Fig 1.
Effects of rhinacanthin C on osteoclastogenesis and cell viability in BMM cultures.
A, Chemical structure of rhinacanthin C. B, BMCs were cultured for 3 days with M-CSF (10 ng/mL), then with M-CSF alone or M-CSF plus RANKL (10 ng/mL) in the presence or absence of rhinacanthin C (0.25–2.0 μM). The cells were stained for TRAP activity. Bar, 100 μm. C, Dose-dependent effects of rhinacanthin C on RANKL-induced TRAP-positive multi-nuclear (>3 nuclei) cell (MNC) formation from BMCs. D, Dose-dependent rhinacanthin C inhibition of TRAP activity in the medium of BMM cultures in the presence of RANKL and M-CSF. E, Viability was determined by MTT assay after 3 days. Data are expressed as means ± SD of three experiments. **P < 0.01 vs. untreated controls.
Fig 2.
Effects of rhinacanthin C on bone resorption in BMM cultures.
A, BMCs were cultured on dentin slices for 3 days with M-CSF (10 ng/mL), then for 6 days in the presence or absence of rhinacanthin C in the presence of RANKL (10 ng/mL). After removal of the cells, areas of resorption pits were stained with hematoxylin. Bar, 100 μm. B, total resorption pit area was measured and the results are shown as % of RANKL treatment. Data are expressed as means ± SD of three experiments. **P < 0.01 vs. untreated control.
Fig 3.
Effects of rhinacanthin C on induction of osteoclast-related proteins by RANKL.
A, BMCs were cultured for 3 days with M-CSF (10 ng/mL), then for 2 days in the presence or absence of rhinacanthin C (RC, 1 μM) and RANKL (R, 10 ng/mL). Whole-cell extracts were analyzed by western blotting with primary antibodies raised against NFATc1, c-Fos, c-Src, and integrin β3. β-Actin was used as an internal control. B, Proteins were normalized to β-actin and expressed as the fold change relative to the untreated control (Ctl). Values are the means ± SD of three to four independent experiments. **P < 0.01.
Fig 4.
Effects of rhinacanthin C on RANKL-stimulated signaling in BMM.
A, BMCs were cultured for 3 days with M-CSF (10 ng/mL), then with RANKL (R, 10 ng/mL) for 10 min in the presence or absence of rhinacanthin C (RC, 1 μM). Whole-cell extracts were analyzed by western blotting. B, The levels of phosphorylated proteins were quantified, normalized to the total levels of corresponding proteins, and expressed as the fold change vs. the untreated control (Ctl). Values are the means ± SD of three to four independent experiments. *P < 0.05, **P < 0.01.
Fig 5.
Effects of rhinacanthin C on complex formation of TRAF6 and TAK1.
A, BMCs were cultured for 3 days with M-CSF (10 ng/mL), then pretreated with rhinacanthin C (RC, 1 μM) or DMSO (0.1%) for 20 min and stimulated with RANKL (R, 10 ng/mL) for 5 min with or without rhinacanthin C. Cell lysates were immunoprecipitated (IP) with anti-TRAF6 or anti-TAK1 and immunoblotted with anti-TAK1 or anti-TRAF6, respectively (upper panel). Expression of TRAF6 and TAK1 in cell lysates was determined by immunoblotting (lower panel). B, The level of co-immunoprecipitated TAK1 or TRAF6 was quantified and normalized to total TRAF6 or TAK1, respectively. Data are expressed as the fold change vs. the untreated control. Values are the means ± SD of three independent experiments. *P < 0.05, **P < 0.01.
Fig 6.
Protective effects of rhinacanthin C on RANKL-induced mouse calvarial osteolysis.
Vehicle (30% DMSO) or rhinacanthin C (RC, 2 mg/kg body weight) with or without RANKL (0.7 mg/kg body weight) was daily injected into the subcutaneous tissue overlying the calvaria of 8-week-old ddy mice. The mice were sacrificed on day 5. A, TRAP staining of whole calvaria (upper) and high magnification of TRAP stain on calvaria (lower). Bar, 400 μm. B, TRAP+ area was measured by densitometry using Image J.C, Three-dimensional micro-CT image of calvaria. D, Bone volume/total tissue volume ratio (BV/TV). E, Trabecular separation (Tb.Sp). F, Trabecular bone pattern factor (TBPf). *P < 0.05, **P < 0.01.
Fig 7.
Rhinacanthin C inhibits LPS-induced osteoclastogenesis in RANKL-primed BMMs.
A, BMMs were primed with RANKL (1 ng/mL) for 24 h, then the culture medium were washed away and the cells were cultured with or without LPS (200 ng/mL) or LPS plus rhinacanthin C (RC, 1 μM). After 3 days, cells were stained with TRAP. Bar, 100 μm. B, TRAP activities were measured. C, TRAP-positive multinucleated osteoclasts. *P < 0.05, **P < 0.01.
Fig 8.
Protective effects of rhinacanthin C on LPS-induced bone destruction in vivo.
Vehicle (30% DMSO) or LPS (3.3 mg/kg body weight) with or without rhinacanthin C (2 mg/kg body weight) was daily injected into the subcutaneous tissue overlying the calvaria of 8-week-old wild type (normal) and OPG-/- mice. The mice were sacrificed on day 5. (A) TRAP staining of whole calvaria and 3D-images. (B) Quantitative data of calvarial bone by μCT analysis. BV/TV: Bone volume/total tissue volume ratio. Tb.Sp: Trabecular separation. TBPf: Trabecular bone pattern factor. *P < 0.05, **P < 0.01.