Serum-Derived Extracellular Vesicles Protect Against Acute Myocardial Infarction by Regulating miR-21/PDCD4 Signaling Pathway

Figure 1. Human serum extracellular vesicles inhibit OGD/R induced apoptosis in NRCMs. (A) Western blot for CD63 in supernatant and EVs of human serum (n = 4). (B) Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay for apoptosis in NRCMs treated with OGD/R (n = 4). Scale bar: 100 μm. (C) Analysis for percentage of TUNEL positive cells (n = 4). (D–F) Western blot for Bax, Bcl-2, Caspase 3, and Cleaved caspase 3, and quantitative analysis for Bax/Bcl-2 and Cleaved caspase 3/Caspase 3 ratio. β-actin was used as a loading control (n = 6). **P < 0.01; ***P < 0.001. EVs, extracellular vesicles.

Figure 2. Mouse serum extracellular vesicles inhibit OGD/R induced apoptosis in NRCMs. (A,B) Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining of NRCMs treated with EVs extracted from mouse serum under the condition of OGD/R (n = 4). Scale bar: 100 μm. (C–E) Western blot for Bax, Bcl-2, Caspase 3, and Cleaved caspase 3, and quantitative analysis for Bax/Bcl-2 and Cleaved caspase 3/Caspase 3 ratio. β-actin was used as a loading control (n = 6). *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001. EVs, extracellular vesicles.

Figure 3. Knockdown of miR-21 partially rescue the inhibition of OGD/R-induced NRCM apoptosis by human serum extracellular vesicles. (A) qRT-PCRs for expression of miR-21 in supernatant and EVs extracted from mouse serum. n = 4. (B) Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay for apoptosis in NRCMs (n = 4). Scale bar: 100 μm. (C) Analysis for percentage of TUNEL positive cells (n = 4). (D–F) Western blot for Bax, Bcl-2, Caspase 3, and Cleaved caspase 3, and quantitative analysis for Bax/Bcl-2 and Cleaved caspase 3/Caspase 3 ratio. β-actin was used as a loading control (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001. EVs, extracellular vesicles.

Figure 4. PDCD4 is a target gene of miR-21 in OGDR-induced NRCM apoptosis. (A–C) Western blot and quantitative analysis for PDCD4 after miR-21 mimic and miR-21 inhibitor treatments in NRCMs (n = 6). (D) mRNA expression levels of PDCD4 was decreased in NRCMs after siRNA-PDCD4 treatment (n = 6). (E,F) Western blot and quantitative analysis for PDCD4 after siRNA-PDCD4 treatments in NRCMs (n = 4). (G) Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay for apoptosis in NRCMs (n = 4). Scale bar: 100 μm. (H) Analysis for percentage of TUNEL positive cells (n = 4). (I–L) Western blot for PDCD4, Bax, Bcl-2, Caspase 3, and Cleaved caspase 3, and quantitative analysis for PDCD4, Bax/Bcl-2, and Cleaved caspase 3/Caspase 3 ratio. β-actin was used as a loading control (n = 6). *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001.

Figure 5. Knockdown of PDCD4 inhibited the protection of human extracellular vesicles against OGDR-induced NRCM apoptosis. (A,B) Western blot for PDCD4 and quantitative analysis for PDCD4/β-actin ratio (n = 6). (C) Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay for apoptosis in NRCMs treated with OGD/R (n = 4). Scale bar: 100 μm. (D) Analysis for percentage of TUNEL positive cells (n = 4). (E–H) Western blot for PDCD4, Bax, Bcl-2, Caspase 3 and Cleaved caspase 3, and quantitative analysis for PDCD4, Bax/Bcl-2 and Cleaved caspase 3/Caspase 3 ratio. β-actin was used as a loading control (n = 6). *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001. EVs, extracellular vesicles.

Figure 6. Mouse serum extracellular vesicles increased miR-21 levels, reduced the AMI-induced infarct area and attenuated cardiomyocytes apoptosis in mouse. (A) Photographs of TTC staining of six slices from mice cardiac tissues (n = 4). Scale bar, 1,000 μm. (B,C) Analysis of AAR/LV and INF/AAR ratio to define the infarct area of hearts from mice treated with MI and EVs extracted from mice serum (n = 4). (D) qRT-PCRs for expression of miR-21 in mouse heart tissues at 24 h after the injection of EVs. (E,F) Western Blot for expression of PDCD4 in mouse heart tissues at 24 h after the injection of EVs. n = 4; (G) Quantitative real time-polymerase chain reactions (qRT-PCRs) for expression of miR-21 (n = 4). (H–K) Western blot of border tissues for PDCD4, Bax, Bcl-2, Caspase 3, and Cleaved caspase 3, and quantitative analysis for PDCD4, Bax/Bcl-2 and Cleaved caspase 3/Caspase 3 ratio. β-actin was used as a loading control (n = 4). (L–O) Western blot of infarct tissues for PDCD4, Bax, Bcl-2, Caspase 3 and Cleaved caspase 3, and quantitative analysis for PDCD4, Bax/Bcl-2, and Cleaved caspase 3/Caspase 3 ratio. β-actin was used as a loading control (n = 4). *0.01 < P < 0.05; **0.001 < P < 0.01;…