Hederagenin Alleviated Ovariectomy-Induced Bone Loss through the Regulation of Innate Immune Signaling in Mice

Objectives: Postmenopausal osteoporosis is the most common form of osteoporosis in clinical practice, affecting millions of postmenopausal women worldwide. Postmenopausal osteoporosis demands safe and effective therapies. This study aimed to evaluate the potential of hederagenin (Hed) for treating osteoporosis and to elucidate its underlying mechanisms of action. Methods: The anti-osteoporotic potential of Hed was assessed by investigating its effects on ovariectomy (OVX)-induced bone loss in mice and on receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation in RAW264.7 cells. Network pharmacology analysis and molecular docking were employed to identify key targets, which were subsequently validated experimentally. Results: In vitro, Hed suppressed osteoclastogenesis by inhibiting the formation of osteoclasts and F-actin rings and by down-regulating osteoclast-specific genes (Atp6v0d2 and Acp5). In vivo, Hed significantly ameliorated OVX-induced bone loss, restoring trabecular bone volume fraction (BV/TV) and trabecular number (Tb.N), while reducing trabecular separation (Tb.Sp). Network pharmacology analysis identified 142 overlapping targets linking Hed to osteoporosis, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1β, with enrichment in innate immune signaling and osteoclast differentiation. Molecular docking analysis indicated strong binding affinities between Hed and targets such as TNF-α, IL-6, and IL-1β. Experimentally, Hed was found to decrease RANKL, elevate osteoprotegerin (OPG), and suppress intestinal mRNA levels of pro-inflammatory cytokines such as IL-1β, IL-6, IL-17A, and TNF-α. Conclusion: Hed exerts significant anti-osteoporotic effects in OVX-induced osteoporosis through a dual mechanism involving the suppression of both osteoclastogenesis and innate immune signaling pathways. These findings highlighted Hed’s novel role in modulating immune-bone crosstalk, offering a promising strategy for treating osteolytic diseases without estrogenic side effects.