3.2 NVP-BHG712 inhibited RANKL-induced osteoclastogenesis and bone resorption in vitro.
To investigate the cytotoxicity of NVP-BHG712, we observed the effects of NVP-BHG712 at 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 μM on the proliferation of BMMs and RAW264.7 cells. After 72 hours of treatment with 0~0.4 μM NVP-BHG712, the ratio of BMMs and RAW264.7 cells was 138% and 154%, respectively. When the concentration was 0.8 to 1.6 μM, NVP-BHG712 promoted cell proliferation. However, when the concentration was increased to 3.2 μm, NVP-BHG712 significantly inhibited the proliferation of both cell lines and caused obvious cytotoxicity. The results indicated that there was no significant cytotoxicity when the concentration of NVP-BHG712 was lower than 1.6 μM (Fig. 2A). To further assess the effects of NVP-BHG712 on osteoclastogenesis, a RANKL-induced osteoclastogenesis assay was carried out in BMMs that were exposed to various concentrations of NVP-BHG712 (0, 0.1, 0.2, 0.4, 0.8, and 1.6 µM). The numbers of TRAP-positive multinucleated cells were counted to assess RANKL-induced osteoclast differentiation. As the results showed, the number of TRAP-positive multinucleated cells was increased when treated with NVP-BHG712 at 0~0.2 µM and dramatically decreased when treated with NVP-BHG712 at 0.4~1.6 µM (Fig. 2B, C).
Furthermore, a pit-formation assay was carried out to determine whether osteoclast functions were impaired. Bone resorption pits on bone slices are the direct result of bone resorption and are also the most reliable indicator of bone resorption function in vitro. BMMs exposed to RANKL were treated with different concentrations of NVP-BHG712 (0, 0.1, 0.2, or 0.4 μM), and then, the area of bone lacunae absorption was observed. The results showed that bone resorption activity was attenuated by NVP-BHG712 (Fig. 2D, E).