Nardosinone (Figure 1A), purchased from Must Biotechnology (Chengdu, China), was dissolved in dimethyl sulfoxide (DMSO). Alpha modification of Eagle’s minimum essential medium (α-MEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco BRL (Gaithersburg, MD, United States). Recombinant murine M-CSF and RANKL were purchased from R&D Systems (Minneapolis, MN, United States). Tartrate-resistant acid phosphatase (TRAP) staining solution, Triton X-100, and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, United States). FITC phalloidin was obtained from Yeasen biotech Co., Ltd (Chengdu, China). Primary antibodies targeting GAPDH, IκBα, phospho-Akt, Akt, phospho-ERK1/2, ERK1/2, phospho-JNK1/2, JNK1/2, phospho-p38, p38, phosphor-PLCγ2, PLCγ2, c-Fos and NFATc1 were purchased from Cell Signaling Technology (Danvers, MA, United States). Dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay kits were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). LPS from P. gingivalis was purchased from InvivoGen (San Diego, CA, United States).

Bone marrow macrophages were prepared as previously described (Xiao et al., 2015). Briefly, monocyte/macrophage precursors were prepared by flushing the marrow from the long bones of 6-week-old C57BL/J6 mice and differentiated into BMMs in α-MEM containing 10% FBS, 100 U/mL penicillin/streptomycin (complete α-MEM) and 30 ng/mL M-CSF. The OC-like RAW264.7 cell line (TIB-71, ATCC, Manassas, VA, United States) was cultured in complete α-MEM. All cell cultures were maintained in a humidified environment of 95% air/5% CO₂ at 37°C.

The anti-proliferative effect of Nd on BMMs was measured with a CCK-8 kit (Dojindo Molecular Technology, Kumamoto, Japan) according to the manufacturer’s instructions. In brief, BMMs plated into 96-well plates at a density of 1 × 10⁴ cells/well were treated for 48 or 96 h with serial dilutions of Nd (0–800 μM). Next, 10 μL CCK-8 mixed with 90 μL complete α-MEM was added to each well. After incubation for 2 h, the optical density (OD) was measured with an ELX800 absorbance microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, United States) at a wavelength of 450 nm (650 nm reference).

Bone marrow macrophages were treated with Nd (0, 12.5, 25, or 50 μM) for 48 h. The cells were washed three times with PBS, then treated for 15 min with Triton X-100 to disrupt the cell membrane integrity. Nuclei were stained with 0.1 μg/mL DAPI in PBS at 37°C for 10 min in the dark. The cell nuclei were observed and photographed using an LSM5 confocal microscope (Carl Zeiss, Oberkochen, Germany).

Bone marrow macrophages were seeded into 96-well plates at a density of 8 × 10³ cells/well, in triplicate, in complete α-MEM containing M-CSF (30 ng/mL), and allowed to adhere overnight. Next day BMMs were treated with RANKL (50 ng/mL) and various concentrations of Nd (0, 12.5, 25, or 50 μM) over a 5 day period. Conversely, to investigate Nd addition-dependent effects on osteoclastogenesis at a particular stage, Nd (50 μM) was added to BMMs cultured with M-CSF and RANKL at different stages of the 5 day culture period as follows: pre-treatment (12 h before RANKL); early treatment (day 1); late treatment (day 3), early + late treatment (days 1 and 3); and pre + early + late treatment (12 h before RANKL, then days 1 and 3). After 5 days, cells were fixed with 4% paraformaldehyde and stained for TRAP activity. TRAP^+ve cells that contained three or more nuclei were counted as mature OCs, and their cell spread areas were measured.

For real-time polymerase chain reaction (PCR), 2 × 10⁵ BMMs were plated into each well of a 12-well plate and cultured in complete α-MEM containing M-CSF (30 ng/mL), and RANKL (50 ng/mL). Cells were then treated with or without Nd at a range of concentrations (0, 12.5, 25, or 50 μM). Total RNA was extracted from cultured cells using an RNeasy Mini kit (Qiagen, Valencia, CA, United States) according to the manufacturer’s instructions, and cDNA was synthesized from 1 μg of total RNA using reverse transcriptase (TaKaRa Biotechnology, Otsu, Japan). Real-time PCR was performed using a SYBR Premix Ex Taq kit (TaKaRa Biotechnology) and an ABI 7500 Sequencing Detection System (Applied Biosystems, Foster City, CA, United States). PCR was performed under the following conditions: 40 cycles each involving 5 s of denaturation at 95°C and 34 s of amplification at 60°C. All PCRs were performed in triplicate and levels were normalized to those of the gene Gapdh. The following primer sets were used as previously described: mouse Gapdh: forward, 5′-ACC CAG AAG ACT GTG GAT GG-3′ and reverse, 5′-CAC ATT GGG GGT AGG AAC AC-3′; mouse Nfatc1: forward, 5′-CCG TTG CTT CCA GAA AAT AAC A-3′ and reverse, 5′-TGT GGG ATG TGA ACT CGG AA-3′; mouse cathepsin K: forward, 5′-CTT CCA ATA CGT GCA GCA GA-3′ and reverse, 5′-TCT TCA GGG CTT TCT CGT TC-3′; mouse c-Fos: forward, 5′-CCA GTC AAG AGC ATC AGC AA-3′, reverse, 5′-AAG TAG TGC AGC CCG GAG TA-3′; and mouse V-ATPase d2: forward, 5′-AAG CCT TTG TTT GAC GCT GT-3′ reverse, 5′-TTC GAT GCC TCT GTG AGA TG-3′.

RAW264.7 cells were seeded into 6-well plates at a density of 6 × 10⁵ cells/well. When the cells were confluent, they were pretreated with or without Nd for 4 h. Cells were then stimulated with 50 ng/mL RANKL for 0, 5, 10, 20, 30, and 60 min. Cellular proteins were extracted from cultured BMMs or RAW264.7 cells using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, United States) supplemented with 10 mg/mL phenylmethylsulfonyl fluoride (PMSF), and the protein concentration was determined using a BCA protein assay (Thermo Fisher Scientific). Lysate proteins (25 μg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were then blocked with 5% skimmed milk in TBS-Tween (TBS: 0.05 M Tris, 0.15 M NaCl pH 7.5; with 0.2% Tween-20) for 1 h, and incubated with primary antibodies diluted in 1% (w/v) skimmed milk powder in TBS-Tween overnight at 4°C. Experiments were repeated independently at least three times.

Equivalent amounts of BMM-derived pre-OCs (after 4 days of RANKL stimulation) were seeded onto 100-mm bovine bone disks, treated with Nd (0, 12.5, 25, or 50 μM) for 48 h and then fixed and stained for TRAP activity. Resorption pits were visualized under a scanning electron microscope (FEI Quanta 250), and the bone resorption area was quantified using Image J software (National Institutes of Health, Bethesda, MD, United States).

Osteoclast cells differentiated from BMMs were cultured on glass coverslips and fixed for 15 min at RT with 4% paraformaldehyde, permeabilized for 5 min with 0.1% Triton X-100 in PBS, and non-specific antibody binding was blocked by incubating for 30 min in 5% skimmed milk in PBS. F-actin was stained with FITC-conjugated phalloidin and nuclei were stained with DAPI. Actin ring distribution was visualized using an LSM5 confocal microscope (Carl Zeiss, Oberkochen, Germany).

The DCFDA cellular ROS detection assay kit was used to detect intracellular ROS levels. BMMs (8 × 10³ cells/well in 96-well plates) were treated with RANKL (50 ng/mL), M-CSF (30 ng/mL), and Nd (25 or 50 μM) for 72 h. Intracellular ROS levels were determined using 2′,7′-dichlorofluorescein diacetate (DCFH), which oxidizes into fluorescent DCF in the presence of ROS. Cells were washed in PBS and incubated in the dark for 60 min with 10 μM DCFH-DA. Images were obtained using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Animal studies were conducted in accordance with the principles and procedures approved by the Animal Care Committee of Shanghai Jiao Tong University, China. Twenty specific-pathogen-free 8-week-old male C57BL/6J mice were randomly divided into four groups: phosphate-buffered saline (PBS; control), LPS (1 mg/kg body weight; Sigma-Aldrich), LPS + Nd (5 mg/kg body weight, low dose group), and LPS + Nd (15 mg/kg body weight, high dose group). Nd was given by intraperitoneal injection on days 1, 4, and 7. On days 4 and 7, mice were sedated via light anesthesia and injected with 1 mg/kg of P. gingivalis LPS at the gingiva of the second molar in the lower left and right jaw (Kim Y.G. et al., 2015). Mice were sacrificed on day 10 and their jaws were collected for further analysis. Left jaws were dissected for micro-CT analysis and right jaws for bone histology and histomorphometry.

Left jaws were fixed in 4% paraformaldehyde for 1 day at 4°C and stored in 70% ethanol at 4°C. They were then scanned using high-resolution micro-computed tomography (μCT) (Scanco microCT100, Brüttisellen, Switzerland). Each jaw was then imaged with the following instrument settings: 70 kV, 200 μA, 0.5 mm Al filter, 300 ms exposure, pixel size 5 μm. After scanning, the data were reconstructed using Scanco μ100 Evaluation software V6.5-3 with a constant threshold value. An image of a precise ruler was captured at the same magnification and used for calibration area measurements, which were performed with Olympus Microsuite 3.2 imaging software. A volume of interest was generated covering the first to third molars and the relevant alveolar bone. The volume of alveolar bone included in the region of interest (bone volume/total volume) (ROI [BV/TV]) was measured for each sample, and comparisons were made among different groups. The polygonal area enclosed by the cementoenamel junction (CEJ), the lateral margins of the exposed tooth root, and the alveolar bone crest (ABC) were also measured, and results are expressed in millimeters squared (mm²).

Right jaws were fixed in 4% paraformaldehyde for 1 day at 4°C, subsequently decalcified in 10% EDTA for approximately 1 month, and then embedded in paraffin. Histological sections (7 μm thick) were prepared for hematoxylin and eosin as well as TRAP staining. The specimens were then examined and photographed using a high quality microscope. The number of TRAP^+ve multi-nucleated OCs (N.Oc/BS, 1/mm) and the percentage of OC surface per bone surface (OcS/BS, %) were assessed for each sample.

Data are shown as mean ± standard deviation (SD) from at least three independent experiments. Student’s t-test was used to determine statistical significance between the results of test and control groups, with ^∗P < 0.05 and ^∗∗P < 0.01 considered statistically significant.

The CCK-8 assay was used to examine the potential cytotoxicity of Nd on BMMs. Treatment with Nd for 48 or 96 h at concentrations less than 400 μM did not induce cytotoxicity (Figure 1B). BMMs were exposed to M-CSF and RANKL in the absence or presence of different concentrations of Nd for 5 days. The formation of TRAP^+ve OCs was suppressed by Nd (Figure 1C) and the number of OCs and the area of TRAP-positive staining were reduced to approximately 9% of control levels by treatment with 50 μM Nd (Figures 1D,E). To determine the stage at which Nd suppressed osteoclastogenesis, BMMs were treated with 50 μM Nd at several time points. Neither treatment for 12 h prior to RANKL stimulation (pre-treatment) nor on the third day of RANKL stimulation (late treatment) altered the number or size of TRAP^+ve OCs induced after 5 days of culture, whereas treatment on the first day of RANKL stimulation (early treatment) led to an almost 35% reduction in OC number and area of TRAP (Figures 1F–H). Continuous exposure to early and late treatment or pre-, early and late treatment potently suppressed OC formation (Figures 1F–H). To exclude the possibility that Nd induced BMM apoptosis during the course of differentiation, BMMs were stained with DAPI following Nd treatment (Figure 1I), and were found to exhibit normal intact nuclei, confirming that the suppressory effects of Nd on osteoclastogenesis were not attributable to the induction of BMM apoptosis. Furthermore, western blotting was carried out to examine whether the apoptotic cell death pathways were activated after Nd treatment. There was no alteration in the levels of the anti-apoptotic protein Bcl-2, nor any activation of the Bax and caspase-3 apoptotic pathways following treatment with up to 50 μM of Nd (Figure 1J).

Next, the effects of Nd on OC bone resorption were assessed. SEM results verified the bone resorptive ability of OCs differentiated from BMMs on the bone surface (Figure 2A) Meanwhile, most of the bone resorption activity was suppressed and was almost completely blocked at the higher concentration of Nd (≥50 μM) (Figures 2A,B). The establishment of F-actin-rich podosomes-polarized cytoskeletal structures known as F-actin rings-indicates the maturity of OCs and is also essential for OC bone resorption (Wu et al., 2015; Zhu et al., 2016). The results showed that osteoclastic bone resorption is suppressed by Nd; further experimentation to verify whether Nd suppresses F-actin ring formation was carried out. Confocal microscopy revealed clearly the formation and morphology of the F-actin ring of mature OCs stained with phalloidin in the control group (Figure 2C). However, in the group treated with Nd the size and morphology of the F-actin ring were reduced, with dose-dependent reductions. Together, these findings clearly indicate that the application of Nd reduced osteoclastic bone resorption and formation of OCs in vitro.

To elucidate the inhibitory effects of Nd on OC formation, intracellular ROS was semi-quantitatively measured using DCFDA. As shown in Figure 3A, intracellular ROS was at a low level before RANKL stimulation but increased sharply to a high level when BMMs were treated with RANKL. This effect could be markedly attenuated by Nd. Both the number of ROS (+) cells and the level of ROS were decreased by Nd (Figures 3B,C). Ca²⁺ signaling is indispensable for osteoclastogenesis and its trigger is the phosphorylation of PLCγ2. Thus, we investigated the inhibitory effects of Nd on the phosphorylation of PLCγ2. As expected, the phosphorylation of PLCγ2 was clearly blocked compared with the control group (Figures 3D,E).

To detect the underlying mechanisms by which Nd mediated OC formation, the bearing of RANKL-induced signaling pathways on osteoclastogenesis were examined. The phosphorylation of MAPK family members (EKR, JNK, and p38) and Akt occurred within 10 min of RANKL stimulation, and continued for 30 min in the control group (Figures 4A–C). In the meantime, by comparison, Nd attenuated the phosphorylation of ERK and JNK, without affecting that of p38 (Figure 4A). The results also showed that Nd did not reduce phosphorylation of Akt or of IκBα, an inhibitory subunit of NFκB, indicating that it does not influence the Akt and NFκB signaling pathway (Figure 4A). Overall, these data suggest that the suppressive influence of Nd on osteoclastogenesis could be put down to the attenuation of RANKL-induced JNK and ERK signaling cascades.

It has been shown that activation of NFATc1, the master regulator of osteoclastogenesis which relies on c-Fos, results from phosphorylation of the MAPK signaling pathway (Nakashima et al., 2012). BMMs were stimulated with RANKL in the absence or presence of Nd for 0, 24, 48, or 72 h. As shown in Figures 4D–F, c-Fos protein expression increased after 24, 48, and 72 h, and NFATc1, the downstream transcriptional targets of c-Fos, showed steep increases at 48 and 72 h. By comparison, the situation was potently suppressed by Nd at various time-points. (Figures 4G–J On the other hand, Nd also suppressed the expression of OC marker genes including c-Fos, VATPase d2, ctsk, and Nfatc1), shown by quantitative PCR.

To evaluate the role of Nd in bone resorption in vivo, we created the LPS-induced mouse alveolar-bone-loss model. No fatalities were recorded after LPS and Nd administration, and all the animals maintained normal activity throughout the experiment. Micro-CT, morphometric and histomorphometric analyses were performed. Micro-CT confirmed that LPS-injected mice exhibited significantly greater areas of exposed roots than the control and Nd groups (Figures 5A,B). No significant differences in areas of exposed roots were observed among control, low dose, and high dose groups (Figures 5A,B). A volumetric quantitative analysis of alveolar bone loss with micro-CT verified the results obtained by macroscopic analysis. BV/TV in the LPS group was also significantly lower than those of the control or Nd groups (Figure 5C). The LPS-mediated decrease in BV/TV values was restored up to a level similar to the control group by treatment with Nd at 5 or 15 mg/kg (Figure 5C).

To further explore whether OCs were involved in the inhibitory effects of Nd on LPS-induced alveolar bone loss, TRAP staining was performed to calculate the number of OCs in tissue sections. TRAP-positive multinucleated cells lining the alveolar bone surface were visually enumerated (black arrows in Figure 5D) in a specific AOI with standardized dimensions and surfaces. The LPS group showed the highest number of OCs compared with the control group. However, the administration of 5 mg/kg of Nd significantly reduced OC number. Furthermore, the administration of 15 mg/kg of Nd almost completely prevented an LPS-induced increase in OC number and bone erosion (Figure 5D). Histomorphometric analysis of Oc.S/BS and OC number confirmed that Nd treatment attenuated LPS-induced alveolar bone loss and reduced OC numbers (Figures 5E,F). Collectively, these results indicated that Nd effectively prevented LPS-induced alveolar bone loss in vivo. Tissue toxicities of Nd on the liver and kidney were confirmed by histological analyses (Supplementary Figure S1). Compared to control animals, administration of Nd at 5 or 15 mg/kg body weight/day for 10 days did not induce visible hepatotoxicity or nephrotoxicity.