Figure 5. NVP-BHG712 did not affect the expression of osteoclast differentiation-related receptors.
A-H, Representative quantification of the mRNA expression of IP3R1 (A), IP3R2 (B), IP3R3 (C), NFATc1 (D), OC-STAMP (E), DC-STAMP (F), αV-integrin (G) and Atp6v1c1 (H) in osteoclasts derived from RANKL-treated BMMs treated with or without 0.4 μM NVP-BHG712 by qPCR. I, J Representative immunofluorescence staining images of c-Fms in RANKL-treated BMMs treated with or without 0.4 μM NVP-BHG712 (I) and the quantification of c-Fms (J) expression. K, L Representative immunofluorescence staining images of RANK in RANKL-treated BMMs treated with or without 0.4 μM NVP-BHG712 (K) and the quantification of RANK (L) expression. The data are presented as the mean ± SD (n=3) (*P <.05, *P <.01, ***P <.001)
Figure6. NVP-BHG712 attenuated bone loss in ovariectomized mice.
C, Serum ALP (B) and TRACP-5b (C) levels in the Sham group, ovariectomized group and ovariectomized groups treated with different concentrations of NVP-BHG712 (5, 10, 20, or 40 mg kg-1) for 5 weeks. C-E, Representative microcomputed tomography (μCT) 2D (C) and 3D (D) reconstruction images of distal femurs from the Sham group, ovariectomized group and ovariectomized groups treated with different concentrations of NVP-BHG712 (5, 10, 20, or 40 mg kg-1) for 5 weeks. Quantitative morphometric properties of distal femurs showing bone mineral density (BMD), bone volume (BV), bone surface/volume ratio (BS/BV), bone surface density (BS/TV), fractional bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp) and trabecular pattern factor (Tb.Pf) (F). n=5. G, H H&E staining images (G) and statistical analysis (H) of mouse femur slices. Scale bars = 40 μm. All the experiments were performed with five biological replicates per group without independent repetition. The data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA. (*P <.05, *P <.01, ***P <.001)
Figure 7. NVP-BHG712 was not toxic in ovariectomized mice.
A-F, Changes in the body weights of mice before each feeding (A), heart index (B), spleen index (C), kidney index (D), forelimb grip-strength index (E), and heart weight/tibial length (F) in the Sham group, ovariectomized group and ovariectomized groups treated with different concentrations of NVP-BHG712 (5, 10, 20, or 40 mg kg-1) for 5 weeks. G, Schematic diagram of the mechanism by which NVP-BHG712 inhibits osteoclast function. With skeletal aging and menopause, osteoclastic bone resorption activity increases relatively or absolutely. During osteoclast bone resorption, CTSK is released (purple dots) from the bone matrix into the bone marrow, where CTSK and other cytokines promote bone resorption. NVP-BHG712 inhibits osteoclast bone resorption by inhibiting the function of the CTSK protein, thereby slowing the loss of bone mass associated with aging or postmenopausal bone. All the experiments were performed with five biological replicates per group without independent repetition. The data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA.