Active components and mechanisms of total flavonoids from Rhizoma Drynariae in enhancing cranial bone regeneration: An investigation employing serum pharmacochemistry and network pharmacology approaches

Rhizoma Drynariae, as the dried rhizome of Drynaria fortunei (Kunze ex Mett.) J. Sm., is a traditional Chinese medicine for treating the injury and bone broken of falling and beating. Total flavonoids is considered as the major and effective compounds for the therapeutic efficacy of Rhizoma Drynariae. To explore the effect of total flavonoids from Rhizoma Drynariae (TFRD) on bone regeneration and the underlying mechanisms. The effect of TFRD in various doses on bone reconstruction in cranial bone defect rats was explored in vivo. The active ingredients in TFRD-medicated serum were characterized by serum pharmacochemistry and integrated by network pharmacology analysis and target prediction. To elucidate the underlying mechanism of TFRD on bone regeneration, experimental validation in vitro was executed to assess the influence of different concentrations of TFRD-medicated serum on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Micro-CT, histological examination, immunohistochemical analysis, and ELSA demonstrated that administration of TFRD could promote bone reconstruction in a rat cranial defect model. We identified 27 active components of TFRD using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Results from CCK8, ALP, and Alizarin Red S staining revealed that TFRD-medicated serum notably enhanced BMSCs proliferation and osteogenic differentiation. qRT-PCR and Western blot harvested results consistent with those predicted by network pharmacology, providing further evidence that TFRD activated the TGF-β signaling pathway to benefit bone regeneration. The active components of TFRD modulate the TGF-β signaling pathway to facilitate osteogenesis, thereby repairing cranial bone defects.