The RAW264.7 mouse macrophage (osteoclast precursor) cell line was obtained from the American Type Culture Collection. Foetal bovine serum (FBS) and the alpha modification of Eagle's medium (α-MEM) were purchased from Gibco-BRL (Thermo Fisher Scientific, Inc.). The cell counting kit (CCK-8) was purchased from Dojindo Laboratories. ZOL (dissolved in purified water) and a TRAP staining kit (387A, Sigma-Aldrich) were obtained from Sigma-Aldrich (Merck KGaA). DAPI and TRITC phalloidin were purchased from Beijing Solarbio. The recombinant human soluble RANK ligand (sRANKL) was purchased from R&D Systems. Polyvinylidene difluoride (PVDF) membranes were obtained from EMD Millipore. Specific antibodies against p38 (#8690), phospho-p38 (p-p38, #4511) (Thr180/Tyr182), IκBα (#4812), phospho-IκBα (p-IkBa, #2859) (Ser32), extracellular signal-regulated kinase 1/2 (ERK1/2, #9102), phospho-ERK (p-ERK, #4370) (Thr202/Tyr204), c-Jun N-terminal kinase (JNK, #9252), phospho-JNK (p-JNK, #4668) (Thr183/Tyr185), p65(#8242), phospho-p65 (p-p65, #3033) were obtained from Cell Signaling Technology. HRP-conjugated goat- anti-rabbit IgG secondary antibody (#014-090P) was purchased from Bioprimacy.

The RAW264.7 cells were cultured in α-MEM supplemented with antibiotics (100 units of penicillin and 100 µg/ml streptomycin, purchased from HyClone) and 10% heat-inactivated FBS at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The cells were used after 3-5 passages in α-MEM.

The effects of various concentrations of ZOL on the growth and viability of RAW264.7 cells in the presence or absence of RANKL were evaluated using a CCK-8 kit. In brief, the RAW264.7 cells were seeded into 3 96-well plates, at a density of 3×10³ cells/well and cultured in α-MEM supplemented with 10% FBS and 1% penicillin and streptomycin for 24 h. The medium was discarded and serially diluted ZOL (0, 0.1, 1, 5, 15, 30 and 50 µM) with or without 100 ng/ml RANKL were added to the cells at the same time, followed by further incubation at 37°C for 24, 48 or 72 h, respectively. A total of 10 µl CCK-8 reagent was added to each well, and the cells were incubated for an additional 2 h at 37°C with 5% CO₂. The optical density at 450 nm was read on an ELX800 microplate reader (BioTek Instruments), and the background reading (medium) was subtracted. Six replicates were used for each condition, and the experiments were repeated at least 3 times. The cell growth curves of the ZOL-treated cells were generated using GraphPad Prism 6.0 (GraphPad Software, Inc).

RAW264.7 cells differentiate into osteoclast-like cells in the presence of RANKL. The cells were seeded in 96-well tissue culture plates at a density of 1.5×10³ cells/well with α-MEM (supplemented with 10% FBS and 1% penicillin-streptomycin) and incubated at 37°C overnight to allow the cells attach to the inner surface of a 6-well plate, and the cell culture was then supplemented with (RANKL group) or without (RANKL-free and ZOL-free, denoted vehicle group) 100 ng/ml RANKL and various concentrations of ZOL (0, 0.1, 1 or 5 µM) for 5 days at 37°C, and the cell culture medium were replaced with fresh complete medium every 2 days until a large number of mature osteoclasts formed in the group treated with RANKL only. In addition, RAW264.7 cells treated with or without 1 µM ZOL and 100 ng/ml RANKL were cultured for 3, 5 or 7 days at 37°C. To determine osteoclast differentiation at the end of each incubation, the cells were washed twice and fixed with 4% paraformaldehyde for 20 min. The TRAP staining kit was then used to stain for TRAP, an osteoclast marker, according to the manufacturer's instructions. TRAP-positive multinucleated cells with >3 nuclei identified under an inverted microscope (Olympus IX 51) were considered as osteoclast-like cells.

RAW264.7 cells, cultured on glass coverslips, were treated with or without 100 ng/ml RANKL and 0, 0.1 or 5 µM ZOL until mature osteoclasts appeared in the control wells. To detect the formation of F-actin rings and nuclei, the cells were stained with TRITC phalloidin and DAPI, and analysed according to the manufacturer's instructions. In brief, the osteoclasts were fixed with 4.0% paraformaldehyde in PBS for 20 min. After washing with PBS 3 times, the cells were permeabilized using 0.25% (v/v) Triton X-100 for 5 min, followed by blocking in blocking buffer (3% bovine serum albumin in PBS, Thermo Fisher Scientific) for 1 h and then washed 3 times with PBS again. F-actin rings were stained with TRITC rhodamine-conjugated TRITC phalloidin and the nuclei with DAPI at room temperature for 30 min or 30 sec, respectively. Finally, the cells were washed with PBS and observed under a fluorescence microscope (BX51; Olympus) and the fluorescence images were obtained using Zeiss ZEN software (Zen 2.6, Zeiss AG).

The resorptive function of the mature osteoclasts derived from the RANKL-differentiated RAW264.7 cells was analysed on sterile bovine bone slices (IDS Nordic), which were placed in 96-well plates with 3 replicates for each condition. The RAW264.7 cells were plated at a density of 1.5×10³ cells/well onto the bovine bone slices. The cells were treated with 100 ng/ml RANKL and 0, 0.1 or 5 µM ZOL to induce osteoclast differentiation. After 10 days of culture (medium was changed every 48 h), all cells were removed from the bone slices, and the resorption pits were then visualised under a scanning electron microscope (Hitachi E-1010). The total number and area of resorption pits was quantified and compared using Image J software 6.0 (National Institutes of Health).

The RAW264.7 cells were seeded onto 6-well plates at a density of 1×10⁵ cells per well and cultured in complete α-MEM in the presence or absence of 100 ng/ml RANKL. These cells were then incubated with 0, 0.1, 1 or 5 µM ZOL at 37°C for 3 days until mature osteoclasts formed. The cells were transferred into a tube containing TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and total RNA was isolated according to the manufacturer's instructions. Complementary DNA was synthesised from 1 mg total RNA using PrimeScript™ reverse transcriptase (2690A, Takara Bio Inc.) and stored at −70°C until further use. RT-qPCR was performed to verify the differential expression of the specific genes during osteoclast formation or of GAPDH using the SYBR^® Premix Ex Taq™ kit (Takara Bio Inc.). For the analysis of mRNAs encoding osteoclastogenic proteins and osteoclast-specific markers, TRAP, CTR, RANK, NFATc1, c-Fos, DC-STAMP and GAPDH were amplified. The specific primer sequences are listed in Table I.

The thermocycling conditions for PCR were as follows: Initial denaturation for 1 min at 95°C, followed by 40 cycles of 95°C for 15 sec and extension at 60°C for 1 min. The 2^-∆∆Cq method (29) was used to calculate relative mRNA expression as described previously, and each sample was run and analysed in triplicate. The expression levels of each gene in all experimental groups were normalised to the endogenous reference gene (GAPDH) and indicated as relative fold changes of the control.

The RAW264.7 cells were seeded in 6-well plates at a density of 1×10⁶ cells/well, and following incubation at 37°C overnight, the cells were pre-treated with or without 5 µM ZOL for 4 h, and then cultured with 100 ng/ml RANKL for a further 0, 5, 10, 20, 30 or 60 min. Whole-cell lysates were prepared from harvested cells using radioimmunoprecipitation assay buffer consisting of 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium vanadate, 1 mM sodium fluoride, 1% deoxycholate and protease inhibitors. Cell debris was removed by centrifugation at 12,000 × g at 4°C for 10 min. The lysates were boiled in the loading buffer for 10 min and the protein concentrations in the whole-cell extracts were quantified using the bicinchoninic acid method (Beijing Solarbio). Total protein (30 µg per lane) was then separated by 10% SDS-PAGE and transferred onto PVDF membranes. After blocking in 5% non-fat milk in Tris-buffered saline containing Tween-20 at room temperature for 2 h, the membranes were incubated with a 1:1,000 dilution of the indicated primary antibodies at 4°C overnight, followed by horseradish-peroxidase-conjugated secondary antibodies diluted at 1:10,000 in the blocking buffer at room temperature for 1 h. After washing, the membranes were soaked in enhanced chemiluminescence solution (ECL, Millipore) for 1 min, and the bands were detected using the Gene Gnome Imaging System (Syngene). The band intensities were quantified using ImageJ software 1.48 q (NIH). Phosphorylated proteins (e.g., p-p65, p-IkBa, p-ERK1/2, p-p38 and p-JNK) were visualized by its specific primary antibody and corresponding secondary antibody. To detect total protein in the same membrane, antibodies, detecting each phosphorylated protein, were stripped from the membranes by using Stripping Buffer purchased from Solarbio^® LIFE SCIENCE (SW3020, Beijing). Antibody-free membranes were then re-incubated with antibodies binding to total proteins (e.g., p65, IκBa, p38, JNK). The GAPDH protein was used as an internal reference.

All experiments were performed 3 times and values are expressed as the means ± standard deviation. The groups were compared using one-way or two-way ANOVA analysis of variance followed by Tukey's post-hoc test for multiple comparisons. All data analyses were performed with GraphPad Prism 6.0 (GraphPad Software, Inc) and differences between means were considered statistically significant at P<0.05.

The effect of ZOL on the viability of RAW264.7 cells in the presence or absence of RANKL was assessed using a CCK-8 assay. First, the cytotoxicity of ZOL (0, 0.1, 1, 5, 15, 30 and 50 µM) on RAW264.7 cells without RANKL was analysed. The results indicated that the cell growth was suppressed by ZOL at concentrations of 15, 30 and 50 µM (Fig. 2A). Subsequently, the cytotoxicity of ZOL (at the same concentrations) in the presence of 100 ng/ml RANKL was assessed. As expected, the result was similar to that observed in the RANKL-free group (Fig. 2B).

After excluding the possibility that the inhibitory effects of ZOL on TRAP activity were due to cytotoxicity at concentrations of up to 5 µM, the effects of ZOL on RANKL-induced osteoclastogenesis were assessed. In the RANKL group, the RAW264.7 cells exhibited characteristic morphological changes toward osteoclasts at day 3 of RANKL-induced differentiation, with increasing cell-cell fusion into large and multinucleate TRAP-positive osteoclast cells, reaching completion at day 5 (Fig. 2C). However, the number of osteoclasts (Fig. 2D) and percentage of the osteoclast area (Fig. 2E) was significantly suppressed by incubation with 1 µM ZOL for different periods of time (3, 5 and 7 days). Furthermore, ZOL suppressed osteoclastogenesis in a dose-dependent manner (Fig. 2F-H). Taken together, these results suggest that ZOL inhibits osteoclast formation.

Mature osteoclasts contain actin ring structures that create sealing zones between the cells and bone matrix, and it is a prerequisite for osteoclast bone resorption (30,31). Thus, immunofluorescence analysis was performed to examine the effects of ZOL on F-actin rings and cell nuclei. Well-structured F-actin rings were observed by confocal fluorescence microscopy in the sealing zones of RANKL induced osteoclasts (Fig. 3A). However, the formation of the F-actin ring and the gathering of nuclei was markedly inhibited by ZOL in a concentration-dependent manner. Therefore, osteoclast morphology appeared abnormal or immature (Fig. 3A).

We then investigated whether ZOL modulates mature osteoclast activity by performing a resorption pit assay. RAW264.7 cells were plated on bone slices, which were treated with various concentrations of ZOL in the presence or absence of 100 ng/ml RANKL. The results indicated that the area of osteoclast bone resorption pits was markedly decreased by ZOL in a dose-dependent manner compared with the ZOL-free group. Furthermore, almost no resorption pits were observed in the groups treated with 5 µM ZOL (Fig. 3B-D). These results suggested that treatment with ZOL markedly attenuates the bone-resorption activity of osteoclasts. This may, at least partially, be explained by the effect of ZOL to impair osteoclastogenesis.

To further elucidate the effects of ZOL on osteoclast formation and resorptive function, the expression of specific osteoclast differentiation-associated genes in ZOL-treated cells was assessed by RT-qPCR. It was indicated that treatment with RANKL markedly increased the expression levels of CTR, RANK, TRAP, DC-STAMP, NFATc1 and c-Fos. However, this upregulation was significantly suppressed by ZOL in a dose-dependent manner during osteoclastogenesis compared with that in the ZOL-free group (Fig. 4). These results suggest that ZOL inhibits the expression of RANKL-induced genes involved in osteoclast differentiation and function.

Previous studies have revealed that NF-κB, p38, ERK1/2 and JNK play critical roles in osteoclast differentiation (Fig. 1) (32-34). To explore the pathways through which ZOL regulates osteoclastogenesis, the protein levels of RANKL-induced signalling pathways were investigated by western blot analysis. As presented in Fig. 5A, the rapid activation of NF-κB was detected by the phosphorylation of IκBα, the inhibitor of NF-κB, at 5 min following RANKL exposure. As expected, RANKL treatment induced a significant increase in the RANKL-induced phosphorylation of p65. In addition, induction with RANKL markedly increased the phosphorylation levels of p38, ERK and JNK, which exhibited a maximum increase at 10 or 20 min (Fig. 5A).

To further examine the influence of ZOL on NF-κB and MAPK mediated osteoclast differentiation, the intensity of each phosphorylated protein was divided by the intensity of corresponding total protein in both RANKL and RANKL+ZOL treated groups. The results revealed that ZOL significant inhibited RANKL-dependent phosphorylation of IκBα (Fig. 5B) and p65 (Fig. 5C). Among the MAPK family proteins, the phosphorylation of JNK (Fig. 5D) was significantly inhibited by ZOL. However, the phosphorylation of p38 and ERK proteins were not significantly affected by ZOL (Fig. 5E and F). Thus, these results indicate that ZOL may inhibit NF-κB and JNK signalling by reducing the levels of p-IκBα, p-p65 and p-JNK. In other words, ZOL may reduce the formation of osteoclasts by suppressing the RANKL-induced activation of the NF-κB and JNK signalling pathways.

As reported previously, JNK and NF-κB signalling play a vital role at the early stage of osteoclast differentiation (35-37). Thus, it was further explored whether ZOL also suppresses early-stage osteoclast formation. Following the addition of RANKL, the RAW264.7 cells were treated with ZOL on days 0, 1, 2, 3 and 4. It was observed that treatment with ZOL at the early stage resulted in a prominent decrease in osteoclastogenesis in the RAW264.7 cells (Fig. 5G). However, the extent of osteoclastogenesis was comparable to that in the control group if ZOL was added at a later stage (Fig. 5H and I). These results thus indicate that ZOL suppresses the early stage of osteoclast formation, which is consistent with the findings of western blot analysis, according to which ZOL suppressed NF-κB and JNK signalling at the early stage of osteoclast differentiation.