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.