Extracellular vesicles-delivered circDB promotes ischemic muscle repair through the miR-34a/USP7/Notch1 signaling pathway.

INTRODUCTION: The incidence of lower limb ischemic diseases has been rising steadily in recent years, often leading to severe outcomes such as limb amputation. Given the limited availability of effective treatments, there is a critical need for novel therapeutic strategies. This study explores the reparative role and underlying mechanisms of extracellular vesicles derived from human umbilical cord mesenchymal stem cells (UMSC-EVs) in promoting ischemic hindlimb recovery through the delivery of circular RNA circDB. METHODS: A hindlimb ischemia model was established in C57BL/6 mice via femoral artery ligation, followed by intramuscular injections of extracellular vesicles derived from either untreated UMSCs (NC-EVs) or UMSCs transfected with si-circDB (si-EVs). Functional recovery was assessed using Laser Doppler imaging for blood flow, grip strength tests, and treadmill endurance evaluations. Molecular analyses included Western blot and qRT-PCR for USP7 and Notch1 expression, EdU assays for myoblast proliferation, and co-immunoprecipitation to confirm USP7-Notch1 interactions. In vitro, C2C12 myoblasts were cultured under hypoxic conditions for 48 h to mimic ischemia, and their proliferation and signaling were studied using similar techniques. Bioinformatics tools (CircBank, TargetScan) were used to analyze circDB-miR-34a interactions. RESULTS: We found that circDB expression is markedly reduced in ischemic hindlimb tissues and is closely associated with tissue repair. In a murine hindlimb ischemia model, localized injection of UMSC-EVs into ischemic muscle significantly enhanced blood flow recovery, improved muscle function, and increased expression of USP7 and Notch1. Additionally, a hypoxia-induced myoblast injury model in vitro revealed that UMSC-EVs delivering circDB promoted myoblast proliferation via the miR-34a/USP7/Notch1 signaling axis. CONCLUSION: Extracellular vesicles circDB enhances ischemic muscle repair by modulating the miR-34a/USP7/Notch1 pathway. These findings highlight a novel mechanism by which UMSC-derived extracellular vesicles facilitate muscle regeneration and suggest a promising therapeutic approach for lower limb ischemic diseases.