Blockage Of RAC1/NOX2 Pathway Protects Photoreceptors Against Glucose Deprivation-induced Cell Death

PurposeThe objective of this study was to investigate the molecular mechanisms that NSC/APO,RAC1/NOX2 pathway inhibitors,protects 661 W cells from glucose deprivation-induced injury.MethodsWe mimicked acute energy failure by using glucose deprivation in photoreceptor cells(661W cells).Cell viability and death were monitored with MTT and PI/Hoechest staining.661 W cells damaged by glucose deprivation were treated with NSC/APO,RAC1/NOX2 pathway inhibitors,then we examined the changes of cell viability and death.GD-induced oxidative stress was evaluated by measuring ROS with the DCFH-DA assay and HO-1 expression by western blot analysis.The activation of RAC1/NOX2/MAPK/NF-?B signal was assessed by western blot at the protein level.ResultsGlucose deprivation leads to remarkable up-regulation of ROS,HO-1 and cell death rate,down-regulation of cell viability.NSC/APO,RAC1/NOX2 pathway inhibitors,protects 661 W cells from glucose deprivation-induced injury.Glucose deprivation leads to remarkable up-regulation of NOX2 core subunits gp91,pro-apoptotic protein Bax,apoptosis-associated protein caspase3,down-regulation of anti-apoptotic protein Bcl-2.Glucose deprivation leads to abnormal mitochondrial membrane potential.Blockage of RAC1/NOX2 pathway protects photoreceptors against glucose deprivation-induced cell death.In addition,GD led to the activation of ERK,P38 and JNK in the three sub-pathways of MAPK signaling pathway,increased expression of p-ERK,p-P38 and p-JNK.Blockage of the three sub-pathways of MAPK protects photoreceptors against glucose deprivation-induced cell death.In addition,GD caused the activation of NF-?B signal and inhibiting NF-?B significantly protected 661 Wcells.ConclusionsAcute energy depletion induced caspase-dependent cell death,but NOX2 as its upstream signal regulated the cell death cascade.Activation of NOX2 produced massive ROS,which further caused the activation of the MAPK and NF-?B signals.Blockage of either MAPK family members or NF-?B could effectively rescue photoreceptors from GD-induced cell death?It can provide new ideas and methods for seeking new drugs for ischemic retinal diseases caused by acute energy failure.