treated with conditioned medium from P. endodontalis LPS-treated siIL-23, compared with siCont cells (Fig. 4D).