Gp91phox (NOX2) in Activated Microglia Exacerbates Neuronal Damage Induced by Oxygen Glucose Deprivation and Hyperglycemia in an in Vitro Model

Abstract

Background/Aims: Peri-operative cerebral ischemia reperfusion injury is one of the most serious peri-operative complications that can be aggravated in patients with diabetes. A previous study showed that microglia NOX2 (a NADPH oxidase enzyme) may play an important role in this process. Here, we investigated whether increased microglial derived gp91phox, also known as NOX2, reduced oxygen glucose deprivation (OGD) after induction of hyperglycemia (HG). Methods: A rat neuronal-microglial in vitro co-culture model was used to determine the effects of gp91phox knockdown on OGD after HG using six treatment groups: A rat microglia and neuron co-culture model was established and divided into the following six groups: high glucose + scrambled siRNA transfection (HG, n = 5); HG + gp91phoxsiRNA transfection (HG-gp91siRNA, n = 5); oxygen glucose deprivation + scrambled siRNA transfection (OGD, n = 5); OGD + gp91phoxsiRNA transfection (OGD-gp91siRNA, n = 5); HG + OGD + scrambled siRNA transfection (HG-OGD, n = 5); and HG + OGD + gp91phoxsiRNA transfection (HG-OGD-gp91siRNA, n = 5). The neuronal survival rate was measured by the MTT assay, while western blotting was used to determine gp91phox expression. Microglial derived ROS and neuronal apoptosis rates were analyzed by flow cytometry. Finally, the secretion of cytokines, including IL-6, IL-8, TNF-α, and 8-iso-PGF2α was determined using an ELISA kit. Results: Neuronal survival rates were significantly decreased by HG and OGD, while knockdown of gp91phox reversed these rates. ROS production and cytokine secretion were also significantly increased by HG and OGD but were significantly inhibited by knockdown of gp91phoxsiRNA. Conclusion: Knockdown of gp91phoxsiRNA significantly reduced oxidative stress and the inflammatory response, and alleviated neuronal damage after HG and OGD treatment in a rat neuronal-microglial co-culture model.