IL‐38 attenuates myocardial ischemia–reperfusion injury by inhibiting macrophage inflammation

Figure 1. Reperfusion injury stimulated interleukin‐38 (IL‐38) expression. (A) Relative messenger RNA (mRNA) expression of IL‐38 in the border zone of infarcted hearts in C57BL/6 at different time points after ischemia/reperfusion (I/R). (B) Representative image of a western blot for IL‐38 protein expression levels in infarcted hearts in C57BL/6 at different time points after I/R. (C) Representative images of infarcted heart sections (obtained from I/R mice on Day 1) stained with antibodies against F4/80 (green), CD3 (green), α‐actinin (green), vimentin (green), and the cytokine IL‐38 (red), and a nuclear stain (blue). DAPI, 4′,6‐diamidino‐2‐phenylindole. Scale bar: 50 μm; magnification: ×200 (each time point n = 8). *p < .05 vs. sham; **p < .01 vs. sham.

Figure 2. Interleukin‐38 (IL‐38) improves left ventricular function after ischemia/reperfusion (I/R). (A) Representative images of Evans blue and 2, 3, 5‐triphenyl tetrazolium chloride (TTC) staining of myocardium slices and calculation of area at risk and infarct size (relative to left ventricular [LV] area) on Day 1 after I/R. (B) Representative M‐mode echocardiography images of the left ventricle on Day 1 after I/R in different groups as indicated above. (B–E) LV ejection fraction (LVEF), LV fractional shortening (LVFS), LV end‐diastolic diameter (LVEDD), and LV end‐systolic diameter (LVESD) on Day 1 after I/R (each group n = 8). *p < .05 vs. sham; **p < .01 vs. sham; # p < .05 vs. I/R + phosphate‐buffered saline [PBS]; ## p < .01 vs. I/R + PBS.

Figure 3. Interleukin‐38 (IL‐38) inhibits inflammatory response in the infarcted heart postreperfusion. (A) Representative images of hematoxylin and eosin (HE) staining, infiltration of myeloperoxidase (MPO+) neutrophils and mouse inos+ cells in the border area of infarct hearts. Images for HE staining, neutrophils, and INOS+ cells are from Day 3 after ischemia/reperfusion (I/R). (B) Infiltration of neutrophils and inos+ cells were compared between the different groups at set time points (each group n = 6). (C–F) Analysis of messenger RNA (mRNA) levels of tumor necrosis factor‐α (TNF‐α), IL‐23, IL‐1β, IL‐17A, and IL‐6 on Day 3 after I/R. Data are depicted as fold changes versus sham and shown as the mean ± SEM of three to six independent experiments. *p < .05; **p < .01.

Figure 4. Interleukin‐38 (IL‐38) inhibited cardiomyocyte apoptosis in vivo. (A) Representative images of terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL)‐stained heart sections from different experimental groups 1 day after ischemia/reperfusion (I/R). TUNEL (green) and 4′,6‐diamidino‐2‐phenylindole (blue) staining of nuclei in apoptotic cardiomyocytes (red) in the peri‐infarct zone. Scar bar:100 μm; magnification: ×400. (B) Quantitative analysis of the percentages of TUNEL‐positive nuclei (each group n = 6). (C) Real‐time polymerase chain reaction determined messenger RNA expression levels of Bax and Bcl‐2 in the in infarct heart on Day 1 in I/R. The results were also expressed as the ratio of Bax/Bcl‐2 (n = 6). *p < .05; **p < .01.

Figure 5. Interleukin‐38 (IL‐38) inhibited lipopolysaccharide (LPS)‐induced macrophage inflammation. Macrophages were incubated with 1 μg/mL LPS and/or 100 ng/mL IL‐38 for 24 h. (A) IL‐38 expression level was assessed by enzyme‐linked immunosorbent assay (ELISA) (in cultured supernatants) and real‐time polymerase chain reaction (PCR) in cultured macrophages. (B) Real‐time PCR determined messenger RNA (mRNA) expression level of IL‐38 receptors. The expression levels of IL‐6 (C), tumor necrosis factor‐α (TNF‐α) (D), IL‐1β (E), and IL‐10 (F) were measured by ELISA in cultured supernatants of different treatment groups (each group n = 6–8). *p < .05; **p < .01; NS, not significant.

Figure 6. Interleukin‐38 (IL‐38) inhibited macrophage inflammation in the presence of troponin I (TnI). Macrophages were incubated with 10 ng/mL lipopolysaccharide (LPS) in the presence/absence of 1 μg/mL TnI and/or 50 ng/mL IL‐38 for 24 h. The expression levels of IL‐6 (A), tumor necrosis factor‐α (TNF‐α) (B), IL‐1β (C), and IL‐10 (D) were measured by enzyme‐linked immunosorbent assay (ELISA) in cultured supernatants of different treatment groups. (E) Protein was extracted from cultured cardiomyocytes. Representative images of a western blot for expression levels of p65NF‐κB and NOD‐like receptor pyrin domain‐related protein 3 (NLRP3) (n = 6). *p < .05 vs. sham (LPS+TnI−IL‐38−), **p < .01 vs. sham (LPS+TnI−IL‐38−); # p < .05 vs. (LPS+TnI+IL‐38−), ## p < .01 vs. (LPS+TnI+IL‐38−).

Figure 7. Macrophages induced by interleukin‐38 (IL‐38) and troponin I (TnI) alleviated cardiomyocyte apoptosis. The supernatant of macrophages induced by TnI(TnI‐M) and IL‐38+TnI(IL‐38/TnI‐M) was used for coculture with cardiomyocytes. The mixed cells were cultured in anaerobic conditions and maintained at 37°C for 6 h and then transferred to the normoxic incubator for 6 h to undergo reoxygenation (hypoxia [H]). Cells without anaerobic conditions served as a control (normoxia [N]). (A) Representative images of terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL)‐stained sections from cultured cardiomyocytes for the indicated times. TUNEL (green) and 4′,6‐diamidino‐2‐phenylindole (blue) staining of nuclei in apoptotic cardiomyocytes (red). Scar bar: 50 μm, magnification: ×400. (B). Representative images and quantitative analysis of apoptotic rate were assessed as (Annexin V (+) PI (−) cells+ Annexin V (+) PI (+) cells)/total cells × 100% using flow cytometry (n = 6). **p < .01.