Mechanistic Integration of Network Pharmacology and In Vivo Validation: TFRD Combat Osteoporosis via PI3K/AKT Pathway Activation

In the context of osteoporosis closely linked to bone metabolism imbalance caused by estrogen deficiency, total flavonoids of Rhizoma Drynariae (TFRD) exhibit potential anti-osteoporotic activity, yet their multicomponent synergistic mechanism and association with the PI3K/AKT signaling pathway remain unclear. This study aimed to systematically elucidate the molecular mechanisms by which TFRD regulate bone metabolism and improve osteoporosis in ovariectomized (OVX) rats through the PI3K/AKT pathway, integrating network pharmacological predictions with animal experimental validation. Methods involved identifying TFRD’s active components using UPLC/MS-MS, predicting targets with SwissTargetPrediction, constructing a “component-target-disease” network, and performing GO/KEGG enrichment analysis with MetaScape (v3.5). In vivo experiments established an OVX rat model, randomized into sham, OVX, low-/high-dose TFRD, and sim groups, assessing bone mineral density (BMD) and mandibular Micro-CT parameters after 12 weeks. Western blot analyzed PI3K, p-AKT1, and related protein expressions. Results showed the high-dose TFRD group significantly increased BMD, improved trabecular bone quantity and structure, and upregulated PI3K, p-PI3K, and p-AKT1 protein expressions compared to the OVX group. Molecular docking confirmed stable binding energy between core components and PI3K/AKT targets. TFRD may ameliorate estrogen deficiency-induced osteoporosis by activating the PI3K/AKT signaling pathway, inhibiting bone resorption, and promoting osteogenic differentiation, providing pharmacological evidence for multitarget treatment of osteoporosis with traditional Chinese medicine.