Protective effect of ischaemic postconditioning combined with nicorandil on myocardial ischaemia‒reperfusion injury in diabetic rats

Fig. 1. Body weight and blood glucose of rats in each experimental group. A Plasma glucose (n = 20), B Body weight (n = 20). C A representative nondiabetic rat. D A representative diabetic rat. E Glucose levels during OGTT. F Glucose area under the curve (AUC) during OGTT. G Glucose levels during ITT. H Glucose area under the curve (AUC) during ITT. **P < 0.01, ***P < 0.001. NG: normal group. DMG: diabetes mellitus group. Data are expressed as mean ± standard error of the mean (SEM) and were analysed by T test

Fig. 2. Diabetes impairs the cardioprotective effects of I-Post and nicorandil, and the cardioprotective effect of the combination treatment on myocardial I/R injury in diabetic rats. A Representative sections of TTC/Evans Blue stained heart tissue subjected to 30 min myocardial ischemia followed by 2.5 h of reperfusion and infarct area histogram analysis (n = 5). B CK-MB plasma levels in rats treated with I-Post or nicorandil (n = 6). D ROS levels in myocardial tissue in rats treated with I-Post or nicorandil (n = 6). Compared with I/R: †††P < 0.001; Compared with DM I/R: ‡‡‡P < 0.001; Comparison between nondiabetic and diabetic groups applying the same treatment: ***P < 0.001. Data represent the mean ± SEM. Data were analysed using one-way ANOVA and Bonferroni multiple comparisons test. Sham, sham surgery; I/R, ischaemia‒reperfusion; I-Post, ischaemia postconditioning; Nic, nicorandil; DM, diabetes mellitus

Fig. 3. Alterations in myocardial tissue structure in diabetic rats after I-Post or nicorandil. A Representative cross-sectional HE-stained pathological section of the heart, and comparison of pathological damage scores (n = 6). Red arrows indicate cellular oedema as well as myocardial fibre swelling; black arrows represent myofiber lysis; blue arrows represent inflammatory cell infiltration. B Representative TUNEL-stained sections of the heart, and apoptotic index of cardiomyocytes in each group (n = 5). Brown nuclei indicate TUNEL-positive nuclei (marked by red arrow). C Representative cross-sectional WGA staining of the heart, and quantitative data on the cross-sectional area of cardiomyocytes (n = 6). Compared with I/R: †††P < 0.001; Compared with DM I/R: ‡‡‡P < 0.001; Comparison between nondiabetic and diabetic groups applying the same treatment: ***P < 0.001, **P < 0.01, *P < 0.05,. Data represent the mean ± SEM. Data were analysed using one-way ANOVA and Bonferroni multiple comparisons test. Sham, sham surgery; I/R, ischaemia‒reperfusion; I-Post, ischaemia postconditioning; Nic, nicorandil; DM, diabetes mellitus

Fig. 4. Alteration of PI3K/Akt signalling pathway in diabetic rats after I-Post or nicorandil treatment alone A Representative western blot showing total expression and the phosphorylation state. Comparison of p-PI3K expression levels in myocardial tissue of rats treated with I-Post or nicorandil (n ≥ 3). B Comparison of p-Akt expression levels in myocardial tissue of rats treated with I-Post or nicorandil (n ≥ 3). C Comparison of p-GSK3βexpression levels in myocardial tissue of rats treated with I-Post or nicorandil (n ≥ 3). D Comparison of p-mTOR expression levels in myocardial tissue of rats treated with I-Post or nicorandil (n ≥ 3). E Comparison of p-eNOS expression levels in myocardial tissue of rats treated with I-Post or nicorandil (n ≥ 3). F NO plasma levels in rats treated with I-Post or nicorandil (n = 6). Compared with I/R: †††P < 0.001; Compared with DM I/R: ‡‡‡P < 0.001; Comparison between nondiabetic and diabetic groups applying the same treatment: ***P < 0.001, **P < 0.01, *P < 0.05, nsP > 0.05. Data represent the mean ± SEM. Data were analysed using one-way ANOVA and Bonferroni multiple comparisons test. Sham, sham surgery; I/R, ischaemia‒reperfusion; I-Post, ischaemia…

Fig. 5. Protective effect of I-Post combined with nicorandil on myocardial I/R injury in diabetic rats A Representative sections of TTC/Evans Blue stained heart tissue subjected to 30 min myocardial ischemia followed by 2.5 h of reperfusion and infarct area histogram analysis (n = 5). B CK-MB plasma levels in rats treated with I-Post combined with nicorandil (n = 6). C ROS levels in myocardial tissue in rats treated with I-Post combined with nicorandil (n = 6). D Representative cross-sectional HE-stained pathological section of the heart, and comparison of pathological damage scores (n = 6). Red arrows indicate cellular oedema as well as myocardial fiber swelling; black arrows represent myofiber lysis; blue arrows represent inflammatory cell infiltration. E Representative TUNEL-stained sections of the heart, and apoptotic index of cardiomyocytes in each group (n = 5). F Representative cross-sectional WGA staining of the heart, and quantitative data on the cross-sectional area of cardiomyocytes (n = 6). Compared with I-Post + Nic: ***P < 0.001, **P < 0.01, *P < 0.05. Data represent the mean ± SEM. Data were analysed using one-way ANOVA and Bonferroni multiple comparisons test. I/R,…

Fig. 6. Alteration of PI3K/Akt signalling pathway in diabetic rats after I-Post combined with nicorandil treatment alone A Representative western blot showing total expression and the phosphorylation state. Comparison of p-PI3K expression levels in myocardial tissue of rats treated with I-Post combined with nicorandil (n ≥ 3). B Comparison of p-Akt expression levels in myocardial tissue of rats treated with I-Post combined with nicorandil (n ≥ 3). C Comparison of p-GSK3βexpression levels in myocardial tissue of rats treated with I-Post combined with nicorandil (n ≥ 3). D Comparison of p-mTOR expression levels in myocardial tissue of rats treated with I-Post combined with nicorandil (n ≥ 3). E Comparison of p-eNOS expression levels in myocardial tissue of rats treated with I-Post combined with nicorandil (n ≥ 3). F NO plasma levels in rats treated with I-Post combined with nicorandil (n = 6). Compared with I/R: †††P < 0.001; Compared with DM I/R: ‡‡‡P < 0.001; Compared with I-Post + Nic: ***P < 0.001, **P < 0.01, *P < 0.05, nsP > 0.05. Data represent the mean ± SEM. Data were analysed using one-way ANOVA and Bonferroni multiple comparisons test. Sham, sham surgery; I/R,…

Fig. 7. Activation of PI3K/Akt signalling pathway by I-Post combined with nicorandil is hindered by PI3K phosphorylation inhibitor wortmannin A Representative sections of TTC/Evans Blue stained heart tissue subjected to 30 min myocardial ischemia followed by 2.5 h of reperfusion and infarct area histogram analysis (n = 5). B Representative western blot showing total expression and the phosphorylation state, Comparison of p-PI3K expression levels in myocardial tissue of rats (n ≥ 3). C Comparison of p-Akt expression levels in myocardial tissue of rats (n ≥ 3). D Comparison of p-GSK3βexpression levels in myocardial tissue of rats (n ≥ 3). E Comparison of p-mTOR expression levels in myocardial tissue of rats(n ≥ 3). F Comparison of p-eNOS expression levels in myocardial tissue of rats (n ≥ 3). Compared with I-Post + Nic + wortmannin: ***P < 0.001. Data represent the mean ± SEM. Data were analysed using T test, one-way ANOVA and Bonferroni multiple comparisons test. I/R, ischaemia‒reperfusion; I-Post + Nic, ischaemia postconditioning, combined with nicorandil; wortmannin, PI3K phosphorylation inhibitor; DM, diabetes mellitus