Zuo Gui Wan Restores Bone Metabolism in Postmenopausal Osteoporosis through HIF-1 and PI3K–Akt Pathway Modulation: Evidence from UPLC– MS/MS and Network Analysis

<title>Abstract</title> <bold>Ethnopharmacological Relevance:</bold> Zuo Gui Wan (ZGW), a traditional Chinese medicine (TCM) formula, shows potential for treating postmenopausal osteoporosis (PMOP), combining traditional herbal knowledge with modern scientific validation. <bold>Background:</bold> Postmenopausal osteoporosis (PMOP) is a metabolic bone disorder caused by estrogen deficiency, leading to decreased bone mass and increased fracture risk. ZGW has shown promise in managing PMOP, but its active metabolites and mechanisms remain unclear. <bold>Methods:</bold> Ovariectomized (OVX) rats were used to model osteoporosis. ZGW's efficacy was evaluated through micro-CT, HE staining, and serum ELISA. Active metabolites in serum were identified by UPLC-MS/MS. A "botanical drug-metabolite-target-disease" network was built using network analysis. Pathway enrichment was performed using GO and KEGG in R. Molecular docking of key metabolites and targets was conducted using AutoDock Vina and PyMOL. In vitro assays, including MTT, ALP, Alizarin Red S staining, PCR, and Western blotting, validated osteogenic effects. <bold>Results:</bold> ZGW improved bone microstructure and serum bone metabolism in OVX rats. UPLC-MS/MS identified 209 metabolites, with 20 transferring into the serum. PPI analysis revealed 144 key targets, and molecular docking showed strong binding between active metabolites (e.g., Remycin A, Farnesecin) and their targets, such as ALB and EGFR. GO and KEGG analyses identified pathways like HIF-1, estrogen signaling, and PI3K-Akt. In vitro, ZGW activated these pathways, enhancing osteogenic marker expression and promoting osteoblast proliferation and differentiation. <bold>Conclusion:</bold> ZGW treats PMOP through multiple mechanisms involving active metabolites, targets, and pathways. It restores normal gene expression and modulates pathways such as HIF-1 and PI3K-Akt, while also inhibiting inflammation. This study highlights the power of combining UPLC-MS/MS with network analysis for exploring TCM formulations in PMOP treatment.