Nox4-mediated Oxidative Stress And TF/TfR-mediated Iron Overload Promote Neuronal Iron Death After Intracerebral Hemorrhage

BackgroundIntracerebral hemorrhage(ICH)is a common cerebrovascular disease with extremely high fatality and disability rate,making it a huge challenge for clinical doctors.The pathological process after ICH is complicated,and nerve cell death is considered to be the main factor of nerve function defect after ICH.Ischemia reperfusion and hemoglobin disintegration in the hematoma area cause irreversible death of neurons,and the mechanism of neuronal death after ICH is still not completely clear.Therefore,the explanation of the form and mechanism of neuronal death has become an important direction of the preclinical research on ICH.This study aims to clarify the role and mechanism of oxidative stress and iron overload mediated by NADPH oxidase 4(Nox4)and Transferrin/Transferrin Receptor(TF/TfR)in neuronal ferroptosis after ICH.In order to reduce the neuronal death and improve the prognosis of neural function after ICH,relevant intervention methods were sought.MethodsThe rat model of ICH was established by collagenase injection.In vivo transfection of Nox4-specific siRNA was used to knock down the expression level of Nox4,Deferoxamine(DFO)was used to inhibit iron overload after ICH,and Ferrostatin 1(Fer-1),a ferroptosis inhibitor,was used to save neuronal ferroptosis.Lateral ventricular injection of ferrous chloride was used to induce excess iron accumulation.Neurological function score and forelimb placement test were used to evaluate the degree of neurological impairment.The expression of Nox4,TF/TfR,Excitatory Amino Acid Transporter 3(EAAT3)and other related proteins were analyzed by immunofluorescence and Western-blot.4-HNE and other markers of oxidative damage were determined by immunofluorescence or immunohistochemistry.Prussian blue staining was used to analyze iron overload.Neuron death and tissue damage were evaluated by cresyl violet/fast blue staining.The levels of related REDOX products(hydrogen peroxide,malondialdehyde,reduced/oxidized glutathione)were determined by various commercial kits.The characteristic cell structure of ferroptosis were observed by transmission electron microscopy.ResultsICH can induce oxidative stress and iron overload and lead to the occurrence of neuronal ferroptosis.However,the accumulation of iron and excessive hydrogen peroxide(H2O2)in neurons induced by TF/TfR activation and up-regulation of Nox4 can mediate the occurrence of oxidative stress and iron overload after ICH.According to the Fenton reaction principle,these mechanisms can induce the production of a large number of lethal reactive oxygen species(ROS)after ICH.In addition,astrocyte derived glutathione synthesis is upregulated after ICH,and neuronal glutathione depletion may be associated with down-regulation of EAAT3 expression due to primary lipid peroxidation.GPX4 expression is inhibited by the depletion of reduced glutathione(GSH)induced by downregulation of EAAT3 expression,which implies that the lipid peroxide clearance ability of neurons is inhibited.All the abovementioned pathological processes eventually lead to the occurrence of irreversible neuronal ferroptosis caused by lipid peroxide accumulation.Our study found that Nox4 knockdown and the application of iron chelating agent DFO can effectively inhibit oxidative stress and iron overload after ICH and restore the expression of EAAT3GSH-GPX4 signal axis,so as to save the ferroptosis of neurons after ICH and improve the corresponding neurological deficit symptoms.ConclusionThis study clarified that the abnormal up-regulation of Nox4 and TF/TfR mediated oxidative stress and iron overload are important molecular mechanisms of neuronal ferroptosis after ICH,and indicated that GSH deficiency and GPX4 inhibition induced by down-regulation of EAAT3 expression are the main causes of ferroptosis in neurons.However,the synthesis capacity of astrocyte derived glutathione was up-regulated to some extent after ICH.In addition,this study suggested that Nox4-siRNA and the application of iron-chelating agent DFO can save the ferroptosis of neurons by relieving oxidative stress and iron overload,and thus improve the early brain injury and neurological function prognosis after ICH in rats,which supports the necessity of the development of relevant antioxidant and iron-chelating therapeutic means and strategies,and provides corresponding therapeutic targets.