Protective Effects of MicroRNA‐200b‐3p Encapsulated by Mesenchymal Stem Cells–Secreted Extracellular Vesicles in Myocardial Infarction Via Regulating BCL2L11

Figures

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  Figure 1. Identification of mesenchymal stem cells (MSCs) and extracellular vesicles (EVs). A, Observation of MSCs. B, Flow cytometry detection of MSCs surface markers. C, Transmission electron microscope observation of EVs. D, Nanoparticle tracking analysis of EVs. E, Western blot detection of EVs protein markers in MSCs debris and MSCs‐EVs. F and G, miR‐200b‐3p expression in MSCs and MSCs‐EVs. N=3. Data are shown as mean±SD and evaluated by t‐test. *P<0.05. NC indicates negative control.

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  Figure 2. Mesenchymal stem cells (MSCs)–extracellular vesicles (EVs) effectively reduce pathological damage and cardiomyocyte apoptosis in myocardial infarction (MI) mice through miR‐200b‐3p. A, Dil‐labeled EVs were injected into the infarcted heart of mice for 6 hours. B, Reverse transcription quantitative polymerase chain reaction detection of miR‐200b‐3p expression. C, Infarction area of mice observed by 2,3,5‐triphenyltetrzoliumchloride staining. D, Pathological characteristics of mice observed by hematoxylin‐eosin staining. E, Collagen volume fraction detected by Masson staining. F, Cardiomyocyte apoptosis rate detected by terminal deoxynucleotidal transferase–mediated biotin–deoxyuridine triphosphate nick‐end labeling) staining. n=6 each. Data are shown as mean±SD and evaluated by ANOVA. *P<0.05. NC indicates negative control.

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  Figure 3. Extracellular vesicles (EVs) can effectively inhibit inflammatory response and oxidative stress and improve cardiac function in myocardial infarction (MI) mice through miR‐200b‐3p. A and B, Interleukin (IL)‐1β and IL‐18 levels in myocardial tissues of MI mice. C, Reactive oxygen species (ROS) content in myocardial tissues of mice. D and E, Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in myocardial tissues of mice. F, Left ventricular end‐diastolic diameter (LVEDD) and left ventricular end‐stage systole diameter (LVESD) in mice. G, Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in mice. H, Heart rate in mice. n=6 each. Data are shown as mean±SD and evaluated by ANOVA. *P<0.05. NC indicates negative control.

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  Figure 4. BCL2L11 (Bcl‐2–like protein 11) is targeted by miR‐200b‐3p and binds to NLR family pyrin domain containing 1 (NLRP1). A, Target relationship prediction diagram. B, Dual luciferase experimental analysis results. C, RNA pull‐down experimental results. D, Western blot detection of BCL2L11 expression. E, Co‐immunoprecipitation (IP) experimental results. F, Western blot detection of NLRP1 expression. N=3; n=6 each. Data are shown as mean±SD and evaluated by t‐test or ANOVA. EVs indicate extracellular vesicles; MI, myocardial infarction; and NC, negative control. *P<0.05.

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  Figure 5. Downregulating BCL2L11 (Bcl‐2–like protein 11) enhances mesenchymal stem cells–extracellular vesicles (EVs)–mediated effect on improving myocardial pathology and reducing cardiomyocyte apoptosis in myocardial infarction mice. A, Western blot detection of BCL2L11 expression. B, Infarction area of mice observed by 2,3,5‐triphenyltetrzoliumchloride staining. C, Pathological characteristics of mice observed by hematoxylin‐eosin (HE) staining. D, Collagen volume fraction (CVF) detected by Masson staining. E, Cardiomyocyte apoptosis rate detected by terminal deoxynucleotidal transferase–mediated biotin–deoxyuridine triphosphate nick‐end labeling staining. n=6 each. Data are shown as mean±SD and evaluated by ANOVA. *P<0.05. NC indicates negative control.

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  Figure 6. BCL2L11 (Bcl‐2–like protein 11) silencing enhances extracellular vesicles (EVs)–mediated reduction in myocardial inflammatory response, oxidative stress, and improvement of cardiac function in myocardial infarction (MI) mice. A and B, Interleukin (IL)‐1β and IL‐18 levels in myocardial tissues of mice. C, Reactive oxygen species (ROS) content in myocardial tissues of mice. D and E, Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in myocardial tissues of mice. F, Left ventricular end‐diastolic diameter (LVEDD) and left ventricular end‐stage systole diameter (LVESD) in mice. G, Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in mice. H, Heart rate in mice. n=6 each. Data are shown as mean±SD and evaluated by ANOVA. *P<0.05. NC indicates negative control.

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  Figure 7. Upregulation of BCL2L11 (Bcl‐2–like protein 11) negates the role of miR‐200b‐3p–modified mesenchymal stem cells (MSCs)–extracellular vesicles (EVs) in myocardial infarction (MI) mice. A, Infarction area of mice observed by 2,3,5‐triphenyltetrzoliumchloride staining. B, Pathological characteristics of mice observed by hematoxylin‐eosin staining. C, Collagen volume fraction (CVF) detected by Masson staining. D, Cardiomyocyte apoptosis rate detected by terminal deoxynucleotidal transferase–mediated biotin–deoxyuridine triphosphate nick‐end labeling staining. E, Interleukin (IL)‐1β and IL‐18 levels in mice. F, Malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and reactive oxygen species (ROS) content in myocardial tissues of mice. G, Left ventricular end‐diastolic diameter (LVEDD), left ventricular end‐stage systole diameter (LVESD), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) in mice. H, Heart rate in mice. n=6 each. Data are shown as mean±SD and evaluated by t‐test. *P<0.05.

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  Figure 8. Molecular mechanism diagram. Mesenchymal stem cells (MSCs)–extracellular vesicles (EVs) secrete miR‐200b‐3p, thereby inhibiting the transcription of BCL2L11 (Bcl‐2–like protein 11) , further inhibiting the activation of NLR family pyrin domain containing 1 (NLRP1) inflammasomes, thus treating cardiomyocyte apoptosis and myocardial inflammatory response in myocardial infarction mice.