Fetal bovine serum and α-modification of Eagle's medium (α-MEM) were obtained from Gibco-BRL (Sydney, Australia). The cell counting kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Soluble human recombinant macrophage-colony stimulating factor (M-CSF) and bacteria-derived recombinant mouse RANKL were supplied by R&D Systems (Minneapolis, MN, USA). AS-IV (purity >98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide prior to dilution to the appropriate concentrations in culture medium (34). Western blot-specific antibodies were obtained from Cell Signaling Technology (Cambridge, MA, USA). RAW264.7 cells were purchased from the American Type Culture Collection (Rockville, MD, USA).

The cytotoxic effects of AS-IV were determined using a CCK-8 assay. Bone marrow macrophages (BMMs) of C57/BL6 mice were seeded in 96-well plates at a density of 8×10³ cells/well, and cultured for 24 h. Cells were subsequently treated with different concentrations of AS-IV for 24, 48, 72 and 96 h. CCK-8 buffer (10 µl) was added to each well, and the cells were incubated at 37°C for an additional 2 h. The absorbance was subsequently measured at a wavelength of 450 nm (650 nm reference). The half-maximal inhibitory concentration (IC₅₀) value was calculated.

For primary cell culture, BMMs were obtained from the femurs and tibias of 6-week-old C57/BL6 mice. Cells were cultured in a T75 flask for 24 h. Floating cells were removed, and the adherent cells were cultured for another 3 days. The BMMs were subsequently seeded in a 96-well plate at a density of 8×10³ cells/well in complete α-MEM supplemented with 30 ng/ml M-CSF, 50 ng/ml RANKL and different concentrations of AS-IV (0, 25, 50 or 100 µM). Culture media were replaced every 2 days until mature osteoclasts had formed. Osteoclasts were identified by positive staining for tartrate-resistant acid phosphatase (TRAP). TRAP-positive cells with >3 nuclei were counted under a microscope.

For F-actin ring immunofluorescent staining, osteoclasts were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized for 5 min with 0.1% v/v Triton X-100. Cells were incubated with rhodamine-conjugated phalloidin (1:100; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Cells were subsequently incubated with Hoechst 3342 dye (1:5,000) for visualization of the nuclei, washed with phosphate-buffered saline (PBS), and mounted with ProLong Gold anti-fade mounting medium (both from Invitrogen). Fluorescence detection was performed on Nikon A1Si spectral detector confocal system equipped with 20X (dry) lenses (Nikon, Tokyo, Japan). Images were collected using NIS-C elements software and analyzed using ImageJ Software (National Institutes of Health, Bethesda, MD, USA).

BMMs were seeded on bone slices in 96-well plates at a density of 8×10³ cells/well and stimulated (in triplicate) with M-CSF (30 ng/ml), RANKL (50 ng/ml) and AS-IV (0, 25, 50 or 100 µM). Bone slices were imaged using field-emission scanning electron microscopy (FESEM, Hitachi S-4800, CamScan; Hitachi, Tokyo, Japan) with a magnification of 10 kV.

RT-qPCR was used to measure specific gene expression. BMMs were plated in 6-well plates at a density of 1×10⁵ cells/well and cultured in complete α-MEM supplemented with 30 ng/ml M-CSF and 50 ng/ml RANKL. For the concentration gradient assay, cells were incubated with AS-IV (0, 25 or 50 µM) for 5–7 days until mature osteoclasts formed. For the time gradient assay, cells were incubated with 50 µM AS-IV for 0, 1, 3 or 5 days. Total RNA was extracted using the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA) and cDNA was synthesized from 1 µg of total RNA using reverse transcriptase (Takara Biotechnology, Otsu, Japan). qPCR was performed using the SYBR Premix Ex Tag kit (Takara Biotechnology) and an ABI 7500 Sequencing Detection system (Applied Biosystems, Foster City, CA, USA) with 40 cycles of denaturation at 95°C for 5 sec and amplification at 60°C for 24 sec. GAPDH was used as the reference gene, and all the reactions were run in triplicate. The mouse primer sequences for cathepsin K (CtsK), TRAP, dendritic cell-specific trans membrane protein (DC-STAMP), V-ATPase d2, c-fos, NFATc1 and GAPDH were as follows: CtsK forward, 5′-CTTCCAATACGTGCA GCAGA-3′ and reverse, 5′-TCTTCAGGGCTTTCTCGTTC-3′; TRAP forward, 5′-CTGGAGTGCACGATGCCAGCGACA-3′ and reverse, 5′-TCCGTGCTCGGCGATGGACCAGA-3′; DC-STAMP forward, 5′-AAAACCCTTGGGCTGTTCTT-3′ and reverse, 5′-AATCATGGACGACTCCTTGG-3′; V-ATPase d2 forward, 5′-AAGCCTTTGTTTGACGCTGT-3′ and reverse, 5′-TTCGATGCCTCTGTGAGATG-3′; c-fos forward, 5′-CCA GTCAAGAGCATCAGCAA-3′ and reverse, 5′-AAGTAG TGCAGCCCGGAGTA-3′; NFATc1 forward, 5′-CCGTTG CTTCCAGAAAATAACA-3′ and reverse, 5′-TGTGGGATG TGAACTCGGAA-3′; and GAPDH forward, 5′-ACCCAG AAGACTGTGGATGG-3′ and reverse, 5′-CACATTGGG GGTAGGAACAC-3′.

RAW264.7 cells were stably transfected with a p-NF-κB-TA-Luc luciferase reporter construct. Briefly, cells were plated in a 24-well plate at a density of 1×10⁵ cells/well. The cells were treated 24 h later with different AS-IV concentrations (0, 12.5, 25, 50, 100 or 200 µM) for 1 h, prior to incubation with 50 ng/ml RANKL for a further 8 h. Cells were subsequently lysed and luciferase activity was measured using the Promega Luciferase Assay system (Promega, Madison, WI, USA).

RAW264.7 cells were seeded in 6-well plates at a density of 5×10⁵ cells/well. After pretreatment with or without AS-IV (200 µM) for 4 h, the cells were stimulated with RANKL for 0, 5, 10, 20, 30 or 60 min. The cells were subsequently washed twice in PBS and lysed in radioimmuno-precipitation assay lysis buffer. The lysates were centrifuged at 12,000 x g for 15 min and supernatants were collected.

Protein concentrations were determined using the bicinchoninic acid assay. Protein from each lysate (20 µg) was resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were subsequently blocked with 5% skimmed milk in TBS-Tween-20 for 1 h, and incubated with primary antibodies overnight at 4°C. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) for 1 h. Antibody reactivity was detected using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

An in vivo wear particle-induced osteolysis model was generated as previously reported (35). All the animal care and experimental procedures complied with Directive 2010/63/EU revising Directive 86/609/EEC approved by Animal Care and Use Committee of Zhengzhou University (approved on Feb 19th 2014, the approval number is 001381). Twenty healthy 8-week-old C57BL/J6 mice were randomly assigned to four groups: Sham PBS control (sham), Ti particles with PBS (vehicle), and Ti particles with low (10 mg/kg/day) and high (25 mg/kg/day) concentrations of AS-IV (low and high, respectively). AS-IV was used by intraperitoneal injection every other day for 14 days. At the end of the experiment, the mice were sacrificed, and the calvaria were excised and fixed in 4% paraformaldehyde for micro-computed tomography (micro-CT) analysis.

A high-resolution micro-CT scanner (Skyscan 1176; Skyscan; Aartselaar, Belgium) was used with the following settings: X-ray voltage, 50 kV; electric current, 500 mA; and rotation step, 0.7°. Following reconstruction, a square region of interest (ROI) around the midline suture was chosen for further qualitative and quantitative analyses. The bone volume against tissue volume (BV/TV), number of porosities and percentage of total porosity were determined for each sample as described previously (36).

The data Are expressed as mean ± standard deviation. The results were analyzed using the analysis of variance and post hoc tests with the SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference between groups.

CCK-8 cell viability assays were performed to examine the potential AS-IV cytotoxicity. The results showed that AS-IV cytotoxicity was concentration- and time-dependent (Fig. 2). These assays indicated that the maximum concentration used in the subsequent studies (200 µM) showed no cytotoxic effects in BMMs, even after 96-h exposure. Based on these data, the AS-IV concentrations used in subsequent experiments were considered non-cytotoxic.

As osteoclasts have key roles in osteoclast-related diseases (2), the effect of AS-IV on osteoclastogenesis was investigated. BMMs were exposed to AS-IV (0, 25, 50 and 100 µM) during osteoclast formation. The control group formed numerous TRAP-positive multinucleated osteoclasts (Fig. 3A). Osteoclast formation was inhibited by AS-IV (Fig. 3A–C). Furthermore, comparison of the control and 100 µM AS-IV cells after 3, 5 and 7 days produced similar results (Fig. 3D–F).

To further examine the effects of AS-IV on osteoclastogenesis, RANKL-induced osteoclast F-actin ring formation was studied, which is the most well-known characteristic of mature osteoclasts and a prerequisite for osteoclast bone resorption during osteoclastogenesis (37). As expected, confocal microscopy revealed F-actin ring formation and characteristic podosomal condensation in control osteoclasts. However, the size and number of F-actin rings significantly decreased in cells incubated with AS-IV (Fig. 4A), suggesting that AS-IV suppressed F-actin ring formation.

As the formation of a well-polarized F-actin ring is an essential prerequisite for efficient bone resorption by osteoclasts, we inferred that osteoclast bone resorption would also be inhibited by AS-IV. Numerous bone resorption pits were observed on the surface of control bone slices (Fig. 5). In bone slices exposed to AS-IV, the resorption area was decreased. These findings demonstrated that AS-IV impaired osteoclast bone resorption in vitro.

The expression levels of several specific genes are upregulated during osteoclast differentiation. Thus, RT-qPCR was used to examine and quantify the RANKL-induced mRNA expression of osteoclast-related genes (including NFATc1, TRAP, V-ATPase d2, CtsK, DC-STAMP and c-fos). The results showed that the expression of these genes was inhibited by AS-IV in a dose- and time-dependent manner (Fig. 6).

RANKL-induced activation of ERK is essential for osteoclast differentiation and function (38,39). The effects of AS-IV on RANKL-induced signaling pathways were therefore investigated. ERK phosphorylation increased within 5–30 min of stimulation with RANKL in the control group. However, ERK phosphorylation was significantly reduced by exposure to AS-IV (Fig. 7A). Quantitative analysis confirmed these results (Fig. 7C). These results suggested that AS-IV inhibited phosphorylation of ERK during osteoclast differentiation.

NFATc1 is an important master regulator of osteoclastogenesis and osteoclast function (40). Therefore, the effects of AS-IV on RANKL-induced NFATc1 expression were investigated. The data presented in Fig. 6A and B indicated that NFATc1 transcriptional activity increased when the cells were stimulated by RANKL. AS-IV inhibited this activity in a dose- and time-dependent manner. To confirm this effect of AS-IV on NFATc1 expression, the NFATc1 protein level was examined using western blot analysis. NFATc1 protein levels increased when cells were exposed to RANKL, and AS-IV attenuated this increase (Fig. 7D and E), suggesting that AS-IV suppressed RANKL-induced NFATc1 expression.

RANKL-induced NF-κB activation is also a dominant mediator of osteoclast differentiation, resorption function and survival (41–43). Western blot analysis and luciferase assays were used to investigate the NF-κB signaling pathway. Similar levels of IκBα phosphorylation and degradation were observed in control and AS-IV groups (Fig. 7A). This observation was supported by luciferase reporter gene assays (Fig. 7B). These data indicated that AS-IV inhibited osteoclastogenesis without affecting the NF-κB signaling pathway.

To explore the effects of AS-IV on pathological osteolysis, a Ti particle-induced mouse calvarial osteolysis model was used. The degree of particle-induced osteolysis was assessed using high-resolution micro-CT. Compared with the sham group (no Ti particles), the vehicle group (administration of Ti particles in PBS) showed significant calvarial osteolysis. When AS-IV (10 mg/kg, low; 25 mg/kg, high) was administered with the Ti particles, this osteolytic bone loss reduced (Fig. 8A). Quantitative analysis of bone parameters further demonstrated that the high or low concentrations of AS-IV significantly increased the BV/TV (Fig. 8B), and decreased the number of porosities and the percentage of total porosity in the ROI in the calvaria (Fig. 8C and D).