Abstract: BACKGROUND: Nimbolide, a triterpenoid bioactive compound, exhibits multiple biological activities including anti-inflammatory, antioxidant, antitumor, and antibacterial effects. However, its potential to alleviate osteoporosis by regulating osteoclast differentiation and apoptosis remains unreported.
OBJECTIVE: To investigate the effects of nimbolide on osteoclast differentiation, osteoclast apoptosis, and osteoporosis.
METHODS: (1) Cell experiments: Mouse bone marrow-derived macrophages were divided into four groups: the cells were cultured in α-MEM complete medium containing macrophage colony-stimulating factor in the control group; the cells were cultured in α-MEM complete medium containing macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand in the osteoclast induction group; the cells were cultured in osteoclast-inducing differentiation medium supplemented with 100 nmol/L or 200 nmol/L nimbolide, respectively in the low- and high-dose nimbolide groups. The effects of nimbolide on osteoclast differentiation and apoptosis were assessed using tartrate-resistant acid phosphatase staining, Annexin V-FITC/PI staining, and RT-qPCR. (2) In vivo experiments: Twenty-four 8-week-old female C57BL/6J mice were randomly divided into four groups: The mice in the sham group underwent only removal of perovarian fat; the mice in the model group underwent bilateral ovariectomy. The low-dose and high-dose nimbolide groups received intraperitoneal injections of 5 mg/kg and 10 mg/kg nimbolide solutions, respectively, every 2 days after modeling. Serum and femoral samples were collected after 8 weeks of modeling for relevant analyses.
RESULTS AND CONCLUSION: (1) Cell experiments: RT-qPCR and tartrate-resistant acid phosphatase staining revealed that nimbolide inhibited the expression of osteoclast differentiation-related genes and suppressed osteoclast differentiation in vitro. RT-qPCR and Annexin V-FITC/PI staining demonstrated that nimbolide suppressed the expression of apoptosis-related genes and induced apoptosis in mature osteoclasts. RT-qPCR results demonstrated that nimbolide promotes osteoclast apoptosis through the Fas/Fasl signaling pathway, with its regulatory effects on osteoclast differentiation and apoptosis exhibiting concentration-dependent properties. (2) Animal experiments: Micro-CT and hematoxylin-eosin staining results demonstrated that nimbolide reduced bone loss in estrogen-deficient osteoporotic mice, with a more pronounced effect at 10 mg/kg; nimbolide had no significant impact on serum estradiol levels in ovariectomized mice. In vitro experiments confirm that nimbolide not only inhibits osteoclast differentiation but also promotes apoptosis in mature osteoclasts. In vivo experiments confirm that nimbolide can mitigate excessive bone loss in estrogen-deficient osteoporosis in mice.

Key words: nimbolide, osteoclast differentiation, osteoclast apoptosis, osteoporosis, postmenopausal osteoporosis, bone destruction, osteoclasts, bone homeostasis