Network pharmacology-guided discovery of quercetin as an anti-osteoporotic agent from cyathulae radix with dual pro-osteogenic and antioxidant effects.

Osteoporosis is a major public health concern characterized by reduced bone mass and increased fracture risk, and Cyathulae Radix has been used in China for its prevention, yet its active constituents and mechanisms remain unclear. This study integrated network pharmacology with in vitro experiments to identify key bioactive components and underlying pathways. Using the TCMSP database, we screened the major compounds of Cyathulae Radix and, through intersection with osteoporosis-related targets from OMIM and GeneCards, obtained 29 overlapping genes. Subsequent protein-protein interaction network analysis via STRING revealed core targets including AKT1, TNF, IL-6, and MMP2, among which quercetin emerged as a principal active component. To validate its effects, we employed H₂O₂ to induce oxidative damage in MC3T3 E1 pre-osteoblasts, and then treated cells with quercetin based on CCK8 and ROS assay results. The CCK8 assay showed that quercetin restored cell viability to near-normal levels. Quercetin treatment significantly improved cell proliferation and restored osteogenic differentiation, as shown by elevated alkaline phosphatase staining intensity, increased mineralized nodules in alizarin red staining, and markedly upregulated mRNA levels of RUNX2, OPN, and BMP-2. Furthermore, quercetin attenuated oxidative stress by increasing SOD and GSH activities, decreasing MDA production, and activating the NRF2/HO-1/GPX4 antioxidant cascade, as confirmed by western blotting. Collectively, these results demonstrate that quercetin, a key constituent of Cyathulae Radix, exerts anti osteoporotic effects through both promoting osteogenesis and enhancing antioxidant defense, offering a mechanistic rationale for its clinical use against osteoporosis.