JKAOH Journal of Korean Acadomy of Oral Health
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Fig. 3. Effects of three bile acids on osteoclast differentiation of RAW 264.7 cells stimulated by RANKL. (A-C) RAW 264.7 cells (2×103 cells/well) seeded in 96-well plates and treated with RANKL (100 ng/ml) combined with M-CSF (30 ng/ml) in the absence or presence of three bile salts in the range of 0-100 or 0-1,000 µM. After 4 days, the fixing of cells and TRAP staining was carried out. TRAP-positive multinucleated cells (with ≥3 nuclei) were regarded as osteoclast or OLCs. The treatment of RANKL and M-CSF (only RANKL group) stimulated the cells to differentiate to multinucleated cells as osteoclast or OLCs. However, the bile salts treatments did not suppress osteoclast differentiation of the cells at most concentrations. (A, B) While sodium taurodeoxycholate and sodium taurocholate were no effect on the inhibition of osteoclastogenesis, (C) sodium glycocholate at 1,000 µM (the highest concentration) significantly suppressed osteoclast differentiation of RAW 264.7 cells (P<0.001) (D) as showed in the pictures of TRAP staining for sodium glycocholate treatment group. The RANKL only group presented many multinucleated giant cells as the cells indicated red arrows, while the cells with 1,000 µM sodium glycocholate were similar to ones of the control. Results were expressed by relative number (ratio) of TRAP positive-OLCs to the only RANKL group. Bar and error bar present the mean±S.D. of relative ratio to the only RANKL without bile salt as a reference value (1.00). ***P<0.001 significantly different from the only RANKL group. Each point is repeatedly measured (n=4). RANKL, activating receptor activator of nuclear factor-κB ligand; M-CSF, macrophage colony-stimulating factor; TRAP, tartrate-resistant acid phosphatase; OLCs, osteoclast-like cells.
J Korean Acad Oral Health 2020;44:205~213 https://doi.org/10.11149/jkaoh.2020.44.4.205
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