Total flavonoids of Rhizoma drynariae targets NRF2-mediated anti-ferroptosis in osteoblasts to promote induced membrane osteogenesis

Abstract Background Induced membrane technique (IMT), a novel approach for reconstructing critical-size bone defect, encounters the challenge of lengthy mineralization time after bone grafting. Total flavonoids of Rhizoma drynariae (TFRD), the extracts from dried rhizome of Drynaria roosii Nakaike, is widely used in the treatment of orthopedic diseases. Purpose This study primarily investigates the impact of TFRD on the NRF2-mediated anti-ferroptosis effect in osteoblasts within the IMT bone grafting area. Methods An IMT model was established in the right femur of rats. After 4 and 8 weeks of treatment with TFRD and DMF (an NRF2 activator) respectively, bone defect repair and ferroptosis-related indicators were evaluated. In vitro, an Erastin-induced ferroptosis model of osteoblasts was constructed to analyze the mineralization capacity of osteoblasts, ferroptosis-related indicators, and factors related to the NRF2/ARE pathway under TFRD and DMF treatment. Additionally, the components of TFRD and TFRD-containing serum were analyzed using UHPLC-Q-Orbitrap HRMS. Finally, the main compounds in TFRD that bind to the NRF2 protein were studied through molecular docking, molecular dynamics simulation (MDS), and CETSA. Results In vivo results demonstrated that excessive iron accumulation occurred in the IMT bone grafting area, accompanied by elevated levels of lipid peroxidation products (MDA and 4-HNE) and decreased levels of antioxidants (GSH), suggesting the presence of ferroptosis during the bone graft mineralization process in IMT. Treatment with TFRD and DMF reduced iron accumulation and the production of MDA and 4-HNE, accelerated bone defect healing, and enhanced expression of osteogenesis-related factors and NRF2/ARE pathway factors. In vitro experiments revealed that Erastin induced ferroptosis in osteoblasts, diminishing cell viability and mineralization capacity. Treatment with TFRD and DMF alleviated mitochondrial damage, reduced production of ROS, MDA and 4-HNE, increased the expression of osteogenesis-related factors, upregulated the NRF2/ARE pathway, and enhanced cell viability and mineralization. Meanwhile, five active components in TFRD and TFRD-containing serum were identified using UHPLC-Q-Orbitrap HRMS. Molecular docking, MDS and CETSA results indicated that the main compounds in TFRD-containing serum could directly bind to the NRF2 protein in osteoblasts and maintain its stability. Conclusion The NRF2-mediated anti-ferroptosis effect in osteoblasts positively regulated the mineralization of osteoblasts. The main components of TFRD targeted NRF2 in osteoblasts within the IMT bone grafting area, maintained its stability, promoted the expression of downstream antioxidant response elements (ARE), enhanced the anti-ferroptosis role of osteoblasts, thereby accelerating the repair of bone defects.