The Role And Molecular Mechanism Of NADPH Oxidase-4 In Mitochondrial Oxidative Stress And Neuronal Pyroptosis After Intracerebral Hemorrhage

BackgroundIntracerebral hemorrhage(ICH)is often accompanied by oxidative stress induced by reactive oxygen species(ROS),which can cause sustained secondary brain injury(SBI),such as neuronal death and damage to the blood-brain barrier.ROS has been reported to directly trigger the death of neurons.Since most ROS are produced in mitochondria,inhibiting the production of ROS at the source is crucial for reducing neuronal death after ICH.NADPH Oxidase 4(NOX4),a member of the NADPH Oxidase family,is the only subtype which can be activated in tissues without other clastral regulatory subunits to produce ROS.It is reported that pyroptosis can be triggered directly by ROS and inhibition of pyroptosis can effectively reduce neuronal death.However,it is still unknown whether NOX4 mediates the occurrence of neuronal pyroptosis after ICH.In this study,we found that ICH induced the increasing expression of NOX4,which was mainly located in neurons.Furthermore,knockdown of NOX4,carried out by Adeno-Associated virus(AAV),can reduce mitochondrial ROS production,helping to reduce mitochondrial damage and neuronal pyroptosis after ICH.Moreover,neuronal oxidative stress was significantly reduced with an enhanced antioxidant response after NOX4 knockdown.Finally,we used mitochondria-specific ROS inhibitors(Mito-TEMPO)as the way in which mitochondria act on NOX4 in reverse to treat ICH and was pleasantly surprised discovering that Mito-TEMPO inhibited NOX4 expression and neuronal pyroptosis,which was similar to AAV-NOX4.This portends that NOX4 may be an important target for inhibiting mitochondrial ROS production.In brief,our study shows that NOX4 knockdown can inhibit mitochondrial ROS production,reducing neuronal pyroptosis accompanied with increased neuronal antioxidant response after ICH.ObjectiveTo clarify the role of NOX4 in neuronal pyroptosis after ICH,starting with NOX4-mediated oxidative stress,the intervention of NOX4 was discussed,starting with mitochondria,the source of ROS production,to reduce NOX4-mediated ROS production and block the possible pathways of neuron pyroptosis caused by ROS.Experiments have proved that knockdown of NOX4 after ICH can effectively reduce ROS production in mitochondria and ultimately reduce neuronal pyroptosis.MethodsNOX4 expression was detected after ICH in rats by stereotactic localization technique.Immunofluorescence confirmed the cellular spatial localization of NOX4 after ICH in rats.Nissl staining was used to detect the neuronal death after ICH in rats.Transmission electron microscopy(TEM)was used to observe the changes of neurons and mitochondria after ICH in rats.Adeno-associated virus(AAV)and ShRNA-NOX4 were used to construct stable and reliable AAV-NOX4,and NOX4 was knocked down.Western Blot,immunofluorescence,Nissl Staining,transmission electron microscopy and other techniques were used to compare the changes of neurons and mitochondria before and after NOX4 knockdown in the brain of rats.Western Blot and immunofluorescence staining were used to explore the related pathways.Results1.NOX4 is significantly elevated in the brain tissue after ICH and reaches its peak at 72 hours.NOX4 increase was directly proportional to the severity of cerebral edema in the tissues around the bleeding lesion,and the increase mainly occurred in neurons.2.Knockdown NOX4 in the brain of rats,cerebral edema was relieved after ICH,and the permeability of blood-brain barrier decreased.3.NOX4 was knocked down in the brain of rats to inhibit the neuronal pyroptosis by inhibiting caspase1/GSDMD-N and Caspase4/11/GSDMD-C pathways.4.After NOX4 was knocked down in the brain of rats,ROS production in the brain tissue decreased significantly after ICH,and Nrf2/Keap-1 was used to enhance the neuronal tolerance on oxidative stress after ICH.5.After NOX4 was knocked down in the brain of rats,NOX4 expression in mitochondria and ROS generation in the neuronal mitochondria were significantly reduced,and mitochondrial damage of neurons was alleviated.6.After the treatment of ICH with Mito-TEMPO,NOX4 production was inhibited,the neuronal pyroptosis was significantly reduced,and the damage of the blood-brain barrier was alleviated.Conclusion1.The expression of NOX4 is increased after ICH and is proportional to the severity of cerebral edema.2.NOX4 elevation is mainly located in neurons after ICH.3.Knockdown of NOX4 in the brain of rats can effectively relieve cerebral edema and protect the blood-brain barrier after ICH.4.Knockdown of NOX4 in the brain of rats can reduce neuronal pyroptosis after ICH.5.Knockdown of NOX4 in the brain of rats can effectively relieve the oxidative stress response and enhance the neuronal tolerance on oxidative stress after ICH.6.Knockdown of NOX4 in the brain of rats can relieve neuronal mitochondrial damage and alleviate the vicious cycle of ROS superposition after ICH.7.Mito-TEMPO inhibits NOX4 elevation and decreases neuronal pyroptosis after ICH,and NOX4 may be its locus of action.