Mechanism of ischemic postconditioning-induced activation of Nrf2-ARE signaling pathway during myocardial ischemia-reperfusion: the relationship with ROS

Abstract

Objective To investigate the relationship between the mechanism of ischemic postconditioning-induced activation of nuclear factor-E2 related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway during myocardial ischemia-reperfusion (I/R) and reactive oxygen species (ROS). Methods Healthy male Sprague-Dawley rats, aged 16-20 weeks, weighing 250-300 g, were heparinized and anesthetized with intraperitoneal 1% pentobarbital sodium 40 mg/kg.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution.Thirty-two isolated rat hearts were randomly divided into 4 groups (n=8 each) using a random number table: control group (group C), group I/R, ischemic postconditioning group (group IPO), and N-(2-mercaptopropionyl)-glycine (a ROS scavenger) + IPO group (group M + IPO). After 20 min of equilibration, group C was continuously perfused with K-H solution for 100 min, and the isolated hearts received the drugs via the perfusion system in the other groups.Group I/R was perfused with cardioplegic solution 4 ℃ St.Thomas, and then was subjected to 40 min of ischemia at 32 ℃ followed by 60 min of reperfusion.In group IPO, ischemic postconditioning was induced by 6 cycles of 10 s reperfusion followed by 10 s limb ischemia starting from the onset of reperfusion, and the hearts were then perfused for 58 min.In group M + IPO, the hearts were perfused with K-H solution containing N-(2-mercaptopropionyl)-glycine 2 mmol/L for 3 min starting from the onset of reperfusion, underwent 2 min of ischemic postconditioning, and then was perfused for 55 min.Heart rate (HR), left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and positive maximal pressure of left ventricular increase (+ dp/dtmax) were recorded at the end of equilibration and of reperfusion.At 5 min of reperfusion and the end of reperfusion, myocardial specimens were obtained from the left ventricle for determination of ROS content by enzyme-linked immunosorbent assay.At the end of reperfusion, myocardial specimens were obtained from the left ventricle for examination of the ultrastructure of myocardial cells and for determination of Nrf2, heme oxygenase-1 (HO-1), quinone oxidoreductase 1 (NQO1), and superoxide dismutase 1 (SOD1) mRNA and protein expression (by using Western blot and real-time polymerase chain reaction). The damage to myocardial mitochondria was assessed using Flameng scoring. Results Compared with group C, HR, + dp/dtmax and LVDP were significantly decreased, and LVEDP was increased at the end of reperfusion in I/R and M+ IPO groups, HR and LVDP were decreased, LVEDP was increased, and no significant changes were found in + dp/dtmax at the end of reperfusion in IPO group, Flameng score was increased in I/R, IPO and M+ IPO groups , the ROS content was increased at the end of reperfusion in I/R, IPO and M+ IPO groups, and Nrf2, HO-1, NQO1 and SOD1 mRNA and protein expression was down-regulated at the end of reperfusion in I/R, IPO and M+ IPO groups.Compared with group I/R, HR, + dp/dtmax and LVDP were significantly increased, and LVEDP and ROS content were decreased at the end of reperfusion, Nrf2, HO-1, NQO1 and SOD1 mRNA and protein expression was up-regulated at the end of reperfusion in IPO and M+ IPO groups, Flameng score was decreased in IPO group, there was no significant change in Flameng score in M+ IPO group.Compared with group IPO, HR, + dp/dtmax and LVDP were significantly decreased, LVEDP and ROS content were increased at the end of reperfusion, Flameng score was increased, and Nrf2, HO-1, NQO1 and SOD1 mRNA and protein expression was down-regulated in M+ IPO group. Conclusion Ischemic postconditioning can regulate ROS level and activate Nrf2-ARE signaling pathway, thus attenuating myocardial I/R injury in rats. Key words: Ischemic postconditioning; Myocardial reperfusion injury; Reactive oxygen species; Nuclear factor-E2 related factor 2; Response elements