3.6 NVP-BHG712 attenuated bone loss in ovariectomized mice.
The decrease in estrogen during postmenopausal osteoporosis leads to an
increase in osteoclast activity, bone resorption rates become greater
than bone formation rates, and the bone turnover rate increases, leading
to bone loss related to high turnover. Ovariectomized female mice are
widely used as a postmenopausal osteoporosis model in
research(C. Y. Chen et al., 2019; K. Chen et al.,
2019). Thus, we established OVX models in 8-week-old female mice and
tested whether the intragastric administration of NVP-BHG712 every 3
days for five weeks could attenuate bone mass loss in these OVX female
mice. (Fig. 6A). The effect of NVP-BHG712 on bone mass in ovariectomized
mice was confirmed by measuring the serum TRACP-5b levels by ELISA and
the serum ALP levels with a microplate reader. The results of TRACP-5b
measurement showed that the serum TRACP-5b levels in the mice in the OVX
group were significantly increased compared with those in the Sham group
(Fig. 6B), which indicated that the model of postmenopausal osteoporosis
was successfully established. Similar results were observed in the
measurement of ALP activity, which was lower in the OVX group than in
Sham group (Fig. 6C). Both of these results proved the success of the
operation. After administration of NVP-BHG712 at a concentration of
10-40 mg kg-1, the serum TRACP-5b levels were
significantly decreased in the mice. The serum ALP levels of the mice
were significantly increased after the administration of NVP-BHG712 at a
concentration of 5~40 mg kg-1.
Together, these results suggest that gavage of NVP-BHG712 can be used in
the treatment of high turnover osteoporosis.
As shown in Fig. 5D-F, the NVP-BHG712-treated OVX mice had the lower
trabecular bone mass phenotypes than the OVX mice, as revealed by the 2D
and 3D
µCT
reconstructed images of distal femurs, the much higher BMD, BS/TV,
BV/TV, Tb.N, Tb.Th values, and the lower BS/BV, Tb. Pf, Tb.Sp values.
These results revealed that NVP-BHG712 increased the bone mass in OVX
mice. The results of HE staining and quantitative analysis of the distal
femur showed that NVP-BHG712 increased the density and number of
trabecular bones in OVX mice (Fig. 6G, H). Together, these findings
indicate that NVP-BHG712 exerts a protective effect against bone loss in
ovariectomized mice, which is related to the decreased osteoclast
resorption induced by NVP-BHG712.
After the operation, we recorded the weight of the mice before each
feeding, and the results showed that the weight of the mice in the OVX
group, different NVP-BHG712 treatment groups and Sham group increased
gradually, and no significant weight loss was observed (Fig. 7A). The
organ index of the mice was calculated as the ratio of the organ weight
to the body weight of the mice. Compared with those of the Sham group,
the heart index, spleen index, kidney index and tibia index of the OVX
group did not change significantly, and gavage with different doses of
NVP-BHG712 did not cause significant differences compared with OVX alone
(Fig. 7B-E). No differences in the heart weight/tibia length results
were observed between the different NVP-BHG712 groups and the OVX group
(Fig. 7F). Based on these results, we found that NVP-BHG712 did not
cause toxicity in ovariectomized mice when administered by gavage at a
dose of 40 mg kg-1.
In conclusion, our findings identified a novel compound for the
prevention of postmenopausal osteoporosis (Fig. 7). With skeletal aging
and menopause, osteoclastic bone resorption activity increases
relatively or
absolutely.
During osteoclast bone resorption, CTSK is released 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
that is associated with aging or postmenopausal bone.
Discussion
Our investigation to understand the role of NVP-BHG712 in osteoclasts
establishes that CTSK is the target of NVP-BHG712 and promotes
RANKL-induced osteoclast differentiation and maturation. This
assertion
stems from the strong binding of NVP-BHG712 to each of the four
molecular conformations of CTSK and its inhibitory effect on
RANKL-induced osteoclast differentiation and maturation. When compared
with other compounds that inhibit osteoclast differentiation and
maturation, several striking features emerge in our results. In
particular, to our knowledge, the magnitude with which NVP-BHG712
inhibits osteoclast differentiation and maturation is matched by
OCN.
Through a literature search, it was found that NVP-BHG712 had not been
previously reported in studies on CTSK targets and osteoclasts.
EphrinB2/EphB4 signaling pathway was activated to inhibit osteoclast
differentiation, while decreased osteoclast maturation and
differentiation was observed when NVP-BHG712, an EphB4-specific
inhibitor, was added, suggesting that EphB4 inhibition was not the
primary role of NVP-BHG712 in this process. Therefore, in this study, we
did not continue to investigate the role of Ephrin signaling pathways.
Therefore, we did not continue to investigate the role of Ephrin
signaling pathways. We observed a decrease in osteoclast maturation and
differentiation with the addition of NVP-BHG712, so we speculate that
inhibition of EphB4 to promote osteoclast maturation is not the primary
effect of NVP-BHG712 in this process. Therefore, we did not continue to
investigate the role of Ephrin signaling pathways. Moreover, we found
that in vitro osteoclast proliferation can be promoted by NVP-BHG712,
but bone resorption can be inhibited by NVP-BHG712, indicating that
NVP-BHG712 can inhibit osteoclast resorption. When we explored the time
at which NVP-BHG712 affected osteoclasts, we found that NVP-BHG712
mainly inhibited osteoclast differentiation and maturation in the early
stage. Additionally, our findings show that the formation of the F-actin
loop, which is used to demonstrate osteoclast function, is mainly
inhibited in the early stage after NVP-BHG712 treatment at different
time periods. However, NVP-BHG712 could not reverse the differentiation
and maturation of osteoclasts because mature osteoclasts were still
formed when NVP-BHG712 was added at a later stage. We hypothesize that
once CTSK-dependent osteoclast maturation is disrupted, the formation of
the bone resorption microenvironment is blocked, and bone resorption
function is lost (Fig. 6G).
CTSK plays a role in bone resorption, the immune system, tumorigenesis
and invasion, the circulatory system, the cardiovascular system and
other systems and participates in extracellular matrix remodeling in
different organs. Under acidic conditions, CTSK can bind to collagen to
degrade it, leading to increased bone fragility(Gruber,
2015; Mujawar et al., 2009). Furthermore, because NVP-BHG712 is a
CTSK-targeting compound that was identified by molecular docking, we
hypothesized that NVP-BHG712 inhibits the function and expression of
CTSK. Interestingly, Western blotting results showed that NVP-BHG712
inhibited the protein expression of CTSK and inhibited the protein
expression of MMP9 and CTR, which are markers related to osteoclast
differentiation.
In addition, we also found an inhibitory effect of NVP-BHG712 on IP3R1,
IP3R3 and OC-STAMP mRNA expression, among which IP3R1 and IP3R3 are
related to intracellular calcium oscillation, and OC-STAMP is related to
osteoclast maturation. c-Fms and RANK receptors play an important role
in the proliferation and differentiation of osteoclast precursor cells.
M-CSF participates in the proliferation of osteoclast precursor cells by
binding to the c-Fms receptor, and RANKL participates in the
differentiation of osteoclast precursor cells by binding to the RANK
receptor. Our immunofluorescence staining results showed that NVP-BHG712
had no inhibitory effect on the expression of c-Fms and the RANK
receptor, which indicated that the regulation of osteoclast
proliferation, differentiation and maturation by NVP-BHG712 did not
occur through the M-CSF/c-Fms and RANKL/RANK signaling pathways.
The pathogenesis of PMOP involves bone remodeling caused by an imbalance
in bone resorption and bone formation(Darby, 1981).
Estrogen withdrawal leads to increased osteoclast production, enhanced
bone resorption, and substantial bone loss(Kameda et
al., 1997; Mundy, 2007). Therefore, targeting overactivated osteoclasts
provides an effective therapeutic option for PMOP. Osteoclast
hyperactivation promotes excessive bone loss in the pathophysiology of
PMOP. We further confirmed our in vitro findings by modelling the
pathological state of PMOP in an ovariectomized mouse model. Before the
start of the experiment, we measured the bone mass of 8-week-old female
mice at 3-6 weeks after ovariectomy by H&E staining of femur sections.
It was found that the trabecular bone density of mice was significantly
reduced 5 and 6 weeks after ovariectomy. Therefore, NVP-BHG712
administration for 5 weeks after the operation can be used to explore
its effect in vivo. By measuring the serum levels of TRACP-5b and ALP,
we found that NVP-BHG712 significantly inhibited osteoclast bone
resorption activity. Using microcomputed tomography and H&E staining of
the distal femur, we found that NVP-BHG712 significantly attenuated the
loss of bone mass in mice after ovariectomy. In conclusion, NVP-BHG712
inhibited osteoclast maturation and bone resorption in vivo and
prevented bone loss caused by ovariectomy. Additionally, it was observed
that the body weights of the mice in the Sham group, OVX group and
different NVP-BHG712 treatment groups were continuously increased and
did not decrease. The cardiac index, spleen index, kidney index, tibial
index, and heart weight to tibial length ratio were not significantly
different in OVX mice treated with different concentrations of
NVP-BHG712 (Fig. 6B-E), suggesting that 0~40 mg
kg-1 NVP-BHG712 was not toxic to mice in vivo. Our
results suggest that NVP-BHG712 at 0~40 mg
kg-1 concentration has a protective effect on bone
loss in ovaries excised mice, but the effect and toxicity of NVP-BHG712
at higher concentration need further investigation.
In summary, our work identifies a previously unknown small molecule
compound with anti-osteoporosis effects, namely, NVP-BHG712, which
inhibits osteoclast activation by inhibiting CTSK function. In the
ovariectomized female mouse model, NVP-BHG712 slowed the loss of bone
mass.Although the molecular mechanism of the effect of NVP-BHG712 on
osteoclast differentiation needs further investigation, this study may
provide new insights into the effect of NVP-BHG712 on bone metabolism in
osteoporosis.
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legends.