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Abstract
Osteoclasts, derived from multipotent myeloid progenitor cells, play homeostatic roles in skeletal modeling and remodeling, but may also destroy bone in pathological conditions such as osteoporosis and rheumatoid arthritis. Osteoclast development depends critically on a differentiation factor, the receptor activator of NF-κB ligand (RANKL). In this study, we found that the hexane soluble fraction of the common fig Ficus carica (HF6-FC) is a potent inhibitor of osteoclastogenesis in RANKL-stimulated RAW264.7 cells and in bone marrow-derived macrophages (BMMs). HF6-FC exerts its inhibitory effects by suppression of p38 and NF-κB but activation of ERK. In addition, HF6-FC significantly decreased the expression of NFATc1 and c-Fos, the master regulator of osteoclast differentiation. The data indicate that components of HF6-FC may have therapeutic effects on bone-destructive processes such as osteoporosis, rheumatoid arthritis, and periodontal bone resorption.
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Fig. 2.
Effect of HF6-FC on RANKL-induced osteoclastogenesis in RAW 264.7 cells. (A) RAW 264.7 cells were cultured in the presence of RANKL (50 ng/ml) for 6 days. HF6-FC was added to the culture medium at final concentrations of 0.1, 1, and 10 μg/ml, and cells were stained for TRAP activity on day 6. (B) TRAP-positive multi-nucleated osteoclasts were counted. Data represent the mean±SD of three independent experiments. ∗p<0.05 and ∗∗p<0.01, as compared to the control without HF6-FC, respectively. (C) RAW 264.7 cells were seeded into 96-well plates and incubated with HF6-FC for 24 h. Cell proliferation was evaluated with the MTT assay.
Fig. 3.
Effect of HF6-FC on RANKL-induced osteoclastogenesis in bone marrow-derived macrophages (BMM). (A) BMMs were cultured in the presence of M-CSF (20 ng/ml) and RANKL (100 ng/ml) for 3 days. HF6-FC was added to the culture medium at final concentrations of 0.1, 1, and 10 μg/ml. (B) After three days, cells were fixed and stained for TRAP, and TRAP-positive multinuclear cells (MNC) were counted. Data represent the mean±SD of three independent experiments. ∗∗p<0.01 versus the control without HF6-FC.
Fig. 4.
Effect of HF6-FC on MAPK activation by RANKL in RAW 264.7 cells. Cells were serum-starved for 16 h, pretreated with or without HF6-FC (10 μg/ml) for 30 min, and stimulated with RANKL (100 ng/ml) for times indicated. Whole cell lystes were used for western blotting with MAPK-specific antibodies. Blots were stripped and reprobed with other antibodies.
Fig. 5.
Effect of HF6-FC on NF-κB activation by RANKL in RAW 264.7 cells. Cells were serum-starved for 16 h, pretreated with or without HF6-FC (10 μg/ml) for 30 min and stimulated with RANKL (100 ng/ml) for indicated times. Whole cell lysates were immunoblotted with antibodies specific for phospho-IκB-α and phospo-p65. Blots were stripped and reprobed with control antibodies. The histograms represent the level of the phospho-IκB-α (A) and phospo-p65 (B). The asterisk (∗) indicates a significant difference (p<0.05) compared with the control (#) at same time period.
Fig. 6.
Effects of HF6-FC on c-Fos protein and NFATc1 mRNA in RANKL-stimulated RAW 264.7 cells. Cells were serum-starved for 16 h, pretreated with or without HF6-FC (10 μg/ml) for 30 min and stimulated with RANKL (100 ng/ml) for times indicated. (A) Whole cell lysates were analyzed by immunoblotting with c-Fos-and β-actin-specific antibodies. (B) NFATc1 mRNA level was determined by RT-PCR and compared with that of GAPDH. The asterisk (∗) indicates a significant difference (p<0.05) compared with the control (#) at same time period.
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