Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

Figures

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  Fig. 1.. Dexmedetomidine (DEX) has a protective effect on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury. (A) Cell -Counting Kit 8 (CCK-8) was used to measure cell viability. (B, C) Apoptosis and apoptotic rate were determined using flow cytometry. (D) GSE126104 showed that DEX decreased ectodysplasin-A2 receptor (EDA2R) expression in the left ventricle of rats. ***, P<0.001 vs. con. (E) The expression of EDA2R in cardiomyocytes was detected by RT-qPCR and western blotting. **P<0.01; ***P<0.001; ****P<0.0001 vs. con. ##P<0.01; ####P<0.0001 vs. H/R. n=3.

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  Fig. 2.. Ectodysplasin-A2 receptor (EDA2R) knockdown suppresses hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis. (A) The knockdown efficiency of EDA2R in cardiomyocytes was detected by RT-qPCR and western blotting. (B) Cell -Counting Kit 8 (CCK-8) was applied to detect cell viability. (C, D) Apoptosis was determined using flow cytometry, and apoptotic rate was calculated. ***P<0.001; ****P<0.0001 vs. con. ##P<0.01; ###P<0.001; ####P<0.0001 vs. H/R+shNC. n=3.

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  Fig. 3.. Ectodysplasin-A2 receptor (EDA2R) knockdown represses mitochondria-mediated apoptosis. (A) Mitochondrial morphology of cardiomyocytes was observed by transmission electron microscopy (the arrows represented mitochondrial morphology, scale bar=500 nm). (B) Mitochondrial membrane potential (MMP) was detected by JC-1 (Scale bar=100 µm, Red: ****P<0.0001 vs. con. or hypoxia/reoxygenation (H/R)+shNC; Green: ####P<0.0001 vs. con. H/R+shNC). (C, D) The levels of cytochrome C in mitochondria and cytoplasm of cardiomyocytes were determined by western blotting. (E) The expressions of Bax and Bcl-2 were determined in cardiomyocytes by western blotting. (F, G) Caspase-3 and Caspase-9 activity in cardiomyocytes was detected. ***P<0.001; ****P<0.0001 vs. con. #P<0.05; ##P<0.01; ###P<0.001; ####P<0.0001vs. H/R+shNC. n=3.

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  Fig. 4.. Ectodysplasin-A2 receptor (EDA2R) knockdown relieves hypoxia/reoxygenation (H/R)-induced oxidative stress. (A, B) Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in cardiomyocytes. (C) Dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to examine reactive oxygen species (ROS) levels in cardiomyocytes. ***P<0.001; ****P<0.0001 vs. con. #P<0.05; ##P<0.01; ###P<0.001; ####, P<0.0001 vs. H/R+shNC. n=3.

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  Fig. 5.. Ectodysplasin-A2 receptor (EDA2R) knockdown inhibits the activation of the NF-κB signaling pathway. (A) The levels of IκBα, p-IκBα (Ser32), NF-κB p65 and p-NF-κB p65 (Ser536) were detected in cardiomyocytes by western blotting. (B) The expression distribution of NF-κB p65 in cardiomyocytes was determined by immunofluorescence, the fluorescence intensity of p65 in the nucleus was quantified in three fields of three sections (the arrows represented the distribution of p65 in the nucleus, scale bar=50 µm). (C) The transcriptional activity of NF-κB in cardiomyocytes was measured by the electrophoretic mobility shift assay (EMSA). ****P<0.0001 vs. con. ####P<0.0001. n=3.

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  Fig. 6.. Ectodysplasin-A2 receptor (EDA2R) knockdown weakens myocardial ischemia/reperfusion (I/R) injury in mice. (A) The knockdown efficiency of EDA2R was determined by western blotting. (B, C) The left ventricular end systolic diameter (LVESD), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), left ventricular end diastolic dimension (LVEDD), LVEF and LVFS were evaluated, and representative echocardiograms were presented. (D) Heart rate in each group. (E) H&E staining was used to detect histopathological changes of myocardial tissues (Scale bar=100 µm). Heart weight was also measured. (F) The infarct size was determined by triphenyl tetrazolium chloride (TTC) staining, and infarct percentage was measured. (G) Myocardial tissue apoptosis was detected by TUNEL staining (Scale bar=50 µm). (H) mitochondrial membrane potential (MMP) was determined by JC-1 in myocardial tissues (Scale bar=50 µm). JC-1 monomer and dimer were the same cell samples and field of view for each experimental condition. (I) Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in myocardial tissues. Dichlorodihydrofluorescein diacetate (DCFH-DA) probe was…

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  Fig. 7.. A scheme of ectodysplasin-A2 receptor (EDA2R) role in myocardial ischemia/reperfusion (I/R) injury. Dexmedetomidine (DEX) downregulates the expression of EDA2R, and decreasing EDA2R inhibits the activation of the NF-κB signaling pathway, thus suppressing cell apoptosis and oxidative stress, further alleviating myocardial I/R injury.