Total Flavone of Flowers of Abelmoschus manihot (L.) Medic Suppress Nfatc1 Expression to Reduce Osteoclastogenesis Thereby Alleviating Ovariectomy-Induced Osteoporosis

Background: Osteoporosis is a systemic skeletal disorder characterized by excessive osteoclast-mediated bone resorption and progressive bone loss. The clinical application of current anti-osteoporotic agents is often limited by adverse effects, necessitating the development of safer and more effective therapeutic strategies. Total flavonoids of Abelmoschus manihot (TFA) possess potent anti-inflammatory and renoprotective activities. However, their potential role in osteoporosis remains unclear. This study aimed to investigate the protective effects of TFA against osteoporosis and elucidate its underlying mechanisms in osteoclastogenesis. Methods: In vivo bone mineral density, bone histomorphology, biomechanical properties, and serum biochemical markers were assessed in ovariectomy (OVX) rats following TFA treatment. In vitro osteoclast differentiation, actin ring formation, bone resorption, and osteoclast-related gene expression were examined in receptor activator of nuclear factor-κB ligand (RANKL)-stimulated bone marrow-derived macrophages (BMDMs). Transcriptomic sequencing was performed to identify potential key genes and pathways regulated by TFA. Results: TFA markedly attenuated OVX-induced bone loss, improved bone microarchitecture and biomechanical properties, and restored serum bone turnover markers. In vitro, TFA significantly inhibited RANKL-induced osteoclast formation, actin belt assembly, and bone resorptive activity without affecting cell viability. Mechanistically, TFA suppressed the expression of the master osteoclastogenic transcription factor Nfatc1 and its downstream genes, including Acp5, Ctsk, Mmp9, and Atp6v0d2. Transcriptomic analysis revealed that TFA coordinately regulated multiple genes associated with monocyte commitment and early osteoclast differentiation. Conclusions: TFA protects against pathological bone loss by inhibiting the Nfatc1 signaling axis and reprogramming the transcriptional network governing osteoclastogenesis. These findings suggest that TFA is a promising therapeutic candidate for the prevention and treatment of osteoporosis.