Abstract: BACKGROUND: Vascular calcification is a type of bone metabolic disorder, and its pathological mechanism is highly similar to the process of bone mineralization. However, there is a lack of effective evidence regarding the application of Epimedium Sagittatum Maxim. (a traditional Chinese medicine) in cardiovascular diseases and how to improve its bioavailability.
OBJECTIVE: To investigate the effects and molecular mechanisms of Epimedium Sagittatum Maxim.-extracellular vesicles on vascular calcification.
METHODS: (1) Epimedium Sagittatum Maxim.-extracellular vesicles were isolated from using density gradient centrifugation, and characterized by transmission electron microscopy, nanoparticle size analyzer, and nanoparticle tracking analysis. (2) The effects of Epimedium Sagittatum Maxim.-extracellular vesicles on the viability of aortic vascular smooth muscle cells were evaluated by CCK-8 assay. The cellular uptake of Epimedium Sagittatum Maxim.-extracellular vesicles by aortic vascular smooth muscle cells was observed by confocal laser scanning microscopy. (3) Proteomic analysis was performed to identify and characterize the protein composition of Epimedium Sagittatum Maxim.-extracellular vesicles, and potential functional proteins were screened. (4) Aortic vascular smooth muscle cells were divided into blank, model, and Epimedium Sagittatum Maxim.-extracellular vesicle intervention groups (low, medium, and high doses). After co-culturing for 48 hours, cytoskeletal changes were observed by confocal microscopy. Apoptosis was assessed by flow cytometry. Ras homolog family member A/Rho-associated coiled-coil containing protein kinase pathway proteins were measured by western blot assay. Ras homolog family member A-GTP levels were assessed by ELISA. After 7 days of co-culture, calcification areas were detected by Alizarin Red S staining. The expression levels of osteogenic phenotype proteins were detected using western blot assay. 
RESULTS AND CONCLUSION: (1) Epimedium Sagittatum Maxim.-extracellular vesicles exhibited saucer-shaped morphology with particle sizes ranging from 50 to 200 nm. (2) CCK-8 assay showed that Epimedium Sagittatum Maxim.-extracellular vesicles had no significant effect on cell viability and were efficiently taken up by aortic vascular smooth muscle cells, indicating good bioactivity. (3) Proteomic analysis identified 37 specific proteins in Epimedium Sagittatum Maxim.-extracellular vesicles. Gene Ontology analysis suggested that Epimedium Sagittatum Maxim.-extracellular vesicles may exert anti-calcification effects by participating in cellular metabolism, signal transduction, and stress response via complex protein interaction networks. (4) Alizarin Red S staining revealed that Epimedium Sagittatum Maxim.-extracellular vesicle intervention significantly reduced calcification areas compared with the model group. Flow cytometry indicated that Epimedium Sagittatum Maxim.-extracellular vesicles exerted notable anti-apoptotic effects. Confocal microscopy results showed that compared with the model group, Epimedium Sagittatum Maxim.-extracellular vesicles dose-dependently restored cytoskeletal structure in the Epimedium Sagittatum Maxim.-extracellular vesicle intervention groups (low, medium, and high doses). Western blot analysis showed that, compared with the model group, the expression levels of Runt-related transcription factor 2, bone morphogenetic protein 2, and phosphorylated Rho-associated coiled-coil containing protein kinase 1 were dose-dependently decreased in the low, medium, and high-dose Epimedium Sagittatum Maxim.-extracellular vesicle groups, while the expression levels of α-smooth muscle actin and smooth muscle 22α were dose-dependently increased. ELISA results showed a dose-dependent decrease in Ras homolog family member A-GTP. The results indicate that Epimedium Sagittatum Maxim.-extracellular vesicles can effectively improve vascular calcification, possibly by regulating the Ras homolog family member A/Rho-associated coiled-coil containing protein kinase pathway.  

Key words: Epimedium Sagittatum Maxim., extracellular vesicles, Ras homolog family member A/Rho-associated coiled-coil containing protein kinase (RhoA/ROCK), vascular calcification, proteomics, cytoskeleton