Heat stress and ischemia/reperfusion cause oxidative stress via NADPH oxidase in hypothalamic neurons

Abstract

Ischemia/reperfusion-related injuries are associated with a wide range of diverse disorders including but not limited to aging, heat stroke, diabetes and Alzheimer’s disease. Ischemia/reperfusion is known to cause oxidative stress through the generation of reactive oxygen species (ROS) and the resulting oxidative stress is thought to be a contributing factor in the progression of these neurological disorders [345]. In addition to ischemia/reperfusion injury, oxidative stress has also been reported to occur in response to heat stress in the brain and is thought to be one of the main contributors to the progression of heat-related illnesses [353]. In the present study, we examined whether simulated ischemia/reperfusion and mild heat stress increases ROS generation through similar mechanisms in cultured hypothalamic cells. Hypothalamus IVB cells were grown under normal culture conditions and either heat stressed at 43oC for 15 minutes followed by 15 minutes recovery or exposed to simulated ischemia/reperfusion by incubation for 1 hour in ischemic media in the absence of oxygen followed by 2 hours incubation in normal oxygenated media (reperfusion). Heat stress caused a significant increase in HSP70 and HO-1 gene expression as measured by real time RT-PCR. Heat stress also caused an increase in cytoplasmic HO-1 protein expression and nuclear translocation of HSF-1 as measured by western blot. Heat stress and simulated ischemia/reperfusion also increased ROS generation as measured by the fluorescent indicator carboxy-H2DCFDA. The increase in ROS was attenuated by pretreatment with the NOX inhibitor apocynin and the PKC inhibitors Gö6976 and Ro-31-8220. To further investigate the generation of ROS, we measured NOX activity using chemiluminescence. Similar to what was seen with ROS generation, both mild heat stress and simulated ischemia/reperfusion increased NOX activity and these effects were blocked by apocynin, DPI, Gö6976, Ro-31-8220 and calphostin C. Furthermore, using RT-PCR and western blot analysis, NOX4 and PKCα were found to be expressed in IVB cells. These results suggest that both heat stress and simulated ischemia/reperfusion cause oxidative stress through PKCα-mediated NOX4 activation in hypothalamic IVB cells.