Mechanism of notopterol in promoting osteoblast differentiation via the COX2/WNT/β-catenin axis based on network pharmacology and in vitro experiments -data

The osteogenic differentiation-promoting effects and underlying mechanisms were evaluated by assessing alkaline phosphatase (ALP) activity, Alizarin Red S staining, RT-qPCR, Western blot, and validation using the wingless-type MMTV integration site family/beta-catenin (WNT/β-catenin) signaling pathway inhibitor XAV-939. The results showed that network pharmacology screening identified 61 common targets between notopterol and osteoporosis.

Core targets included prostaglandin-endoperoxide synthase 2 (PTGS2) (which encodes the cyclooxygenase-2 (COX2) protein), potassium voltage-gated channel subfamily A member 2 (KCNA2), and hypoxia inducible factor 1 subunit alpha (HIF1A). KEGG analysis suggested a potential role through the WNT/β-catenin signaling pathway. Molecular docking indicated that notopterol exhibited the most stable binding with the COX2 protein (binding energy: -8.5 kcal/mol). In vitro experiments confirmed that at safe concentrations (5, 10, 20 μmol/L), notopterol significantly enhanced ALP activity, promoted calcium nodule formation, and upregulated the mRNA expression of osteogenic-related genes Runt-related transcription factor 2 (Runx2), transcription factor Sp7 (Sp7), and distal-less homeobox 5 (Dlx5).

Western blot analysis further demonstrated that notopterol activates the WNT/β-catenin pathway by upregulating COX2 expression, promoting β-catenin nuclear translocation and the expression of downstream proteins G1/S-specific cyclin-D1 (Cyclin D1) and B-cell lymphoma 2 (BCL-2). These effects were reversed by XAV-939. The findings suggest that notopterol promotes osteoblast differentiation potentially by regulating the COX2/WNT/β-catenin signaling axis, providing a theoretical basis for the application of notopterol in the treatment of osteoporosis.