Deficiency of Interleukin-36 Receptor Protected Cardiomyocytes from Ischemia-Reperfusion Injury in Cardiopulmonary Bypass

Figures

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  Figure 1. IR injury alleviation in the CPB model was characterized by improved systolic cardiac function and decreased levels of myocardial injured biomarkers in the serum of IL-36R−/− rats (Myh6-Cre IL1RL2flox/flox). (A) LVSP was improved in IL-36R−/− rats (Myh6-Cre IL1RL2flox/flox) compared to wild-type rats. (B) Improved systolic function was indicated by instantaneous first derivation of LVP (+dp/dtmax and −dp/dtmax) in rats with IL-36 knockout in CPB models. (C–E) Serum levels of myocardial injury biomarkers, including myoglobin (C), troponin (D), and LDH (E) were decreased in rats with myocardial genetic knockout in CPB models. The t test was used for comparison of 2 groups. All experiments were repeated 3 times. n=8. P<0.05 indicates a significant difference. * P<0.05, ** P<0.01. LDH – lactate dehydrogenase; LVSP – left ventricular systolic pressure; WT – wild-type.

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  Figure 2. Apoptosis and inflammatory infiltration were reduced in the hearts of IL-36R−/− rats (Myh6-Cre IL1RL2flox/flox). (A) Representative figure of TUNEL staining in the hearts of WT and IL-36R−/− rats, respectively. (B) Semi-quantitative analysis of TUNEL staining in the hearts from WT rats and IL-36R−/− rats, respectively, illustrated apoptotic cardiomyocytes were decreased in IL-36R−/− rats. (C) Western blot measurements revealed decreased protein expression relative to apoptosis in hearts of rats with myocardial genetic knockout after reperfusion in CPB models. (D) Decreased chemokine expression in hearts from rats with myocardial genetic knockout after reperfusion in CPB models measured by qPCR. (E) CD68+ macrophage infiltration was reduced in IL-36−/− rat hearts as compared to WT rats. (F) The reduced expression of inflammatory cytokines illustrated by immunohistochemical staining in the hearts of in rats with myocardial genetic knockout after reperfusion in CPB models. The t test was used for comparison of 2 groups. Comparison for multiple groups was performed by ANOVA. All experiments were repeated 3 times, n=8. P<0.05 indicates a significant difference. * P<0.05, ** P<0.01.…

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  Figure 3. Oxidative stress was inhibited and the expression of iNOS/eNOS ratio was significantly decreased in IL-36R−/− compared to WT rats. (A) The inhibition rate of xanthine oxidase by SOD in heart tissue extracts from IL-36−/− rats was increased compared to WT rats, demonstrating the increased activity of SOD in the heart tissue of IL-36−/− rats in IR injury induced by CPB. (B) Significantly decreased MDA levels in the hearts from IL-36R−/− rats compared to WT rats suggests inhibition of lipid peroxidation. (C) Representative figures and semi-quantitative analysis of expression of iNOS and eNOS measured by immunoblot assays in the hearts from IL-36R−/− and WT rats. (D) Ratio of iNOS/eNOS expression in the hearts from IL-36R−/− and WT rats. The t test was used for comparison of 2 groups. Comparison for multiple groups was performed by ANOVA. All experiments were repeated 3 times, n=5. P<0.05 indicates a significant difference. * P<0.05, ** P<0.01. eNOS – endothelial NO synthetase; iNOS – inducible NO synthetase; WT – wild-type.

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  Figure 4. Inhibition of oxidative stress and expression of inflammatory cytokines was accompanied by activation of SIRT1-FOXO1-p53 signaling. (A) Western blot results of SIRT1, FOXO1, and p53 expression in human cardiomyocytes (AC16) with or without silencing of IL-36R in vitro. (B) Representative figures of ROS production evaluated by DCFA-FH fluorescent probes in the human AC16 cell line with or without silencing of IL-36R in vitro. (C) Quantitative analysis of relative fluorescent intensity of DCFA-FH probes in AC16 with or without silencing of IL-36R in vitro. (D) Expression of inflammatory cytokines and chemokines in AC16 with or without silencing of IL-36R in vitro. The t test was used for comparison of 2 groups. All in vitro experiments were repeated 5 times. P<0.05 indicates a significant difference. * P<0.05, ** P<0.01.

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  Figure 5. Deficiency of IL-36RN worsened cardiac dysfunction and IR injury after cardiopulmonary bypass. (A–C) Serum levels of myocardial injury biomarkers, including myoglobin (A), LDH (B), and troponin (C), were higher in the IL-36RN−/− rats compared to WT rats. (D) LVSP was worse in the IL-36RN−/− rats compared to WT rats. (E) Systolic function manifested by instantaneous first derivation of LVP (+dp/dtmax and −dp/dtmax) was worse in the IL-36RN−/− rats compared to WT rats. The t test was used for comparison of 2 groups. All experiments were repeated 3 times, n=8. P<0.05 indicates a significant difference. * P<0.05, ** P<0.01. LDH – lactate dehydrogenase; LVSP – left ventricular systolic pressure; WT – wild-type.