Anti-IL-20 Antibody Protects against Ischemia/Reperfusion-Impaired Myocardial Function through Modulation of Oxidative Injuries, Inflammation and Cardiac Remodeling

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  Figure 1. Interleukin (IL)-20 antibody reduces ischemia/reperfusion (I/R)-induced oxidative stress through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibition. Sprague Dawley rats receiving sham operation or ischemia/reperfusion (I/R) treatment were studied. In the IL-20 antibody treatment group, animals were injected with 5 mg/kg IL-20 antibody at the phase of reperfusion. The left ventricular tissues were collected for Western blotting assay. Representatives of Western blot (A), and the densitometric analysis of NADPH oxidase-2 (NOX-2) (B), p47phox (C), and Rac-1 (D) were shown. Plasma malondialdehyde (MDA) concentration (E) and superoxide dismutase (SOD) activity (F) were checked. The data were presented as the mean ± standard deviation (SD) of six animals in each group. (G) NADPH oxidase activity in H9C2 cells after hypoxia/reoxygenation (H/R) was presented by NAPDH/NADP+ ratio. H9C2 cells that had been first exposed to hypoxia chamber with oxygen-glucose deprivation (OGD) for 1 h were moved into the normoxia chamber with high glucose medium for further 4 h, a condition abbreviated as H1R4. In some cases, cells were transfected with si-Control or si-IL-20 for 48 h…

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  Figure 2. Interleukin (IL)-20 antibody reduces ischemia/reperfusion (I/R)-induced apoptosis by modulation of the mitochondria-dependent apoptosis pathways. Sprague Dawley rats receiving sham operation or I/R treatment were studied. In the IL-20 antibody treatment group, animals were injected with 5 mg/kg IL-20 antibody at the phase of reperfusion. (A) Histological assessment of infarct area after I/R was shown. Tissue sections were obtained from formalin-fixed heart slices that were treated with hematoxylin and eosin staining. The left ventricular tissues were collected for Western blotting assay. Representatives of Western blot (B), and the densitometric analysis of p-53 (C), Bax (D), Bcl-2 (E), and cytochrome c (F) were shown. (G) Apoptosis in tissue was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. (H) Apoptosis in H9C2 cells after hypoxia/reoxygenation (H/R) was presented by TUNEL assay using flow cytometry. H9C2 cells that had been first exposed to hypoxia chamber with oxygen-glucose deprivation (OGD) for 1 h were moved into the normoxia chamber with high glucose medium for further 4 h, a condition abbreviated as H1R4. In some cases, cells…

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  Figure 3. Interleukin (IL)-20 antibody mitigates ischemia/reperfusion (I/R)-induced inflammation by modulating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity. Sprague Dawley rats receiving sham operation or I/R treatment were studied. In the IL-20 antibody treatment group, animals were injected with 5 mg/kg IL-20 antibody at the phase of reperfusion. The left ventricular tissues were collected for Western blotting assay. Representatives of Western blot (A), and the densitometric analysis of p-p38 (B), p-inhibitor of NF-κB (I-κB) (C), and p-NF-κB (D) were shown. The enzyme-linked immunosorbent assay was used for the investigation of IL-8 concentration in plasma (E). The data were presented as the mean ± standard deviation (SD) of six animals in each group. NF-κB activity (F) and IL-8 release (G) in H9C2 cells after hypoxia/reoxygenation (H/R) intervention were checked. H9C2 cells that had been first exposed to hypoxia chamber with oxygen-glucose deprivation (OGD) for 1 h were moved into the normoxia chamber with high glucose medium for further 4 h, a condition abbreviated as H1R4. In some cases, cells were transfected with si-Control or si-IL-20 for 48 h…

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  Figure 4. The effect of interleukin (IL)-20 antibody on ischemia/reperfusion (I/R)-induced upregulation of fibrosis factors. Sprague Dawley rats receiving sham operation or I/R treatment were studied. In the IL-20 antibody treatment group, animals were injected with 5 mg/kg IL-20 antibody at the phase of reperfusion. The left ventricular tissues were collected for Western blotting assay. Representatives of Western blot (A), and the densitometric analysis of transforming growth factor beta 1 (TGFβ1) (B), p-extracellular regulated protein kinases (ERK) (C), transcription factor specificity protein 1 (Sp1) (D), and connective tissue growth factor (CTGF) (E) were shown. (F) The mRNA levels of fibrosis factors in ischemic area (anterior ventricular wall) and nonischemic area (posterior ventricular wall) were investigated by quantitative real-time polymerase chain reaction assays. The data were presented as the mean ± standard deviation (SD) of six animals in each group. (G–I) The mRNA expression of fibrosis factors in H9C2 cells after hypoxia/reoxygenation (H/R) was examined. H9C2 cells that had been first exposed to hypoxia chamber with oxygen-glucose deprivation (OGD) for 1 h were moved into…

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  Figure 5. The effect of interleukin (IL)-20 antibody on ischemia/reperfusion (I/R)-induced upregulation of cardiac remodeling-related factors and fibrosis. Sprague Dawley rats receiving sham operation or I/R treatment were studied. In the IL-20 antibody treatment group, animals were injected with 5 mg/kg IL-20 antibody at the phase of reperfusion. The left ventricular tissues were collected for Western blotting assay. Representatives of Western blot (A), and the densitometric analysis of fibroblast growth factor 2 (FGF2) (B), urokinase-type plasminogen activator (uPA) (C), matrix metallopoateinase-2 (MMP-2) (D), matrix metallopoateinase-9 (MMP-9) (E), and α-smooth muscle actin (α-SMA) (F) were shown. Masson’s trichrome staining was done to detect fibrotic tissue. Representative images of Masson’s Trichrome staining (G) and quantification (H) of fibrosis areas were shown. (I) The mRNA levels of remodeling-related factors in ischemic area (anterior ventricular wall) and nonischemic area (posterior ventricular wall) were investigated by quantitative real-time polymerase chain reaction assays. The data were presented as the mean ± standard deviation (SD) of six animals in each group. (J–N) The…

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  Figure 6. Examination of cardiac function using echocardiography. The parameters of the left ventricular function were measured using M-mode echocardiography. The ejection fraction (EF) (A) and fractional shortening (FS) (B) of left ventricle were measured to determine the left ventricular contraction ability. The volume of the left ventricle at end systole (C) and at end diastole (D), the internal dimension of left ventricle at end systole (E) and at end diastole (F), as well as the left ventricular posterior wall thickness at end systole (G) and at end diastole (H) were measured to determine the dilatation of left ventricle. Representative images of M-mode echocardiography were shown to demonstrate the rescue of ischemia/reperfusion (I/R)-induced cardiac dysfunction by interleukin (IL)-20 antibody treatment. Red arrows indicate the anterior wall of left ventricle (I). The concentrations of plasma B-type natriuretic peptide (BNP) were checked to determine the degree of cardiac dysfunction (J). The data were presented as the mean ± standard deviation of six animals in each group (* indicating p < 0.05 compared to the sham group; # indicating p < 0.05 compared to the I/R group).

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  Figure 7. The effect of interleukin (IL)-20 antibody on the concentration of biomarkers for cardiac damage. The concentrations of creatine kinase-MB (CK-MB) (A) and lactate dehydrogenase (LDH) (B) were checked to determine the extent of myocardial infarction after ischemia/reperfusion (I/R) and the protective effect of IL-20 antibody treatment. The data were presented as the mean ± standard deviation of six animals in each group (* indicating p < 0.05 compared to the sham group; # indicating p < 0.05 compared to the I/R group).

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  Figure 8. Schematic diagram of the major study findings.