ROS-associated mechanism of different concentrations of pinacidil postconditioning in the rat cardiac Nrf2-ARE signaling pathway

Abstract

Previous studies have confirmed that 50 µmol/l pinacidil postconditioning (PPC) activates the nuclear factor-E2 related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway, which protects the myocardium from ischemia-reperfusion (IR) injury; however, whether this is associated with reactive oxygen species (ROS) generation remains unclear. In the present study, a Langendorff rat model of isolated myocardial IR was established to investigate the mechanism of PPC at different concentrations, as well as the association between the rat myocardial Nrf2-ARE signaling pathway and ROS. A total of 48 rats were randomly divided into the following six groups (n=8 per group): i) Normal; ii) IR iii) 10 µmol/l PPC (P10); iv) 30 µmol/l PPC (P30); v) 50 µmol/l PPC (P50); and vi) N-(2-mercaptopropionyl)-glycine (MPG; a ROS scavenger) + 50 µmol/l pinacidil (P50 + MPG). At the end of reperfusion (T3), compared with the IR group, the P10, P30 and P50 groups exhibited improved cardiac function, such as left ventricular development pressure, heart rate, left ventricular end-diastolic pressure, +dp/dtmax, myocardial cell ultrastructure and mitochondrial Flameng score. Furthermore, the P10 and P50 groups demonstrated the weakest and most marked improvements, respectively. Additionally, in the P10, P30 and P50 groups, the residual ROS content at the end of reperfusion was highly negatively correlated with relative expression levels of Nrf2 gene and protein. Higher pinacidil concentration was associated with higher ROS generation at 5 min post-reperfusion (T2), although this was significantly lower compared with the IR group, as well as with increased expression levels of antioxidant proteins and phase II detoxification enzymes downstream of the Nrf2 and Nrf2-ARE pathways. This result was associated with a stronger ability to scavenge ROS during reperfusion, leading to lower levels of ROS at the end of reperfusion (T3) and less myocardial damage. The optimal myocardial protective effect was achieved by 50 mmol/l pinacidil. However, cardiac function of the P50 + MPG group was significantly decreased, ultrastructure of cardiomyocytes was significantly impaired and the relative expression levels of genes and proteins in the Nrf2-ARE pathway were decreased. The aforementioned results confirmed that different PPC concentrations promoted early generation of ROS and activated the Nrf2-ARE signaling pathway following reperfusion, regulated expression levels of downstream antioxidant proteins and alleviated myocardial IR injury in rats. Treatment with 50 mmol/l pinacidil resulted in the best myocardial protection.