Nimbolide relieves osteoporosis by regulating osteoclast differentiation and apoptosis

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 periovarian fat; the model group underwent bilateral ovariectomy; the low- and high-dose nimbolide groups received intraperitoneal injections of 5 and 10 mg/kg nimbolide solution every 2 days after modeling. After 8 weeks of modeling, serum and femurs were collected for relevant assays. RESULTS AND CONCLUSION: (1) Cell experiments: RT-qPCR and tartrate-resistant acid phosphatase staining results showed that nimbolide inhibited the expression of osteoclast differentiation-related genes and suppressed osteoclast differentiation in vitro; RT-qPCR and Annexin V-FITC/PI staining results showed that nimbolide inhibited the expression of apoptosis-related genes and induced apoptosis of mature osteoclasts; RT-qPCR results showed that nimbolide promoted osteoclast apoptosis via the Fas/FasL signaling pathway, and the regulatory effects of nimbolide on osteoclast differentiation and apoptosis were concentration-dependent. (2) Animal experiments: Micro-CT and hematoxylin-eosin staining results showed that nimbolide reduced bone loss in estrogen deficiency-induced osteoporotic mice, with 10 mg/kg nimbolide showing better effects; nimbolide had no significant effect on serum estradiol levels in ovariectomized mice. These results indicate that in vitro experiments confirmed that nimbolide not only inhibits osteoclast differentiation but also promotes apoptosis of mature osteoclasts; in vivo experiments confirmed that nimbolide alleviates excessive bone loss in estrogen deficiency-induced osteoporotic mice.

1. Introduction

Osteoporosis is a common chronic metabolic bone disease characterized by reduced bone mineral density and deterioration of bone microarchitecture due to multiple factors, leading to increased skeletal fragility and fracture susceptibility [1-3]. Studies have shown that bone health is directly influenced by bone homeostasis, which refers to the balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption during bone metabolism [1,4]. When bone resorption exceeds bone formation, bone homeostasis is disrupted, ultimately leading to osteoporosis [5]. With the acceleration of global population aging, osteoporosis is becoming increasingly prevalent, affecting over 200 million people worldwide [6].

Commonly used clinical drugs for osteoporosis include bisphosphonates, calcitonin, and selective estrogen receptor modulators [7-9]. Although these drugs can restore bone strength, they may also lead to reduced bone strain, and some are expensive with severe adverse effects, requiring long-term use [10-11]. Therefore, the development of novel effective drugs is urgent, and addressing this need requires researchers to deeply analyze the pathological mechanisms of osteoporosis.

Osteoclasts are key cells derived from the bone marrow monocyte/macrophage lineage that mediate bone resorption. When bone marrow monocytes/macrophages are stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL), they can differentiate into osteoclasts [12-13]. Subsequently, differentiated osteoclasts secrete hydrogen ions, matrix metalloproteinases, and cathepsin K to degrade bone matrix, leading to excessive bone resorption and osteoporosis [14-15]. Regulating osteoclast differentiation and apoptosis is considered an important therapeutic strategy for osteoporosis [16-17].

Nimbolide, a triterpenoid bioactive compound extracted from the flowers and leaves of the neem tree (Azadirachta indica), has been demonstrated to possess anti-inflammatory, antioxidant, antitumor, and antibacterial activities [18-19]. Recent studies have shown that nimbolide can alleviate adjuvant-induced arthritis in rats by inhibiting inflammatory responses, and can reduce inflammatory cell infiltration, inhibit rheumatoid factor, and improve erythrocyte sedimentation rate via the Toll-like receptor pathway [20]. However, whether nimbolide can alleviate osteoporosis by inhibiting osteoclast differentiation has not been reported. This study investigates the effects of nimbolide on osteoclasts and validates its anti-osteoclastogenic effects in ovariectomized mice.