(+)-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.