Synergistic and antagonistic modulation of PI3K-Akt, MAPK, and NF-κB pathways by velvet antler in osteoporosis: an integrated network pharmacology, single-cell transcriptomic, and experimental validation study

Background Osteoporosis is increasingly viewed as a disorder of bone-immune crosstalk, in which the balance between osteoclast resorption and osteoblast formation is governed by interacting PI3K-Akt, MAPK, and NF-κB signalling. Velvet antler (Lu Rong, Cervus nippon ) is a traditional Chinese medicine with reported anti-osteoporotic activity but poorly defined molecular targets. Methods Drug targets (BATMAN-TCM, SymMap) and disease targets (GeneCards, OMIM, TTD) were intersected to yield core targets for PPI analysis (STRING), hub-gene identification (CytoHubba), and GO/KEGG enrichment. Diagnostic biomarkers were selected by WGCNA and three machine-learning algorithms (LASSO, SVM-RFE, Random Forest) on GSE56815 and validated in GSE7158, GSE13850, and GSE35959. Immune infiltration (ssGSEA), single-cell profiling (GSE169396; Seurat v5/SingleR), and CB-Dock2 molecular docking were performed. Predictions were then tested in primary mouse BMSCs under osteogenic induction with or without estrone (E1; 10 and 100 nM) using RT-qPCR, Western blotting, Alkaline phosphatase staining, and CCK-8. Results Twenty-eight active compounds and 1,312 core targets were identified, from which eight hub genes emerged (MAPK1, MAPK3, SRC, TP53, PRKACA, PRKACB, PRKACG, PIK3R1). A ten-gene diagnostic signature achieved an AUC of 0.947 in training and 0.838, 0.674, and 1.000 in external cohorts. Single-cell analysis of 18,916 cells localized hub and diagnostic genes to distinct populations, and docking ranked estrone–MAPK1 highest (−9.4 kcal/mol). In BMSCs, estrone promoted osteogenic differentiation, increasing ALP staining and viability with a biphasic dose response (maximal at 10 nM), alongside upregulation of Mapk1/ERK1/2 and dose-dependent downregulation of the NF-κB feedback inhibitor Nfkbia, whereas p-AKT declined rather than increased. Conclusion Velvet antler acts on the PI3K-Akt/MAPK/NF-κB network to favour osteoblast formation; experimentally, its constituent estrone promoted BMSC osteogenesis, most plausibly through MAPK/ERK rather than PI3K-Akt activation, while suppression of osteoclast activity remains a computational prediction awaiting dedicated testing. The ten-gene diagnostic panel and cell-type expression atlas reported here provide a basis for more targeted anti-osteoporotic strategies.