Fig. 5

The impact of miR-133a-3p on endothelial cells and cardiomyocytes. (A) The cell proliferation ability of N-EVs and miR-133a-3p inhibitor on HUVEC and AC16 was assessed using the CCK8 assay. Legend: The CCK8 assay was used to evaluate the impact of N-EVs and the miR-133a-3p inhibitor on the proliferation of HUVEC and AC16 cells. Cells were treated with N-EVs (50 µg/mL) alone, the miR-133a-3p inhibitor (100nM) alone, or a combination of both for 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), N-EVs treatment, miR-133a-3p inhibitor treatment, and combined treatment. (B) The cell proliferation ability of N-EVs and miR-133a-3p inhibitor on HUVEC and AC16 was evaluated by EdU staining. Legend: The EdU (5-ethynyl-2’-deoxyuridine) staining assay was used to assess the impact of N-EVs and the miR-133a-3p inhibitor on the proliferation of HUVEC and AC16 cells. Cells were treated with N-EVs (50 µg/mL) alone, the miR-133a-3p inhibitor (100 nM) alone, or a combination of both for 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), N-EVs treatment, miR-133a-3p inhibitor treatment, and combined treatment. (C, E) Flow cytometry was employed to determine the apoptosis rate and necroptosis rate in different treatment groups. Legend: (C) Flow cytometry was used to analyze the apoptosis rate in HUVEC and AC16 cells after treatment with N-EVs (50 µg/mL) alone, the miR-133a-3p inhibitor (100nM) alone, or a combination of both for 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), N-EVs treatment, miR-133a-3p inhibitor treatment, and combined treatment. Legend: (E) Flow cytometry was used to analyze the necroptosis rate in HUVEC and AC16 cells after treatment with N-EVs (50 µg/mL) alone, the miR-133a-3p inhibitor (100nM) alone, or a combination of both for 24 h. Necroptosis was detected using a specific necroptosis marker (e.g., MLKL phosphorylation). The figure shows the proportions of necroptotic cells in the following groups: control (no treatment), N-EVs treatment, miR-133a-3p inhibitor treatment, and combined treatment. (D, F) Western Blot analysis was conducted to examine the effects of different treatments on the expression of pyroptosis and necroptosis markers. Legend: (D) Western blot analysis was used to examine the effects of N-EVs and the miR-133a-3p inhibitor on the expression of pyroptosis markers (e.g., cleaved caspase-1, GSDMD) in HUVEC and AC16 cells. Cells were treated with N-EVs (50 µg/mL) alone, the miR-133a-3p inhibitor (100nM) alone, or a combination of both for 24 h. The figure shows the protein expression levels of cleaved caspase-1 and GSDMD in the following groups: control (no treatment), N-EVs treatment, miR-133a-3p inhibitor treatment, and combined treatment. Legend: (F) Western blot analysis was used to examine the effects of N-EVs and the miR-133a-3p inhibitor on the expression of necroptosis markers (e.g., phosphorylated MLKL, RIPK3) in HUVEC and AC16 cells. Cells were treated with N-EVs (50 µg/mL) alone, the miR-133a-3p inhibitor (100nM) alone, or a combination of both for 24 h. The figure shows the protein expression levels of phosphorylated MLKL and RIPK3 in the following groups: control (no treatment), N-EVs treatment, miR-133a-3p inhibitor treatment, and combined treatment. (G-H) Cell Viability (G) and Cytotoxicity (H) Assessments. Legend: (G) and (H) respectively assess the cell viability and cytotoxicity of HUVEC cells under the following conditions: treatment with N-EVs alone or in combination with a miR-133a-3p inhibitor for 24 h, along with the addition of the apoptosis inhibitor Z-VAD-FMK (VAD, 25 µM), the necroptosis inhibitor necrostatin (Nec, 20 µM), the ferroptosis inhibitor ferrostatin-1 (Fer, 10 µM), the pyroptosis inhibitors Ac-DMPD/DMLD-CMK (DMPD/DMLD, 20 µM) and disulfiram (dis, 1 µM), and the autophagy inhibitor 3-methyladenine (3-me, 10 µM). Statistical significance was determined as * vs. PBS group, P < 0.05; # vs. N-EVs group, P < 0.05