A1 and A2b adenosine receptors regulate GPX4 against ferroptosis of cardiomyocytes in myocardial infarction rat model and in vitro

Abstract

AimThe regulation of GPX4 by A1AR and A2bAR was investigated, and whether the inhibition of A1AR and A2bAR on ferroptosis of myocardial cell is related to GPX4 was also discussed. Methodswe constructed a rat model of myocardial ischemia and reperfusion (MIR) model and hypoxia/reoxygenation (H/R) model of H9C2 cells, and MIR rats were intraperitoneally injected with A1AR and A2bAR agonists and antagonists. TTC staining, DHE, TUNEL, western blot experiments, immunohistochemistry assay were implemented to analyze the influence of A1AR and A2bAR on ferroptosis and potential role of GPX4. To further authenticate the result of non-specific agonists and antagonists, we transfected siRNA interference or overexpression vectors into cells. CCK8, flow cytometry and western blot were performed to evaluate cell proliferation and apoptosis, and the expression of GPX4 and ferroptosis-related proteins. ResultsThe experimental results showed that reduced expression of A1AR, A2bAR and GPX4 was found after MIR. A1AR and A2bAR activation by agonists increased GPX4 expression and decreased production of lipid ROS, further inhibiting apoptosis of cardiomyocytes. In addition, we also analyzed the effect of A1AR and A2bAR on ferroptosis-related proteins. We found that expression of FIH1 protein increased and expression of ACSL4 and NOX1 proteins decreased. Consistent with results in vivo, cellular data also indicated that A1AR and A2bAR overexpression could increase proliferation ability of H9C2, and inhibit apoptosis and ROS production, upregulate GPX4 and FIH1, and downregulate ACSL4 and NOX1. ConclusionA1AR and A2bAR could regulate GPX4, thereby affecting ferroptosis of cardiomyocytes in a rat model of MIR.