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).