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Introduction

Bone mass homeostasis is regulated by a delicate balance between osteoclast-mediated bone degradation and osteoblast-induced bone formation, a process termed bone remodeling (1). Osteoclasts are large cells with multiple nuclei, which form from hematopoietic progenitors of the mono-cyte/macrophage lineage. The cells are specialized in bone resorption (2). Several pathological bone-related diseases, including postmenopausal osteoporosis, rheumatoid arthritis, periodontitis and lytic bone metastasis, are associated with excessive bone breakdown by an increase in the number and activity of osteoclasts (3–5).

Macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL) are two major cytokines, which regulate osteoclast differentiation (6–8). M-CSF is responsible for the proliferation and survival of osteoclast precursors and stimulates the expression of receptor activator of NF-κB (RANK), a receptor for RANKL, with RANKL inducing osteoclast differentiation (7). The binding of RANKL to its receptor, RANK, triggers several downstream signaling pathways, including the p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) and NF-κB pathways, which lead to the activation of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) (9–14). NFATc1, a master transcription factor of osteoclast formation, induces several genes responsible for osteoclast differentiation and function, including matrix metalloproteinase-9 (MMP-9), tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CtsK) (15,16).

There are many natural compounds that can be exploited in the development of novel drugs (17). Several compounds from natural products suppress osteoclast formation and function. These products have potential therapeutic value for bone-related diseases characterized by the hyperactivation of osteoclast activity (18). Rheum undulatum is a perennial plant that belongs to the Polygonaceae family and is mainly distributed in Korea. The rhizomes of R. undulatum and other Rheum species, commonly known as rhubarb, have been used for the prevention of a number of diseases in traditional medicines (19,20). Anthraquinones and stilbenes are the primary constituents from the rhizomes of R. undulatum. Anthraquinones have a laxative effect (21), whereas the stilbenes from this plant exhibit various pharmacological activities, which include antioxidant, anti-inflammatory and hepatoprotective effects (22,23). Desoxyrhapontigenin (DRG) is a stilbene compound isolated from the rhizomes of R. undulatum and has an anti-inflammatory effect via activating the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway and attenuating the NF-κB and MAPK pathways in macrophages (24). However, the anti-osteoclastogenic effect of DRG and its underlying mechanisms remain to be fully elucidated. On the basis of the association between chronic inflammatory and bone diseases (25), the present study aimed to determine the pharmacological effects of DRG on RANKL-induced osteoclast formation and function in mouse bone marrow macrophages (BMMs) and on bone loss induced by lipopolysaccharide (LPS) in an in vivo animal model. It was demonstrated that DRG suppressed RANKL-induced osteoclast formation at an early stage of osteoclastogenesis in BMMs and prevented LPS-induced bone destruction in the animal model.

Materials and methods

Isolation of stilbene derivatives

Dried R. undulatum L. rhizomes were purchased from the Yakryoung-si folk medicine market in Daegu, Korea, in May 2015. Botanical identification was performed by Professor Byung-Sun Min (College of Pharmacy, Catholic University of Daegu, Hayang, Korea). A voucher specimen (CUD-1188-1) was stored at the College herbarium. The MeOH extract of the dried rhizomes of R. undulatum L. was concentrated to yield a residue (4.5 kg), which was suspended in water and then successively partitioned with n-hexane, ethyl acetate and n-butanol to afford n-hexane-, EtOAc-, and n-BuOH-soluble fractions, and a water (H2O) layer. The EtOAc-soluble fraction (658 g) was subjected to silica gel column chromatography and eluted with n-hexane-EtOAc (7:1→0:1) to yield six fractions (E1-E6). Fraction E2 was rechromatographed on a silica gel column eluted with chloroform (CHCl3)-MeOH (7:1→0:1) to yield six sub-fractions (E2-1-E2-6). Subsequently, the E2-1 sub-fraction was chromatographed on an RP-18 silica gel column with MeOH-H2O (1:1→1:0) as the mobile phase to yield five fractions (E2-1-1-E2-1-5). Sub-fraction E2-1-3 was subjected to silica gel column chromatography using a stepwise gradient elution of CHCl3-MeOH (14:1→0:1), to yield rhapontigenin (2.1 g) and resveratrol (200 mg). Sub-fraction E2-1-4 was rechromatographed on a silica gel column with CH2Cl2-MeOH-H2O (5:1:1) to obtain DRG (500 mg). Fraction E3 was fractionated on a silica gel column using CHCl3-MeOH (20:1→0:1) as the solvent system to obtain piceatannol (3 g). Sub-fraction E3-3 was purified using high-performance liquid chromatography with a stepwise gradient of H2O-MeOH (1:1→3:1) to yield resveratroloside (15 mg). The structures of the stilbene derivatives are shown in Fig. 1. The purity of these stilbenes was assessed using 1H and 13C nuclear magnetic resonance spectra. The spectra revealed signals with a high level of purity with no impurities.

Figure 1 Chemical structures of the isolated stilbenes from R. undulatum and their effects on RANKL-induced osteoclast formation in BMMs. (A) Chemical structures of the isolated stilbenes. (B) Effect of the five isolated stilbenes on the RANKL-induced differentiation of BMMs into osteoclasts. BMMs were induced to differentiate into osteoclasts by incubating with macrophage-colony stimulating factor (30 ng/ml) and RANKL (100 ng/ml) in the presence or absence of the indicated stilbenes (10 μM) for 7 days, and then a TRAP assay was performed. The quantities of TRAP-positive multinucleated (>5 nuclei) osteoclasts were determined following image capture (magnification, ×40). Data are presented as the mean ± standard error of the mean (*P<0.01, compared with Veh; n=3). BMMs, bone marrow macrophages; RANKL, receptor activator of nuclear factor-κB ligand; TRAP, tartrate-resistant acid phosphatase; Veh, vehicle-treated control.

Cell culture

RAW264.7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco’s modified essential medium with 10% heat-inactivated fetal bovine serum (FBS; Cambrex, Charles City, IA, USA) and penicillin (100 U/ml)-streptomycin (100 μg/ml). The cells were cultured at 37°C in a humidified 5% CO2 atmosphere.

Isolation of BMMs and osteoclast differentiation

Bone marrow cells were isolated from the femurs and tibias of 6-week-old male ICR mice weighing 22–25 g (DBL, Emseong, Chungbuk, Korea) housed at a temperature of 24±2°C and humidity of 55±10% controlled colony room under a 12 h light and 12 h dark cycle. All mice were allowed ad libitum access to a standard chow diet and water prior to all experiments. The bone marrow cells were then cultured in α-MEM (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 10 ng/ml M-CSF (Prospec-Tany TechnoGene Ltd., East Brunswick, NJ, USA) overnight in a humidified incubator with 5% CO2 at 37°C. The floating cells were harvested and maintained for 3 days with 30 ng/ml M-CSF. The cells adhered to the culture dish were characterized as BMMs and used for subsequent experiments. The BMMs (5×104 cells/well) were cultured in 96-well plates and maintained with RANKL (100 ng/ml) and M-CSF (30 ng/ml) for 7 days in the presence or absence of the indicated compounds. The medium was replaced every 2 days. The cells were then fixed for 15 min in 3.7% formalin, permeabilized with 0.1% Triton X-100, and stained for TRAP with an acid phosphatase leukocyte kit (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA). TRAP-positive multinucleated cells with more than five nuclei were defined as osteoclasts.

Antibodies and reagents

Recombinant mouse RANKL and M-CSF were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Antibodies targeting NFATc1 (cat no. 8032S), c-Fos (cat no. 2250S), c-Src (cat no. 2019), ERK1/2 (cat no. 4695S), p38 (cat no. 9212), JNK (cat no. 9252), phospho-p38 (cat no. 9216S), phospho-ERK1/2 (cat no. 9101S) and phospho-JNK (cat no. 4668S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Fluorescein isothiocyanate (FITC)-conjugated palloidin (cat no. A12379) was from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Antibodies targeting α-tubulin (cat no. SC-5546) and CtsK (cat no. SC48353) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Cytotoxicity assay

Cytotoxicity was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay. The BMMs were cultured into 96-well plates (1×104 cells/well) and supplemented with 30 ng/ml M-CSF in the presence of the indicated concentrations of DRG. After 72 h, 0.5 mg/ml of MTT was added to each well for 3 h. At the end of the incubation, the insoluble formazan products were dissolved in dimethyl sulfoxide, and absorbance at 540 nm was determined.

Bone resorption assay

The BMMs (5×104 cells/well) were seeded in OsteoAssay Surface 96-well plates (Corning Incorporated, Corning, NY, USA) in α-MEM supplemented with 10% FBS, 1% penicillin and streptomycin, 30 ng/ml M-CSF, 100 ng/ml RANKL, and various concentrations of DRG. The culture medium was replaced every 2 days and culture continued for 7 days. The differentiated BMMs were washed with tap water and images of the surface of resorption pits were captured using a model H550L microscope (Nikon Corporation, Tokyo, Japan) and quantified via ImageJ software [Java 1.6.0_20 (64-bit); NIH, Bethesda, MD, USA].

Actin ring formation and immunofluorescence

The BMMs (106 cells/ml) were seeded on a cover glass and cultured for 7 days with 30 ng/ml M-CSF and 100 ng/ml RANKL in the presence of the indicated concentrations of DRG. The BMMs were washed and fixed in 15 min with 4% paraformaldehyde. Following permeabilization with 0.1% Triton X-100, the BMMs were stained for 10 min with FITC-phalloidin at room temperature. Following washing with phosphate buffered saline (PBS), the BMMs were mounted and images were captured using an LSM510 META NLO inverted confocal laser scanning microscope (Zeiss GmbH, Jena, Germany; Korea Basic Science Institute Chuncheon Center, Chuncheon, Korea) equipped with an external Argon, HeNe laser and HeNe laser II.

Western blot analysis

The cells were harvested and lysed in a lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% NP-40, 5 mM sodium orthovanadate and protease inhibitor cocktail (BD Biosciences, Franklin Lakes, NJ, USA), and then centrifuged for 10 min at 4°C and 22,000 × g. Protein concentration was determined using the Bradford method (26). A total of 30 μg of cellular extracts was separated via 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a Hybond-P membrane (GE Healthcare Life Sciences, Chalfont, UK). The membranes were blocked with 5% nonfat skim milk at room temperature for 1 h and probed for 2 h with the indicated primary antibodies (1:1,000 dilution). Following washing with PBS containing 0.1% Tween-20, the membranes were incubated with the secondary antibody (1:2,000 dilution) at room temperature for 2 h. The signal was detected using the enhanced chemiluminescence system (Thermo Fisher Scientific, Inc.).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

The BMMs were collected and total RNA was extracted using an RNeasy Mini kits according to the manufacturer’s protocol (Qiagen, Inc., Valencia, CA, USA). The first-strand cDNA was synthesized using 1 μg of total RNA. RT-qPCR analysis was performed using TOPreal qPCR 2X PreMIX (SYBR-Green; Enzynomics, Inc., Daejon, Korea) and the Rotor-Gene Q real-time PCR cycler (Qiagen, Inc.). The primers sequence were as follows: MMP-9, forward, 5′-TGGGCAAGCAGTACTCTTCC-3′ and reverse, 5′-AACAGGCTGTACCCTTGGTC-3′; CtsK, forward, 5′-GACACCCAGTGGGAGCTATG-3′ and reverse, 5′-AGAGGCCTCCAGGTTATGGG-3′; TRAP, forward, 5′-ACTTGCGACCATTGTTAGCC-3′ and reverse, 5′-TTCGTTGATGTCGCACAGAG-3′; β-actin, forward, 5′-GGGAAATCGTGCGTGACATCAAAG-3′ and reverse, 5′-AACCGCTCCTTGCCAATAGT-3′. The PCR conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 10 sec, 60°C for 15 sec and 72°C for 20 sec. All reactions were performed in triplicate and β-actin was used as an internal control. Quantification of the relative gene expression was computed using the 2−ΔΔCq method (27).

LPS-induced bone loss in vivo

All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (IACUC approval No. KW-180119-3). To investigate the effect of DRG on inflammation-induced bone loss, the mice were randomly divided into control vehicle-treated, LPS-treated, DRG-treated, and DRG + LPS-treated groups (n=4/group). The mice in the control group were treated with control vehicle (dimethyl sulfoxide: cremophor-EL in PBS; 1:1:8). The mice were injected intraperitoneally with DRG (50 mg/kg) solubilized in dimethyl sulfoxide: Chremophore-EL in PBS (1:1:8 by volume) or control vehicle for 1 h prior to the first LPS (5 mg/kg) injection and then every other day for 8 days. LPS was injected on days 2 and 6. All mice were sacrificed on day 9. Intact left femoral metaphysic regions of each mouse were evaluated by high-resolution micro-computed tomography (micro-CT) analysis using an NFR-Polaris-S160 apparatus (Nanofocus Ray; Korea Basic Science Institute Chuncheon Center) with a 90 μA current, source voltage of 45 kVp and 7 μm isotropic resolution. Femoral scans were performed over 2 mm from the growth plate, with a total of 350 sections per scan. Following three-dimensional (3D) reconstruction, trabecular number (Tb.N), bone volume per tissue volume (BV/TV), trabecular separation (Tb.Sp) and bone surface/bone volume (BS/BV) were examined with quantitative analyses using INFINITT-Xelis software (version 1.7; INFINITT Healthcare Co., Ltd., Seoul, Korea).

Statistical analysis

Data are presented as the mean ± standard error of the mean. Statistical analysis was performed using SPSS (version 14.0; SPSS Inc., Chicago, IL, USA). Statistical significance was assessed by one-way analysis of variance and the difference between the experimental groups was compared. P<0.05 was considered to indicate a statistically significant difference.

Results

Isolation of the stilbene derivatives from R. undulatum and their anti-osteoclastogenic effects in BMMs

In order to identify novel anti-osteoclastogenic compounds from the natural products, the stilbene derivatives were isolated from R. undulatum. Repeated column chromatography of the CHCl3-soluble fraction of R. undulatum on a silica gel and RP-C18 led to the isolation of five stilbenes. These stilbenes were identified as DRG, rhapontigenin, resveratrol, piceatannol and resveratroloside, by comparison with the published spectroscopic data (28–30). Their structures are shown in Fig. 1A. To investigate the effects of these stilbenes on osteoclast formation, the effects of these stilbenes on RANKL-induced osteoclast formation were investigated in mouse BMMs. At 10 μM, DRG, resveratrol, piceatannol and resveratroloside significantly reduced the RANKL-induced formation of osteoclasts as characterized by TRAP-positive multinucleated cells. However, rhapontigenin did not significantly inhibit osteoclast formation (Fig. 1B). As resveratrol and piceatannol are reported to have anti-osteoclastogenic activities (31,32) and DRG exhibited potent anti-osteoclastogenic activity, the present study further investigated the anti-osteoclastogenic effect of DRG.

DRG inhibits RANKL-induced formation in mouse BMMs and RAW264.7 cells

To investigate the effects of DRG on RANKL-induced osteoclast formation in detail, the anti-osteoclastogenic activity of DRG was evaluated using mouse BMMs and RAW264.7 cells. The mouse BMMs were incubated with RANKL and M-CSF for 7 days, and the RAW264.7 cells were stimulated with RANKL for 4 days. Treating BMMs or RAW264.7 cells with DRG reduced the formation of osteoclasts in a dose-dependent manner, as measured by TRAP-positive multinucleated cells (Fig. 2A and B). Subsequently, whether DRG down-regulated the RANKL-induced expression of osteoclast markers, including c-Src and CtsK, in the BMMs was evaluated. Western blot analysis revealed that DRG effectively suppressed the RANKL-induced upregulation of these markers (Fig. 2C), suggesting that DRG suppressed RANKL-induced osteoclast formation. To investigate whether DRG inhibited RANK-induced osteoclastogenesis due to the potential cytotoxicity of DRG to BMMs, the cytotoxic effect of DRG on BMMs was examined. The MTT assay results showed that DRG was not cytotoxic towards BMMs up to 30 μM (Fig. 2D). These results suggested that DRG suppressed RANKL-induced osteoclastogenesis without affecting the viability of BMMs.

Figure 2 DRG inhibits RANKL-induced osteoclast formation at the early stage. (A) BMMs were induced to differentiate into osteoclasts by incubating with M-CSF (30 ng/ml) and RANKL (100 ng/ml) in the presence or absence of the indicated concentrations of DRG for 7 days, and then a TRAP assay was performed. The quantities of TRAP-positive multinucleated (>5 nuclei) osteoclasts were determined following image capture (magnification, ×40). Data are presented as the mean ± standard error of the mean (*P<0.01, compared with vehicle-treated control; n=3). (B) RAW264.7 cells were induced to differentiate into osteoclasts by incubating with RANKL (100 ng/ml) in the presence or absent of the indicated concentrations of DRG for 4 days, and then a TRAP assay was performed. The quantities of TRAP-positive multinucleated (>5 nuclei) osteoclasts were determined following image capture (magnification, ×40). Data are presented as the mean ± standard error of the mean (*P<0.01, compared with vehicle-treated control; n=3). (C) BMMs were stimulated with M-CSF (30 ng/ml) and RANKL (100 ng/ml), in the presence or absence of DRG (30 μM) for 7 days. Total lysates were prepared and the expression levels of c-Src and cathepsin K were determined by western blot analysis. (D) BMMs were seeded into 96-well plates with M-CSF (30 ng/ml), followed by incubation with the indicated concentrations of DRG for 3 days. Cell viability was measured using the MTT assay. Data are presented as mean ± standard error of the mean (*P<0.01, compared with vehicle-treated control; n=3). (E) Effect of DRG on RANKL-induced osteoclast formation at different time points. BMMs were induced to differentiate into osteoclasts by incubating with M-CSF (30 ng/ml) and RANKL (100 ng/ml), and then treated with 30 μM DRG at the indicated periods of time. The cells were cultured for 7 days, and then fixed and the TRAP staining assay was performed. The quantities of TRAP-positive multinucleated (>5 nuclei) osteoclasts were determined following image capture (magnification, ×40). Data are presented as the mean ± standard error of the mean (*P<0.01, compared with Con; n=3). BMMs, bone marrow macrophages; DRG, desoxyrhapontigenin; RANKL, receptor activator of nuclear factor-κB ligand; TRAP, tartrate-resistant acid phosphatase; Con, control; M-CSF, macrophage-colony stimulating factor.

To determine whether DRG inhibits the early or late stage of osteoclast formation, the present study investigated the effects of DRG on RANKL-induced osteoclast precursor differentiation by treating at different times between days 1 and 5 post-RANKL stimulation (Fig. 2E). DRG significantly reduced RANKL-induced osteoclast formation at day 1 of treatment. However, DRG was not effective in the suppression of osteoclast formation at day 5 of treatment, suggesting that DRG suppresses RANKL-induced osteoclastogenesis at an early stage.

DRG inhibits actin-ring formation and bone resorption in RANKL-stimulated BMMs

To further investigate the effect of DRG on osteoclast differentiation, the present study determined whether DRG inhibited F-actin ring formation, which is a critical indicator of the bone resorption activity of osteoclasts and is a characteristic feature of mature osteoclasts (33). Under the stimulation of RANKL, BMMs formed the F-actin ring structures, as evidenced by FITC-phalloidin staining (Fig. 3A). Treating the BMMs with DRG significantly decreased the number and size of actin-ring structures in a concentration-dependent manner, suggesting that DRG inhibited the formation of mature osteoclasts. To confirm whether DRG suppressed the bone-resorbing activity of osteoclasts, the effect of DRG on RANKL-induced bone resorption in BMMs was investigated (Fig. 3B). In the presence of RANKL, osteoclasts formed bone-resorption pits. However, DRG markedly reduced the formation of pits formed in the overall osteoassay surface in a concentration-dependent manner. These results demonstrated that DRG suppressed RANKL-induced bone resorption ability.

Figure 3 DRG suppresses RANKL-induced actin-ring formation and resorption pits in BMMs. (A) BMMs were induced to differentiate into osteoclasts by incubating with M-CSF (30 ng/ml) and RANKL (100 ng/ml) in the presence or absence of the indicated concentrations of DRG for 7 days. Actin-ring positive osteoclasts were visualized with fluorescein isothiocyanate-conjugated phalloidin. Data are presented as the mean ± standard error of the mean (*P<0.01, compared with vehicle-treated control; n=3). (B) BMMs were seeded on Corning OsteoAssay Surface 24-well plates and incubated with RANKL (100 ng/ml) and M-CSF (30 ng/ml) in the presence of various concentrations of DRG for 6 days. The cells were washed and pit areas were analyzed with ImageJ software. Data are presented as the mean ± standard error of the mean (*P<0.01, compared with vehicle-treated control; n=3). BMMs, bone marrow macrophages; DRG, desoxyrhapontigenin; RANKL, receptor activator of nuclear factor-κB ligand; M-CSF, macrophage-colony stimulating factor.

DRG suppresses LPS-induced bone loss in vivo

The involvement of DRG in the inhibition of osteoclastogenesis in vivo was assessed using an inflammation-induced bone loss mouse model. The mice were treated with LPS in the presence or absence of DRG. After 9 days, the femurs were examined by micro-CT. 3D analysis revealed that LPS administration appeared to cause trabecular bone loss in the femurs. However, LPS-induced bone loss was considerably reduced in mice who received DRG (Fig. 4A). In correlation with micro-CT images, the reductions of Tb. N and BV/TV by LPS injection were rescued in DRG-treated mice (Fig. 4B). The LPS-induced changes in Tb.Sp and BS/BV were also attenuated by DRG administration (Fig. 4B). These results indicated that DRG suppressed inflammation-induced osteoclast formation in vivo.

Figure 4 DRG suppresses LPS-induced bone loss in vivo. (A) At 9 days following the first LPS injection, mice were sacrificed, their femurs were collected and their three-dimensional images were generated using micro-computed tomography. (B) BV/TV, Tb. N, Tb. Sp, and BS/BV were obtained using the CTAn software. n=4 (eight legs) in each group. *P<0.05, compared with vehicle-treated control. DRG, desoxyrhapontigenin; LPS, lipopolysaccharide; BV/TV, bone volume per tissue volume; Tb. N, trabecular number; Tb. Sp, trabecular separation; BS/BV, bone surface to bone volume.

DRG inhibits the RANKL-induced activation of ERK, c-Fos and NFATc1

The finding that DRG suppressed RANKL-induced osteoclast formation at an early stage prompted an experiment designed to examine the effect of DRG on RANKL-induced early signaling events, including MAPK pathways. BMMs showed increased phosphorylation levels of ERK, p38 and JNK MAPKs in the BMMs upon stimulation with RANKL. Pretreatment with DRG did not decrease the phosphorylation levels of p38 and JNK. However, DRG suppressed the RANKL-induced phosphorylation of ERK (Fig. 5A). Activation of MAPK signaling pathways induces the expression of NFATc1 via activator protein-1 (AP-1) transcription factor (14), which is a heterodimer of the c-Jun and c-Fos transcription factors. Therefore, the present study determined whether DRG suppresses the RANKL-induced expression of c-Fos and NFATc1. Stimulation of the BMMs with RANKL increased the expression levels of c-Fos and NFATc1. However, the RANKL-induced expression of c-Fos and NFATc1 was attenuated by DRG treatment (Fig. 5B), suggesting that DRG suppressed RANKL-induced osteoclastogenesis, at least in part by inhibiting the MAPK/AP-1 signaling pathway.

Figure 5 DRG inhibits RANKL-induced ERK phosphorylation in BMMs. (A) BMMs were stimulated with RANKL (100 ng/ml) for 10 min in the presence of the indicated concentrations of DRG. Western blot analysis was performed to determine the expression levels of p-ERK, p-JNK and p-p38. (B) BMMs were stimulated with RANKL (100 ng/ml) for the indicated periods of time in the presence or absence of 30 μM DRG. Western blot analysis was performed to determine the expression levels of c-Fos and NFATc1. BMMs, bone marrow macrophages; DRG, desoxyrhapontigenin; RANKL, receptor activator of nuclear factor-κB ligand; ERK, extracellular signal regulated kinase; JNK, c-Jun N-terminal kinase; p-, phosphorylated; NFATc1, nuclear factor of activated T cells cytoplasmic 1.

DRG inhibits the expression of NFATc1 target genes

To further examine the role of DRG in the activation of NFATc1, the present study examined the effects of DRG on the expression of marker genes associated with osteoclastogenesis, including CtsK, MMP-9 and TRAP. These three genes are downstream target genes of the NFATc1 pathway. DRG significantly inhibited the expression of these NFATc1 target genes in a time- and concentration-dependent manner during RANKL-induced osteoclastogenesis (Fig. 6).

Figure 6 DRG impairs the RANKL-induced expression of NFATc1 target genes. (A) BMMs were incubated with RANKL (100 ng/ml) in the presence or absence of 30 μM DRG for the indicated periods of time. RT-qPCR analysis was performed to quantify the mRNA expression levels of CtsK, TRAP and MMP-9. Data are presented as the mean ± standard error of the mean (*P<0.01, compared with the RANKL-only treated control; n=5). (B) BMMs were pretreated with the indicated concentration of DRG under the stimulation of RANKL (100 ng/ml) for 4 days. RT-qPCR analysis was performed to quantify the mRNA expression levels of CtsK, TRAP and MMP-9. Data are presented as the mean ± standard error of the mean (*P<0.01, compared with the RANKL-only treated control; n=5). BMMs, bone marrow macrophages; DRG, desoxyrhapontigenin; RANKL, receptor activator of nuclear factor-κB ligand; TRAP, tartrate-resistant acid phosphatase; CtsK, cathepsin K; MMP-9, matrix metalloproteinase-9; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Discussion

Osteoclasts are specialized multinucleated cells, which are able to resorb the collagen matrix of bone. An aberrant increase in RANKL/RANK signaling results in increased osteoclast formation and activity, as reported in various bone loss-related diseases, including postmenopausal osteoporosis, autoimmune arthritis, bone tumors and periodontitis. Therefore, the inhibition of osteoclast differentiation and/or its function may be a potential approach to treat or prevent pathological bone loss. To this end, attention has turned to stilbenes. Stilbenes are a class of plant polyphenols and, due to the potential in therapeutic applications, their derivatives are promising for drug research and development (34). Resveratrol, a naturally occurring stil-bene, and/or a variety of plant food containing resveratrol has a therapeutic potential for treatment or prevention of age-related degenerative diseases such as osteoporosis (35,36). DRG is a stilbene isolated from R. undulatum and has the anti-inflammatory activity (24). However, the effect of DRG on osteoclast differentiation and its underlying molecular mechanism(s) in osteoclastogeneis remain to be elucidated. In the present study, it was demonstrated that DRG suppressed RANKL-induced osteoclastogenesis at the early stages, but had no cytotoxic effect on BMMs. DRG also prevented inflammation-induced bone destruction in the in vivo model. At the molecular level, DRG suppressed the RANKL-induced activation of ERK and NFATc1, and the expression of NFATc1 target genes, including TRAP, CtsK and c-Src. These collective findings support the therapeutic potential of DRG for bone resorption-associated diseases.

M-CSF regulates the proliferation and survival of BMMs, and the differentiation of BMMs to the osteoclast precursor, whereas the differentiation of osteoclast precursors to mature osteoclasts is regulated by RANKL (9). In the present study, it was demonstrated that DRG did not show cytotoxic activity towards the M-CSF-stimulated BMMs. However, DRG did inhibit the RANKL-induced formation of mature osteoclasts from osteoclast precursors, suggesting that DRG suppressed RANKL-induced osteoclastogenesis at the early stage by modulating the RANKL/RANK signaling pathway.

The ERK, JNK, and p38 MAPK pathways are involved in the RANKL/RANK signaling pathway and are important in osteoclastogenesis through their regulation of the expression of NFATc1 via AP-1 transcription factors (9,37). Treating BMMs with JNK- or p38-specific inhibitors suppresses RANKL-induced osteoclast differentiation (38,39). Activation of the ERK signaling pathway is also important for RANKL-induced osteoclastogenesis. Treatment with PD98059, an ERK inhibitor, attenuates osteoclast differentiation (40). In the present study, DRG suppressed the RANKL-induced phosphorylation of ERK and c-Fos, and the expression of NFATc1, suggesting that DRG suppressed RANKL-induced osteoclast differentiation via inhibiting the MAPK/AP-1 signaling pathway.

The present study demonstrated the protective effect of DRG on LPS-induced bone loss in vivo. The administration of DRG at 50 mg/kg suppressed LPS-induced bone loss in a mouse model, as conformed by micro-CT analysis, suggesting that DRG may be effective in treating or preventing inflammation-induced bone loss in vivo by suppressing osteo-clast formation. R. undulatum is a rich source of stilbenes, including DRG and resveratrol. DRG is a major constituent of R. undulatum (28), and the content of DRG in ethanol extract from the dried rhizomes of R. undulatum is ~0.1% (28). The findings in the present study suggested that DRG may be considered as a novel lead compound for the development of a therapeutic or preventive agent against inflammation-induced bone loss. In addition, the DRG-enriched extracts from the rhizomes of R. undulatum may be applied as a supplemental or functional food, having a beneficial effect on inflammation-induced bone.

In conclusion, DRG, a stilbene compound, can suppress RANKL-induced osteoclast differentiation in BMMs and LPS-induced bone destruction in an in vivo model. DRG impaired the RANKL-induced activation of NFATc1 via the MAPK/AP-1 signaling pathway. These findings indicated that DRG may be a valuable stilbene compound for the prevention and/or treatment of osteoclast-associated bone diseases, including rheumatoid arthritis and osteoporosis.

Acknowledgments

The authors would like to thank Mr. Kim Song-Rae (Korea Basic Science Institute, Chuncheon Center, Chuncheon, Gangwon-Do, Republic of Korea) for technical assistance.

References