Acanthopanax exosome-like nanovesicles promote osteogenic differentiation of human bone marrow mesenchymal stem cells

Zhong Zhuolan; Peng Zhina; Tian Xiaohong; Han Cuifei; Zhang Zihan; Chu Jiaqi

doi:10.12307/2026.245

Abstract：BACKGROUND:Acanthopanax and its extracts exhibit osteogenic effects,but the osteogenic potential and mechanisms of acanthopanax exosome-like nanovesicles remain unclear. OBJECTIVE:To investigate the molecular mechanism by which acanthopanax exosome-like nanovesicles promote osteogenic differentiation of human bone marrow mesenchymal stem cells and their preventive role in osteoporosis. METHODS:(1)Human bone marrow mesenchymal stem cells were extracted by gradient density centrifugation.Exosome-like nanovesicles derived from acanthopanax were isolated by differential centrifugation and sucrose gradient density centrifugation.(2)Human bone marrow mesenchymal stem cells were treated with 0,2.5,and 5 μg/mL acanthopanax exosome-like nanovesicles.Osteogenic differentiation was assessed by alkaline phosphatase staining,Alizarin red staining,qRT-PCR,and western blot assay.(3)Key pathways were identified by transcriptome sequencing and validated with a transforming growth factor β1 receptor inhibitor.(4)An ovariectomized rat model of osteoporosis was established.After 12 weeks of intraperitoneal injection of exosome-like nanovesicles derived from acanthopanax,bone microstructure was analyzed by micro-CT and osteogenic protein expression was assessed by histological staining. RESULTS AND CONCLUSION:(1)Acanthopanax exosome-like nanovesicles exhibited a cup-shaped or discoid morphology.(2)In vitro,acanthopanax exosome-like nanovesicles dose-dependently promoted osteogenic differentiation of bone marrow mesenchymal stem cells,as evidenced by increased alkaline phosphatase activity,enhanced mineralization nodule formation,and upregulated osteogenic gene expression.(3)Transcriptome analysis revealed that exosome-like nanovesicles activated the transforming growth factor-β1/Smad2/3 pathway,upregulating transforming growth factor-β1 and phosphorylated Smad2/3 protein expression.Moreover,the transforming growth factor-β1 receptor inhibitor partially inhibited the osteogenic effects.(4)Animal experimental results demonstrated that treatment with 5 mg/kg exosome-like nanovesicles from acanthopanax significantly increased bone mineral density,bone volume fraction,and trabecular thickness in ovariectomized rats(P<0.05),significantly enhanced collagen fibrillogenesis,and upregulated the expression of Runt-related transcription factor 2,osteocalcin,and transforming growth factor-β1 proteins in bone tissue.No significant toxicity was observed in major organ histological observations.Results showed that exosome-like nanovesicles derived from acanthopanax promote osteogenic differentiation of human bone marrow mesenchymal stem cells by activating the transforming growth factor β1/Smad2/3 pathway and effectively improve osteoporosis.