Nox2/4 inhibition in NB69 during ischemia/reperfusion : Inhibition of ROS-production using M4, M107, and M114

Sammanfattning: Cerebral stroke has become one of the leading causes of death and disability worldwide. During an ischemic stroke, oxygen and nutrient deprivation occurs, which combined lead to cell starvation, anoxia, and eventually cell death. However, when blood flow is restored, reperfusion damage occurs resulting in increased cell death through several mechanisms. One of the main reasons behind ischemia/reperfusion damage is oxidative stress due to elevated production of reactive oxygen species (ROS) during reperfusion. There are several proteins and processes that are thought to be involved in elevated oxidative stress and the formation of ROS during reperfusion, among which the NADPH oxidase (Nox) family is suggested to be the main contributor of ROS.To examine this hypothesis, in the present work, we inhibited activity of the Nox2 and Nox4 enzymes during ischemia/reperfusion with the Glucox Biotech AB (Sweden) inhibitors M4, M107, and M114 to evaluate whether reducing Nox activity could reduce the ischemia/reperfusion-induced cell death, hence be used as a potential stroke treatment, the cell viability was measured with MTS after ischemia/reperfusion induction and treatment with the Nox substances. We also examined the gene expression levels of the Nox enzymes Nox2 and Nox4 with qPCR after induced ischemia/reperfusion in the neuroblastoma cell line NB69.Our results showed a decrease in Nox4 gene expression after 1h ischemia/8h reperfusion and an increased expression after 1h ischemia/24h reperfusion in NB69 cells. Treatment with M114 resulted in increased cell viability after 2h ischemia/72h reperfusion. However, the toxic effect of ischemia/reperfusion-induced response was found to be inadequate, as indicated by extensive proliferation and lack of cell death. This unfavorable outcome is suggested to be excess of oxygen in medium, metabolization of L-glutamine, and effects of growth factors in the serum used in cell culture medium during the ischemic phase. Therefore, the cell culture protocol was modified to the use of PBS instead of glucose-free medium under serum-free condition during the ischemia. The altered ischemic conditions resulted in continuous reduction in cell viability at increasing ischemic time points with total cell death at 2h ischemia, suggesting applicable conditions for ischemia/reperfusion studies. Even though a conclusion could not be made about the inhibitors M4, M107, and M114 as the cell viability assay was performed under insufficient conditions; the Nox inhibitors shows high potential as future ischemic stroke treatments, which may help save lives and improve life quality for affected patients.