Taohong Siwu Decoction Regulates MSC‐Mediated H‐Type Angiogenesis to Accelerate Bone Fracture Healing Through VHL/HIF‐1α Ubiquitination

Background: Bone fracture healing is a multifaceted process that involves different stages and intercellular interactions. In this study, we aimed to investigate the effect of Taohong Siwu decoction (TSD) on bone fracture healing and the underlying mechanisms. Methods: First, a mouse model of femur fracture was constructed, and TSD intervention was administered for durations of 7, 14, and 21 days. Following this, immunofluorescence (IF) was employed to evaluate the expression of CD90 (a marker for mesenchymal stem cells [MSCs]), endomucin (Emcn), and CD31. We also treated MSCs with normal serum and 10% TSD-containing serum to investigate the effects of TSD. Molecular docking was applied to verify the binding of active compounds in TSD to pVon Hippel-Lindau (VHL). Additionally, MSCs were treated with paeoniflorin and 2-methoxyestradiol (2-ME2) to explore the effects of paeoniflorin. Subsequently, mouse aortic endothelial cells were extracted and identified. Furthermore, normally cultured MSCs were cocultured with endothelial cells. MSCs were exposed to control serum, 10% TSD-containing serum, and a combination of 10% TSD-containing serum with 2-ME2. Finally, we administered a combination of 2-ME2 over 21 days to evaluate its effects on the fractured mice. Results: TSD significantly influenced H-type angiogenesis during the healing process of fractured mice. Compared to the sham group, the model group exhibited lower levels of Emcn, CD90, hypoxia-inducible factor-1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF), while there was an increase in pVHL expression. After 7, 14, and 21 days of TSD intervention, the levels of Emcn, CD90, HIF-1α, VEGF, and pVHL gradually increased, whereas HIF-1α expression decreased. In vitro experiments revealed that TSD enhanced the proliferation and migration of MSCs while inhibiting the ubiquitination of pVHL/HIF-1α. Moreover, ferulic acid, amygdalin, hydroxysafflor yellow A, and paeoniflorin demonstrated a strong affinity for binding with pVHL. Notably, paeoniflorin promoted the proliferation and migration of MSCs through the pVHL/HIF-1α pathway to promote angiogenesis. Furthermore, TSD was found to enhance endothelial angiogenesis in MSCs. In summary, TSD affects H-type angiogenesis and MSCs homing during the healing process of fractured mice through the HIF-1α axis. Conclusions: TSD regulated MSC-mediated H-type angiogenesis to accelerate fracture healing through VHL/HIF-1α ubiquitination.