An inhibitory and beneficial effect of chlorpromazine and promethazine (C + P) on hyperglycolysis through HIF-1α regulation in ischemic stroke

Abstract

BackgroundAfter ischemic stroke, the increased catabolism of glucose (hyperglycolysis) results in the production of reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). A depressive or hibernation-like effect of C + P on brain activity was reported to induce neuroprotection. The current study assesses the effect of C + P on hyperglycolysis and NOX activation. MethodsAdult male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion. At the onset of reperfusion, rats received C + P with or without temperature control, or phloretin [glucose transporter (GLUT)-1 inhibitor], or cytochalasin B (GLUT-3 inhibitor). We detected brain ROS, apoptotic cell death, and ATP levels along with HIF-1α expression. Cerebral hyperglycolysis was measured by glucose, protein expression of GLUT-1/3, and phosphofructokinase-1 (PFK-1), as well as lactate and lactate dehydrogenase (LDH) at 6 and 24 h of reperfusion. The enzymatic activity of NOX and protein expression of its subunits (gp91phox) were detected. Neural SHSY5Y cells were placed under 2 h of oxygen-glucose deprivation (OGD) followed by reoxygenation for 6 and 24 h with C + P treatment. Cell viability and protein levels of HIF-1α, GLUT-1/3, PFK-1, LDH, and gp91phox were measured. A HIF-1α overexpression vector was transfected into the cells, and then protein levels of HIF-1α, GLUT-1/3, PFK-1, and LDH were quantitated. In sham-operated rats and control cells, the protein levels of HIF-1α, GLUT-1/3, PFK-1, LDH, and gp91phox were measured at 6 and 24 h after C + P administration. ResultsC + P reduced the protein elevations after stroke in HIF-1α, glycolytic enzymes, as well as in ROS, cell death, glucose and lactate, but raised ATP levels in the brain. In ischemic rats exposed to GLUT-1/3 inhibitors, ROS, cell death, glucose, and lactate were all decreased, as well as GLUT-1, GLUT-3, LDH, and PFK-1 protein levels. C + P decreased ischemia-induced NOX activation by reducing the enzymatic activity and protein expression of the NOX subunit gp91phox, as was observed in the presence of GLUT-1/3 inhibitors. These markers were significantly decreased following C + P administration with the induced hypothermia, while C + P administration with temperature control at 37 °C induced lesser protection after ischemia stroke. In the OGD/reoxygenation model, C + P treatment increased cell viability and diminished protein levels of HIF-1α, GLUT-1, GLUT-3, PFK-1, LDH, and gp91phox. However, in OGD with HIF-1α overexpression, C + P was unable to effectively reduce the upregulated GLUT-1, GLUT-3, and LDH. In normal conditions, C + P reduced HIF-1α and the levels of key glycolytic enzymes depending on its pharmacological effect. ConclusionC + P, partially depending on hypothermia, attenuates hyperglycolysis and NOX activation through HIF-1α regulation.