Effects of the Total Flavonoids from Drynaria on Methyltransferase METTL3 and the Coupling of Osteogenesis and Angiogenesis in a Bone Defect Rat Model

Introduction: This study aimed to investigate the effects of the Total Flavonoids from Rhizoma Drynaria (TFRD) on METTL3-mediated m6A methylation and osteogenic-angiogenic coupling during the repair of large bone defects and to elucidate its role in bone remodeling under the Masquelet technique. Methods: A large femoral bone defect rat model was established in Sprague Dawley (SD) rats using the Masquelet technique. Postoperatively, a total of 24 rats were randomly divided into four groups, namely the model group (MOD), low-dose group (0.11 g/kg/day), mid-dose group (0.22 g/kg/day), and high-dose group (TFRD) (0.44 g/kg/day). These groups were established using the corresponding TFRD dosages or saline interventions. The neobone tissue quality was assessed using X-rays (Lane-Sandhu score), and the bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) were quantified using micro-computed tomography (micro-CT). The localization and expression of type H vessel markers (CD31, EMCN) and measurement of the total RNA m6A methylation levels in the neobone tissues were performed using Immunohistochemistry (IHC). The osteogenic-angiogenic coupling factors (BMP-2, PDGF-BB, S1P) and m6A methylation regulators (METTL3, IGF2BP2) were analyzed using a western blot and qRT-PCR. Results: The micro-CT and X-ray techniques revealed that TFRD significantly enhanced the neobone tissue quality, increased the BV/TV, and thickened Tb.Th compared to the MOD group. IHC showed a dose-dependent upregulation of the CD31 and EMCN expression areas and intensity in the neobone tissues, in addition to elevated m6A methylation levels with increasing TFRD doses. The western blot showed that the osteogenic-angiogenic coupling proteins (PDGFBB, S1P, BMP-2) and m6A regulators (METTL3, IGF2BP2) were upregulated in a dosedependent manner. Notably, while qRT-PCR indicated that TFRD suppressed PDGF-BB mRNA expression, the protein level was elevated, suggesting potential post-transcriptional regulation. The mRNA levels of BMP-2, S1P, METTL3, and IGF2BP2 were enhanced. Conclusion: TFRD facilitated bone regeneration by modulating METTL3-mediated m6A methylation, upregulating osteogenic-angiogenic coupling factors, and stimulating type H vessel formation. These findings support the potential of TFRD as a promising therapeutic agent for bone defect repair. TFRD acted through mechanisms that involve RNA methylation and enhanced the interaction between osteogenesis and angiogenesis. Targeting the epitranscriptomic regulation of bone-vascular interaction may open new avenues for orthopedic rehabilitation.