Nox inhibitors : A potential future medicine for ischemia stroke

Abstract: This study investigated the neuroprotective effects of novel small molecule NOX inhibitors after induction of an ischemic stroke model on Neuroblastoma cells (NB69). Previous findings show that high levels of reactive oxygen species (ROS) have been identified for playing a role in causing inflammation, cellular damage and apoptosis in many cardiovascular diseases. It is believed that by inhibiting the Nox enzymes that generate ROS, the damage caused to the brain (measured as % cell viability) would be decreased.  NOX2 and NOX4 gene expression what validated in NB69 cells using PCR, gel electrophoresis and GAPDH as a reference gene. Stroke was modelled by using the oxygen-glucose depletion model and reperfusion injury was modelling by replenishing glucose and oxygen supplies to the cell and incubator for 24-72hrs. Gene expression of NOX4 expression after 1hr ischemia and 24-48hrs reperfusion showed there was an increase in relative expression after 24hrs and a decrease in expression after 48hrs. No gene expression analysis could be shown for NOX2. Cell viability was measured amongst treatment groups using the MTS assay. Statistical analysis consisted of Shapiro Wilk tests, One-way ANOVAs, Tukey tests and ROUT Outlier analysis. Only M114 was seen to have a statistically significant neuroprotective effect on the cells ( after 1hr ischemia, 24+48hrs reperfusion and 2hr ischemia + 48hr reperfusion). After 72hrs reperfusion all three treatments reduced cell viability significantly from the negative control group (p<0.0001) highlighting the importance of ROS signalling in neural cells and the consequences of eradicating it. Media was changed on plates after 48hrs reperfusion. These findings show that all three substances have some effect on the cells and can target their NOX enzymes and identifies M114 as a potential new treatment for stroke patients.