Ferrostatin-1 Attenuates Oxidative Stress-dependent Neuronal Ferroptosis In N2a/APP695swe Cells

Objective:Alzheimer’s Disease(AD)is one of the leading causes of dementia and is characterized by the accumulation of β-amyloid peptide(Aβ)in the brain and the hyperphosphorylation of the microtubule-associated protein(Tau).Amyloid precursor protein(APP)protein hydrolysis is a key step in the development of AD.Studies have shown that APP is cleaved via the amyloidogenic pathway and forms extracellular Aβfibrillary tangles,which induce oxidative stress and intracellular ROS accumulation in neuronal cells and promote cell death.Neuronal ferroptosis is one of the characteristics of AD pathological changes,and ROS-activated lipid peroxidation products and iron imbalance can trigger ferroptosis in neuronal cells,but the potential relationship between Aβ,oxidative stress and ferroptosis remains unclear.Therefore,in this study,we selected human brain hippocampal sections,N2 a cells and N2a/APP695 swe cells as the experimental subjects.Among them,N2a/APP695 swe cells are N2 a cells stably transfected with APP695 gene,which can directly mimic the molecular pathological changes of APP on neuronal cells in the course of AD.To explore the effects of Ferrostatin-1,a ferroptosis inhibitor,on oxidative stress and Aβlevel in neuronal cells,further clarify the interrelationship between Aβ-induced oxidative stress and ferroptosis in neuronal cells,search for the potential pathogenesis of AD,and provide new prevention and treatment strategies and theoretical guidance for its clinical therapy.Methods:In this study,the co-localization and fluorescence intensity of Neu N,a neuronal marker protein,and GPX4,a ferroptosis marker protein,in the hippocampus of human brain were detected by immunofluorescence to determine whether GPX4 is expressed in neuronal cells and the level of GPX4 expression;the co-localization and fluorescence intensity of GPX4 and Nrf2,a key anti-oxidative stress molecule,were measured in the hippocampus of human brain,and the co-localization and fluorescence intensity of GPX4 and Nrf2 in the hippocampus of human brain were examined to determine the express level of Nrf2 in the hippocampus and whether GPX4 and Nrf2 work together to activate the antioxidant and ferroptosis pathways;the co-localization and fluorescence intensity of Nrf2 and NOX4,a key enzyme of oxidative stress,in the hippocampus of human brain were examined to determine the express level of NOX4 in the hippocampus and whether Nrf2 interacts with NOX4.In the cell experiments,cells were divided into three groups,N2 a group,N2a/APP695 swe group and N2A/APP695swe+ferrostatin-1 group,and the intracellular full-length APP was detected using Western blot,as well as extracellular Aβ1-42 levels using Elisa assay to explore whether the characteristic pathology of AD was changed;synaptic function was detected using western blot,and the expression of postsynaptic density protein 95(PSD95),synaptophysin and 25 k Da synaptosomalassociated protein(SNAP25)were measured by western blot to determine the impact of synaptic function.The ROS level was identified by flow cytometry,the fluorescence intensity of 8Oh DG was detected by immunofluorescence,and the expression of ERK1/2/Nrf2/HO-1 signalling-related proteins(p-ERK1/2,t-ERK1/2,Nrf2 and HO-1)was measured by western blot method,to determine the qualification of oxidative stress in the cell.The ferrous content was detected by flow cytometry and the levels of x CT/GPX4 signaling pathway-related proteins(x CT and GPX4)were measured by western blot,as well as the fluorescence intensity of MDA and 4HNE was measured by immunofluorescence,to investigate neuronal ferroprosis;flow cytometry to detect JC-1 levels and immunofluorescence to detect Tomm20 to calculate the number of fragmented mitochondrial cells and to clarify the altered mitochondrial function.Results:Significantly fewer GPX4-positive neuronal cells were seen in the hippocampal region of AD patients compared to normal human hippocampal tissue,as well as fewer Nrf2-positive neuronal cells,while ectopic Nrf2 nuclei could be observed and Nrf2-localized cells with GPX4 were significantly decreased in AD;as well as NOX4-stained neuronal cells were significantly increased in the hippocampal region of AD patients,and co-localized cells with Nrf2 in AD decreased.N2a/APP695 swe cells were seen to have significantly higher intracellular APP and extracellular Aβ1-42 levels and decreased expression levels of synaptic functionrelated proteins compared to N2 a cells;significantly higher intracellular ROS levels and consequently higher DNA damage marker 8OHd G,and the expression levels of ERK1/2/Nrf2/HO-1 signalling pathway-related proteins p-ERK1/2,t-ERK1/2,Nrf2 and HO-1 were significantly increased;the intracellular ferrous iron content was significantly increased,the expression levels of ferroptosis-related proteins GPX4 and x CT were increased,the fluorescence intensity of lipid peroxidation products 4HNE and MDA were significantly increased;the mitochondrial membrane potential was hyperpolarized,the mitochondrial structure became smaller and round,and the number of mitochondria was changed.In Ferrostatin-1 treated N2a/APP695 swe cells compared to N2a/APP695 swe cells,a significant decrease in intracellular APP and extracellular Aβ1-42 levels were observed,with no significant change in the expression levels of synaptic functionrelated proteins;a significant decrease in intracellular ROS levels,a significant decrease in 8OHd G fluorescence intensity,ERK1/2/Nrf2/HO-1 signalling pathwayrelated proteins,the expression levels of p-ERK1/2,t-ERK1/2,Nrf2 and HO-1 were all decreased;intracellular ferrous iron content was significantly reduced,the expression levels of ferroptosis-related proteins GPX4 and xCT were decreased,the fluorescence intensity of lipid peroxidation products 4HNE and MDA were significantly reduced;mitochondrial membrane potential was restored,mitochondrial structure became filamentous and banding,and the number of mitochondria was significantly increased.Conclusions:Neuronal ferroptosis and oxidative stress play an important role in the pathogenesis of AD.The APP695 gene promotes extracellular Aβ1-42 production in neuronal cells,induces neuronal oxidative stress and activates downstream nonenzymatic-dependent ferroptosis,while compensating for the upregulation of ERK1/2/Nrf2/GPX4 signalling pathway expression.