Fig. 3

Improvement effect of N-EVs on AC16 and HUVEC after H₂O₂ treatment. (A) The cell proliferation ability of N-EVs on HUVEC and AC16 cells after H₂O₂ treatment was assessed using CCK8 assay. Legend: The CCK8 assay was used to evaluate the impact of normal extracellular vesicles (N-EVs) on the proliferation of HUVEC and AC16 cells after H₂O₂-induced oxidative stress. HUVEC and AC16 cells were pretreated with N-EVs (50 µg/mL) for 24 h, followed by exposure to H₂O₂ (200 µM) for another 24 h. Cell viability was assessed by measuring the absorbance at 450 nm. The figure shows the relative cell viability of HUVEC and AC16 cells in the following groups: control (no treatment), H₂O₂ treatment alone, and H₂O₂ treatment with N-EVs. (B) EdU assay was employed to evaluate the cell proliferation ability of EVs on HUVEC and AC16 cells after H₂O₂ treatment. Legend: The EdU (5-ethynyl-2’-deoxyuridine) assay was used to assess the impact of N-EVs on the proliferation of HUVEC and AC16 cells after H₂O₂-induced oxidative stress. HUVEC and AC16 cells were pretreated with N-EVs (50 µg/mL) for 24 h, followed by exposure to H₂O₂ (200 µM) for another 24 h. EdU incorporation into newly synthesized DNA was detected using a fluorescence microscope. The figure shows the percentage of EdU-positive cells in the following groups: control (no treatment), H₂O₂ treatment alone, and H₂O₂ treatment with N-EVs. (C) Flow cytometry analysis was conducted to determine the apoptosis rate in different treatment groups. Legend: Flow cytometry was used to analyze the apoptosis rate in HUVEC and AC16 cells after H₂O₂-induced oxidative stress and treatment with N-EVs. Cells were pretreated with N-EVs (50 µg/mL) for 24 h, followed by exposure to H₂O₂ (200 µM) for another 24 h. Apoptosis was detected using Annexin V-FITC/PI double staining. The figure shows the proportions of early apoptotic (Annexin V + PI-), late apoptotic (Annexin V + PI+), and necrotic cells (Annexin V- PI+) in the following groups: control (no treatment), H₂O₂ treatment alone, and H₂O₂ treatment with N-EVs. (D) Angiogenesis assays were performed to assess the impact of N-EVs on tube formation capability. Legend: Angiogenesis assays were conducted to evaluate the impact of N-EVs on the tube formation capability of HUVEC cells. HUVEC cells were seeded on Matrigel and treated with N-EVs (50 µg/mL) for 6 h. Tube formation was observed and photographed under a phase-contrast microscope. The figure shows representative images of tube formation and quantification of the total tube length and number of branch points in the following groups: control (no treatment), H₂O₂ treatment alone, and H₂O₂ treatment with N-EVs. (E) QRT-PCR analysis was utilized to examine the effects of different treatments on the expression levels of angiogenic markers. Legend: Quantitative real-time PCR (QRT-PCR) was used to examine the effects of N-EVs on the mRNA expression levels of angiogenic markers (VEGF, Ang-1, and HIF-1α) in HUVEC and AC16 cells after H₂O₂-induced oxidative stress. HUVEC and AC16 cells were pretreated with N-EVs (50 µg/mL) for 24 h, followed by exposure to H₂O₂ (200 µM) for another 24 h. The figure shows the relative mRNA expression levels of VEGF, Ang-1, and HIF-1α in the following groups: control (no treatment), H₂O₂ treatment alone, and H₂O₂ treatment with N-EVs. Statistical significance was determined by comparing with PBS group (*P < 0.05), as well as H₂O₂ group (#P < 0.05)