RANKL was purchased from PeproTech (London, UK). Dulbecco's modified Eagle's medium (DMEM) was purchased from Welgene (Daejeon, Korea). Cell medium minimum essential medium-α (α-MEM) and fetal bovine serum (FBS) were both purchased from Gibco (Gaithersburg, NY, USA). Penicillin/streptomycin was purchased from Invitrogen (Carlsbad, CA, USA). Dulbecco's phosphate-buffered saline (DPBS) was obtained from Gibco. An aqueous non-radioactive cell proliferation kit (for MTS assay) was purchased from Promega (Madison, WI, USA). A TRAP staining kit was purchased from Sigma-Aldrich (St. Louis, MI, USA). An Osteo Assay Stripwell plate was purchased from Corning Inc. (New York, NY, USA). We obtained the reverse transcription kit from Invitrogen. Taq polymerase was obtained from Kapa Biosystems (Woburn, MA, USA). PCR primers were from Genotech (Daejeon, Korea). Primary antibodies against c-Fos (Cat. no. sc-447) and β-actin (Cat. no. sc-8432) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and NFATc1 antibody (Cat. no. 556602) was obtained from BD Pharmingen (San Diego, CA, USA). Peroxidase IgG secondary antibody (Cat. no. 115-035-062) was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Protease inhibitor cocktail and phosphatase inhibitor cocktail were both purchased from Sigma-Aldrich.

LRC was purchased from Kyung Hee University Medical Center. The extract was prepared by decocting 300 g of the dried herb using 3 liters of boiling distilled water for 2 h and filtered using filter paper (no. 3; Whatman, Maidstone, UK). The extract was concentrated using a rotary evaporator (Eyela, Tokyo, Japan), lyophilized, and it yielded 11.1 g dried powder (yield ratio 3.7%), and the extract was then stored at −20°C until use.

Murine macrophage RAW 264.7 cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO₂ in air. For the cytoxicity assay, RAW 264.7 cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin and plated in a 96-well plate at 5×10³ cells/well. After 24 h, various concentrations of LRC were added to the medium for 24 h. MTS solution was added at 20 μl/well. The plate was incubated for 2 h at 37°C. Cell viability was measured using an enzyme-linked immunosorbent assay (ELISA reader; Molecular Devices, Sunnyvale, CA, USA) at 562 nm optical density.

In order to study osteoclast differentiation, RAW 264.7 cells were cultured in α-MEM with 10% FBS and 1% penicillin/streptomycin in a 96-well plate 5×10³ cells/well. After 24 h, the α-MEM was changed for 10% FBS and 1% penicillin/streptomycin. RANKL (100 ng/ml) and various concentrations of LRC were added to the media for 5 days. The medium was changed every 2 days. After 5 days, multinucleated osteoclasts were observed. Mature osteoclasts were washed with DPBS and fixed in 10% formaldehyde for 10 min. After being fixed, cells were stained using a TRAP staining kit (Sigma-Aldrich) according to the manufacturer's instructions. Cells were washed with deionized water. We then counted TRAP-positive multinucleated (>3 nuclei) under an inverted microscope (Olympus, Tokyo, Japan). In order to measure TRAP activity, we added 50 μl supernatant to a 96-well plate, dissolved 4.93 mg Pnpp in 850 μl 0.5 M acetate, mixed it with 150 μl tartrate acid solution and separated it (50 μl each) into 96-well plates. After 30 min, we added 50 μl 0.5 M NaOH and measured optical density at 405 nm using an ELISA reader.

RAW 264.7 cells were cultured in α-MEM with 10% FBS and 1% penicillin/streptomycin and plated in an Osteo Assay Stripwell plate at 5×10³ cells/well. After 24 h, α-MEM was changed for 10% FBS and 1% penicillin/streptomycin. RANKL (100 ng/ml) and various concentrations of LRC were added to the medium for 5 days. The medium was changed every 2 days. After 5 days, multinucleated osteoclasts were observed. The mature osteoclasts were then lysed by 4% NaClO and lysates were washed with deionized water. The pit area in each plate was studied and measured under an inverted microscope.

In order to generate osteoclasts, RAW 264.7 cells were cultured with α-MEM, 10% FBS and 1% penicillin/streptomycin. RANKL (100 ng/ml) was added for 4 days in the presence of various concentrations of LRC. Total RNA was prepared using TRIzol (Takara Bio, Otsu, Japan) according to the manufacturer's instructions. The concentration of total RNA was determined (absorbance at 260 and 280 nm) with a NanoDrop 2.0 spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA, USA), and 2 μg total RNA was generated using a reverse transcription kit (Invitrogen) according to the manufacturer's instructions. The PCR cycles were as follows: 22–40 cycles of 1 min at 94°C (denaturation), 1 min at 55–58°C (annealing), and a 1-min 72°C (extension), using Taq polymerase. Primer sequences are shown in Table I. The cDNA samples after reaction were separated on a 1–1.2% agarose gel, stained with SYBR-Green (Invitrogen) and studied using ImageJ software.

The cells incubated with various concentrations of LRC (1, 10 and 100 μg/ml) were washed with cold DPBS, and cells were lysed in lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, protease inhibitor cocktail, phosphatase inhibitor cocktail) and incubated on ice for 30 min. After centrifugation at 13,200 rpm for 20 min at 4°C, the supernatants were stored at −70°C until use. Protein concentration was calculated using a BCA protein assay kit (Thermo Fisher Scientific). The protein samples (30 μg) were separated by 10–15% SDS-PAGE and transferred to a nitrocellulose membrane (Whatman, Dassel, Germany). We then blocked them with 5% skimmed milk for 1 h, and the membrane was then incubated with primary antibodies, namely NFATc1, c-Fos and actin, in 1% BSA solution at 4°C overnight. Subsequently, the membrane was probed with the secondary antibody. The protein was detected using ECL solution (Santa Cruz Biotechnology, Inc.).

The animal experiments were conducted in compliance with the principles of the Institutional Animal Care and Use Committee of Kyung Hee University Laboratory Animal Center: the permission no. is KHUASP (SE)-13-051. Sprague-Dawley (SD) rats were purchased from Nara Biotech (Seoul, Korea). The 17β-estradiol (E₂) was from Sigma-Aldrich. Twelve-week-old female Sprague-Dawley rats weighing 240–250 g were used. Rats were housed at 22±1°C in an atmosphere with 55±10% humidity on a 12 h light/dark cycle with free access to food and water. After acclimatization to the laboratory environment for 1 week, rats were divided into 5 groups (8 rats/group), i) sham-operated rats, ii) OVX rats, iii) OVX rats which received 17β-estradiol (100 μg/kg p.o.), iv) OVX rats treated with low doses of LRC (5 mg/kg p.o.), and v) OVX rats treated with high doses of LRC (50 mg/kg p.o.).

After the operation, sham-operated and OVX rats were administrated distilled water orally. After the ovariectomy, the rats were treated 5 times/week for 8 weeks and weighed each week. At the end of the treatment, the rats were euthanized while under deep anesthesia with high doses of pentobarbital sodium (80 mg/kg) and blood samples were acquired by cardiac puncture for biochemical analyses. The uteri and both sides of the femurs were dissected out and immediately weighed on an electronic scale. The femurs were then fixed in 10% neutral buffered formalin (NBF) for 2 days and decalcified using 10% ethylene-diaminetetraacetate (EDTA) for 3 weeks. The samples, which were cut into 5-μm thick sections using a microtome (Zeiss, Oberkochen, Germany), were embedded in paraffin. Tissues were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). Histopathological changes were observed using light microscopy (at ×40 and ×100 magnification).

At the end of treatment, the rats were sacrificed and the bone density of the right femurs was measured using Archimedes' principle, as previously described (22). Right femurs were briefly placed in vials filled with deionized water. The vials were placed in a vacuum for 90 min to ensure that all trapped air diffused from the bones. Right femurs were removed from the vials, dried with gauze, weighed, and placed in new vials containing deionized water. The bones were reweighed in the water. As previously described, using Archimedes' principle, bone density was calculated (g/cm³ bone volume) (23).

In this study, the data are presented as the means ± SEM. Statistical analysis was performed using the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). One-way ANOVA was used to evaluate the treatment effect, followed by Dunnett's multiple comparison test. A p-value <0.05 was considered to indicate a statistically significant difference.

Prior to the in vitro tests, we measured the effects of LRC on cell viability. As shown in Fig. 1, all concentrations of LRC exerted equivalent effects on cell viability. LRC was not cytotoxic to RAW 264.7 cells. Thus, the following experiments were undertaken using a range of LRC concentrations (1, 10 and 100 μg/ml).

To examine the effect of LRC on osteoclastogenesis, RAW 264.7 cells exposed to RANKL were stained using a TRAP staining kit. RAW 264.7 cells were stimulated with RANKL to differentiate into TRAP-positive cells, and we noted that 100 μg/ml LRC significantly decreased the number of TRAP-positive cells (Fig. 2A and B). Furthermore, as shown in Fig. 2C, it was also clear that LRC exerted an inhibitory effect on TRAP activity.

The effect of LRC on pit formation of mature osteoclasts was studied. As shown in Fig. 3A, the area of resorption pit lacunae was significantly reduced by treatment with LRC. It was clear that the measured areas markedly decreased after treatment with LRC, in a dose-dependant manner (Fig. 3B).

In order to confirm the inhibitory effect of LRC on osteoclastogenesis and bone resorption, we measured the expression of important osteoclast differentiation indicators, NFATc1. NFATc1 is known to be a master transcription factor in osteoclastogenesis (4). Thus, in the present study, mRNA and protein levels of NFATc1 were measured. As shown in Fig. 4, we noted that the levels were significantly upregulated upon exposure to RANKL, and LRC exerted a marked inhibitory effect on mRNA and protein expression levels. In addition, we noted that LRC did not markedly affect the expression of housekeeping genes such as GAPDH and actin.

We measured the expression of c-Fos, which contributes to osteoclastogenesis via the downregulation of NFATc1. We measured the effect of LRC on c-Fos mRNA and protein levels. As shown in Fig. 5A, the mRNA expression of c-Fos was induced slightly in normal cells, and in RAW 264.7 cells exposed to RANKL we noted significantly increased mRNA expression of c-Fos compared with normal cells. LRC exerted a marked inhibitory effect on mRNA expression. As shown in Fig. 5B, the protein levels of c-Fos were also significantly upregulated upon exposure to RANKL, whereas LRC clearly exerted a significant inhibitory effect on the protein levels. Moreover, we noted that LRC did not markedly affect the expression of the housekeeping genes GAPDH and actin.

In the present study, we also examined the effect of LRC on osteoclastogenesis-related genes stimulated by RANKL, namely RANK, TRAP, CTK, MMP-9, CTR and carbonic anhydrase II (CAII). As shown in Fig. 6, the mRNA expression of RANK was induced, at a low level, in normal cells, and in RAW 264.7 cells exposed to RANKL we noted significantly increased mRNA expression of RANK compared with normal cells. LRC exerted significant inhibitory effects on RANK expression at concentrations of 10 and 100 μg/ml. Moreover, the mRNA expression of TRAP, CTK, MMP-9, CTR and CAII were significantly upregulated upon exposure to RANKL. LRC exerted a marked inhibitory effect on TRAP and MMP-9 expression at concentrations of 100 μg/ml. In addition, we noted that LRC markedly reduced the expression of CTK and CTR at all concentrations and significantly inhibited the expression of CAII at concentrations of 10 and 100 μg/ml.

We aimed to investigate whether LRC prevents ovariectomy-induced bone loss. As shown in Fig. 7A, after the rats underwent the ovariectomy, the body weight of the OVX group significantly increased (as measured weekly) compared with the sham-operated group. In the LRC-treated groups, no significant changes in body weight were noted, and in the E₂-treated group body weight was significantly inhibited compared with the OVX group. In the OVX group, a significant decrease in the weight of the uterus compared with the sham-operated group was noted (Fig. 7B). In the groups treated with low and high concentrations of LRC, no significant changes in uterus weight compared with the OVX group were noted. In the E₂-treated group, a significant decrease in uterus weight loss compared with the OVX group was clear. We noted in the OVX group significantly decreased femur weight compared with the sham-operated group (Fig. 7C). In the groups treated with low and high concentrations of LRC and the E₂-treated group, no significant changes in femur weight were noted compared with the OVX group. We also measured the bone density of the femurs using Archimedes' principle (Fig. 7D). The OVX group showed significantly decreased bone density compared with the sham-operated group. Moreover, in the LRC-treated groups, we noted a significant decrease in bone density loss in femurs compared with the OVX group. In the E₂-treated group, we also noted decreased bone density loss compared with the OVX group. Thus, our results indicate that LRC decreased bone density loss without markedly affecting body weight, femur weight or uterine weight.

To determine the in vivo effect of LRC on OVX-induced bone loss, we used histological staining on the femur samples. In the OVX group, we noted a significantly decreased trabecular area compared with the sham-operated group (Fig. 8B–F). In the E₂-treated group the trabecular area loss was decreased compared with the OVX group. In the group treated with low concentrations of LRC, only a small decrease in trabecular area loss was noted compared with the OVX group. However, in the group treated with a high concentration of LRC a significant decrease in trabecular area loss compared with the OVX group was noted.