(+)-Magnoflorine suppresses osteoclastogenesis by regulating CCDC88A-associated signaling networks in estrogen-deficient osteoporosis.

BACKGROUND: Postmenopausal osteoporosis is primarily driven by estrogen deficiency-associated osteoclast hyperactivation, resulting in excessive bone resorption and progressive deterioration of bone microarchitecture. Although current anti-resorptive therapies are clinically effective, their long-term application remains limited by adverse effects. PURPOSE: This study aimed to evaluate the anti-osteoporotic effects of Gengnianshou Formula (GNS), identify its pharmacologically relevant absorbed constituents, and investigate the molecular mechanisms underlying its regulation of osteoclast differentiation under estrogen-deficient conditions. METHODS: An ovariectomized (OVX) rat model was used to evaluate the anti-osteoporotic effects of GNS in vivo. Bone microarchitecture and osteoclast activity were assessed using micro-CT, histological staining, ELISA, and western blotting. Serum and fecal metabolite profiling was performed by UPLC-MS/MS to identify exposure-related compounds. Candidate compounds were screened in RAW264.7 osteoclast differentiation models. Public transcriptomic datasets (GSE230665 and GSE246769) were integrated to identify key regulatory genes associated with osteoclastogenesis. CRISPR/Cas9-mediated knockout, molecular docking, microscale thermophoresis (MST), qPCR, immunofluorescence, and western blot analyses were performed for mechanistic validation. RESULTS: GNS significantly improved trabecular bone microarchitecture and reduced osteoclast activity in OVX rats. Exposure profiling identified adenosine, phellodendrine, and jatrorrhizine as prototype compounds in both serum and feces, whereas (+)-magnoflorine (MAG) was detected as a prototype in feces and as a demethylated metabolite in serum, indicating pharmacologically relevant MAG-related exposure. Functional screening identified (+)-MAG as the most potent inhibitor of osteoclast differentiation in vitro. Integrated transcriptomic analyses revealed that CCDC88A was an early-response gene associated with osteoporosis progression and osteoclast differentiation. CCDC88A deficiency attenuated osteoclastogenesis and altered CCDC88A-associated signaling responses. Moreover, molecular docking and MST analyses supported a direct interaction between (+)-MAG and CCDC88A, while CCDC88A depletion abolished the additional inhibitory effects of (+)-MAG on osteoclast differentiation. CONCLUSION: GNS effectively alleviated estrogen deficiency-associated bone loss by suppressing osteoclast activation. (+)-MAG was identified as an exposure-related bioactive constituent, and its anti-osteoclast effects were mediated through a CCDC88A-dependent regulatory mechanism. These findings provide new insights into osteoclast regulation and highlight GNS-derived compounds as potential therapeutic candidates for postmenopausal osteoporosis.