Integrating UPLC-QE/MS, Transcriptomics, Virtual knockout, Molecular Docking and Molecular Dynamics to Elucidate Bazhen Tang's Multi-target Pharmacology in Osteoclast Regulation and Osteoporosis Inhibition

Background: Osteoporosis is a global public health challenge, with osteoclasts as central drivers and therapeutic targets. Bazhen Tang (BZT) has been reported to improve bone density, but its molecular effects on osteoclast-driven osteoporosis remain unclear. Methods: We evaluated RANKL-induced osteoclast differentiation in vitro by tartrate-resistant acid phosphatase staining. BZT components were profiled by UPLC-QE-MS. An integrated network pharmacology–transcriptomics workflow identified active constituents and osteoporosis-related targets linked to osteoclastogenesis. Single-cell omics corroborated expression of key hub genes. The functional impact of target perturbation was explored via virtual knockouts. Molecular docking and MD simulations characterized interactions between active constituents and core targets. Results: BZT inhibited RANKL-induced osteoclast differentiation in vitro. Fifteen active compounds mapped to 680 targets, with five constituents—butein, esculetin, L-tyrosine, salicylic acid, and trans-cinnamaldehyde—emerging as major players and ten core targets prioritized. Single-cell analysis showed stable expression of CTNNB1, MMP9, NFKB1, and SRC in osteoclasts. Virtual knockouts implicated these genes in pathways governing osteoclast formation and activity. The five principal constituents displayed high binding affinity to the four targets, particularly MMP9, a result supported by MD simulations. Conclusion: An integrative approach reveals that BZT’s active components exert anti-osteoclastogenic effects via multiple targets, providing a mechanistic basis for its potential use in osteoporosis therapy.