Integration of UPLC-MS and Quantitative Proteomics Reveals Key Bioactive Components and Osteoactive Targets of Liuwei Dihuang Wan in Bone Metabolism Modulation

Objective: Liu Wei Di Huang Wan(LWDHW) is a classic traditional Chinese medicine widely used in preventing and treating osteoporosis associated with kidney yin deficiency, with proven clinical benefits. However, its active ingredients, targets, and synergistic mechanisms remain unclear. This study aims to identify the key factors and mechanisms by which LWDHW treats osteoporosis using proteomics, serum pharmacochemistry, molecular docking, pharmacokinetics, and in vitro validation. Methods: Healthy male rat were randomly assigned to a saline control group or an LWDHW-treated group. After seven days of intervention, serum samples were collected to assess bone metabolism by measuring PINP and β-CTX levels via ELISA. Active compounds in the serum were identified using UPLC-MS. Differentially expressed proteins (DEPs) were analyzed through quantitative proteomics, and key targets were determined using GO and KEGG enrichment analyses, molecular docking. Western blot analysis was performed to verify the expression trends of these key proteins. Results: Compared with the control group, the LWDHW group showed a significant increase in PINP levels (P < 0.01) and a significant decrease in β-CTX levels (P < 0.001), indicating that LWDHW not only suppresses bone resorption but also promotes bone formation. Seven categories of active compounds were detected, including glycosides, triterpenoids and derivatives, phenolics, organic acids, fatty acids and lipids, carbohydrates, and others. Proteomic analysis identified 173 DEPs, with 63 proteins upregulated and 114 downregulated in the treated group. Bioinformatics analysis highlighted PTPN11, SRC, GLUL, and ALOX12 as key targets, and molecular docking revealed that compounds such as linolenic acid, Alisol B, adenosine, and swertiamarin had strong binding affinities (all binding energies below –5) with these targets. Moreover, Western blot results confirmed that high concentrations of LWDHW-containing serum significantly inhibited the AKT, ERK1/2, and IκBα pathways (P < 0.05) while markedly increasing osteocalcin (OCN) expression (P < 0.05). Conclusion: LWDHW promotes osteogenic differentiation and effectively treats osteoporosis through multiple synergistic pathways.