Exploring the mechanism of Bushenhuoxue formula acting on postmenopausal osteoporosis via network pharmacology and experimental validation

Abstract Background: Bushenhuoxue (BSHX) formula, a ten-compound herbal decoction, has been used to treat postmenopausal osteoporosis (PMOP) in China for decades. Our previous study also revealed the anti-PMOP effects of BSHX formula in ovariectomized (OVX) mice. However, its pharmacological mechanisms remain unknown. Methods: Network pharmacology followed by animal experiments was used to explore the action mechanism of BSHX formula. Firstly, we obtained the chemical compounds and potential targets of BSHX formula using TCMSP and TCMID databases. Simultaneously, GeneCards and DisGeNET databases were used to determine the targets in PMOP. Based on their overlapping genes between BSHX formula and PMOP, a protein-protein interaction (PPI) network was constructed for screening hub-targets. GO and KEGG enrichment analyses were further performed to identify the critical biological processes and signaling pathways. In vivo experimental study, an OVX mouse model was established to determine the anti-PMOP effects of BSHX formula via Micro-CT assay and ABH staining. The expressions of β-catenin, alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF) and CD31 were detected by immunohistochemistry.Results: A total of 218 active ingredients and 274 related targets were identified in BSHX formula. After matching with 292 therapeutic targets of PMOP, 64 overlapping genes were obtained and further used to build a PPI network in which CTNNB1 and VEGFA played a central role. GO and KEGG enrichment analyses indicated that the anti-PMOP effects of BSHX formula were mainly associated with cell proliferation, angiogenesis and their regulated pathways including β-catenin signaling and VEGF signaling. In mouse experiments, we revealed that bone mass and blood vessels in the OVX mice were significantly enhanced after treated with BSHX formula for 8 weeks. Moreover, down-regulations of β-catenin, ALP, VEGF and CD31 caused by OVX surgery were significantly restored by BSHX formula.Conclusions: Through network pharmacology and an experimental validation, we have revealed that BSHX formula exerts anti-PMOP effects mainly through promoting β-catenin-mediated osteogenesis and VEGF-mediated angiogenesis. BSHX formula can be considered as a new option for the treatment of PMOP.