Cardioprotective Mechanisms Of Ischemic Post-conditioning To Ischemia/Reperfusion Myocardium In Rat

Background and AimIschemic postconditioning (IPO) defined as a serious of repetitive cycles of ischemia/reperfusion applied at the beginning of reperfusion after prolonged ischemia is cardioprotective by attenuating ischemia-reperfusion injury. Lots of studies have confirmed that 5 cycles of ischemia/reperfusion (10 s/10 s) followed by subsequent reperfusion after 30-min left anterior descending coronary artery (LAD) ischemia could reduce infarct size obviously compared with control group. However, most studies focus on IPO performed after a short period of reperfusion (less than 6 h). Little information is about the protective effect of IPO at 24 h of reperfusion. Therefore, using the in vivo I/R and IPO rat model and observing the change of infarct size and the expression of iNOS, we aimed to investigate the duration and protective mechanisms of IPO against I/R injury.MethodsA total of 126 healthy male Wistar rats were divided into following seven groups and 18 rats in each group received 3-h or 24-h reperfusion (n=9 per time point).① I/R group:30-min left anterior descending coronary artery (LAD) ischemia, followed by 3 h or 24 h of reperfusion;②IPO group:30 min of LAD ischemia, followed by 5 cycles of I/R (10 s/10 s) and subsequent reperfusion; ③IPO+1400W (iNOS inhibitor) group:animals received an i.v. infusion of 1400W (1 mg/kg, caudal vein) 5 min before postconditioning;④I/R+1400W group:animals received 1400W (1 mg/kg, caudal vein) 5 min before reperfusion; ⑤IPO+LY294002 (PI3K inhibitor) group: animals received an i.v. infusion of LY294002 (0.3 mg/kg, caudal vein) 5 min before postconditioning; ⑥I/R+LY294002 group:animals received LY294002 (0.3 mg/kg, caudal vein) 5 min before reperfusion; ⑦Sham group:rats were subjected to the same surgical procedures with other group and LAD wears a line without ligation. At R 3 h and R 24 h,3 rats were randomly selected from each group for dyeing by TTC to measure infarct size. Myocardial tissues and serum samples were collected from the remaining animals. The serum levels of CK were measured by elisa assays. The expression of p-Akt was measured by immunohistochemistry and western blot was used for measuring the expression of p-Akt and iNOS. SPSS 16.0 was used for statistical analysis.Results1. Modulation of infarct size.In Sham group, no infarct areas were found at each time point. At reperfusion (R) 3 h and 24 h, IPO decreased infarct size of LV significantly compared with I/R group (p<0.05). There was a significant increase in infarct size at R 24 h compared to that at R 3 h in both I/R group and IPO group. At R 24 h, the IPO-induced reduction in infarct size of LV was significantly reversed by iNOS inhibitor (1400W) and PI3K inhibitor (LY294002). That is, there were no differences of infarct size among IPO+1400W、IPO+LY294002 group and I/R group (p>0.05).2. CK activity in serum.In all groups, CK levels were comparable at baseline. At reperfusion 24 h, CK activity in the IPO group was obviously lower relative to I/R group, but this trend was cancelled by 1400W completely. There were no differences of CK activity in serum between IPO+1400W group and I/R group.3. The expression of p-Akt. Very few p-Akt expressions were found in the sham group animals. At R 3 h, a significant increase was observed in the immunoreactive scores of p-Akt in IPO groups (6-fold highter than in I/R group) (p<0.05). Certainly, the increased expression of p-Akt in IPO group could be prevented by LY294002. However, at R 24 h, there were no significantly different between IPO group and I/R group in the expression of p-Akt (p>0.05).4. Expression of iNOS in the myocardium. At R 24 h, compared with I/R group, the IPO group had higher expression levels of iNOS (p<0.05). With the present of LY294002, the increased expression of iNOS was prevented. At R 3 h, the expression of iNOS among all groups were comparable (p>0.05).ConclusionsAt reperfusion 24 h after ischemia, IPO has obvious cardioprotective effects and iNOS as an important downstream effector of PI3K-Akt pathway contributes to the cardioprotection of IPO.