Proteomics based analysis of the therapeutic mechanism of Yishen Gushu Formula against postmenopausal osteoporosis.

BACKGROUND: YSGSF has shown therapeutic potential in PMOP, but its molecular mechanism remains unclear. OBJECTIVE: This study aimed to identify the protein targets and regulatory pathways underlying the therapeutic effects of YSGSF against PMOP. METHODS: Thirty female SD rats were randomly assigned to sham-operated, OVX model, and treatment groups (n = 10 per group). After 12 weeks of intervention, femoral morphology was evaluated by hematoxylin and eosin staining. Femoral proteins from five rats per group were analyzed using data-independent acquisition quantitative proteomics. DEPs were identified using fold-change thresholds of > 1.20 or < 0.833 and P < 0.05. GO, KEGG, GSEA, and lncRNA-miRNA-mRNA network analyses were performed. Key targets were further assessed using serum ELISA and femoral-tissue qPCR in three rats per group. RESULTS: Ovariectomy caused cortical thinning, trabecular fragmentation, and enlargement of the medullary cavity, whereas YSGSF partially improved cortical structure and trabecular continuity. Proteomic analysis identified 69 DEPs between the model and sham groups, including 35 upregulated and 34 downregulated proteins, and 96 DEPs between the treatment and model groups, including 25 upregulated and 71 downregulated proteins. The model-associated DEPs were enriched in 8 biological processes and 3 KEGG pathways, whereas the treatment-associated DEPs were enriched in 15 biological processes and 16 KEGG pathways. These pathways mainly involved calcium-ion binding, chemokine signaling, ECM-receptor interaction, and metabolic regulation. Intersection analysis identified three core proteins: RNASE3, DDX54, and MINDY2. GSEA associated RNASE3 with nitrogen metabolism and circadian rhythm, DDX54 with extracellular-matrix regulation, and MINDY2 with sulfur metabolism and unsaturated-fatty-acid biosynthesis. The predicted regulatory network contained OIP5-AS1 and 10 target miRNAs. ELISA and qPCR supported the reduction of RNASE3 and the elevation of DDX54 and MINDY2 in model rats, with these changes partially reversed by YSGSF treatment. DISCUSSION: These findings suggest that YSGSF may improve PMOP by coordinating nitrogen metabolism, circadian regulation, extracellular-matrix remodeling, and lipid metabolism through a three-target regulatory network. CONCLUSION: In this 30-rat PMOP model, 12 weeks of YSGSF treatment was associated with 96 treatment-related DEPs compared with the model group. The convergence of 69 disease-associated DEPs, 96 treatment-associated DEPs, three core proteins, and 10 target miRNAs supports a quantitative metabolism-circadian regulatory framework involving RNASE3, DDX54, and MINDY2. The novelty of this study lies in integrating femoral DIA proteomics, pathway analysis, regulatory-network prediction, and independent serum and tissue validation, thereby providing a more direct mechanistic explanation of YSGSF-mediated bone protection than single-omics or computational approaches alone.