The Mechanism Of Perampanel In Mediating Neuronal Ferroptosis Induced By Oxidative Stress Through Mitophagy

Objective:Traumatic brain injury（TBI）is defined as mechanical trauma-induced brain tissue damage that causes functional or pathological changes in the brain and has a high rate of disability and mortality.But there are no effective drugs to reduce neuronal death after TBI.An increasing number of studies have found that mortality and disability rates after TBI are CLosely related to secondary brain injury.Secondary brain injury is a dynamically changing pathophysiological process that occurs hours to weeks after the onset of trauma,in which the earliest and most critical process is the post-traumatic oxidative stress on neurons due to oxygen radical overload.There are several theories about the mechanism of secondary brain injury,such as"excitatory amino acids","calcium overload","reactive oxygen radicals","mitochondrial recession",and"mitochondrial overload"."Perampanel is a non-competitive inhibitor of the novel glutamate receptor subtype AMPAR.We previously demonstrated that Perampanel significantly reduced brain tissue edema,decreased brain contusion volume,and improved motor function scores in mice after TBI,but its mechanism of action is unknown.To explore the mechanism of how Perampanel alleviates neuronal injury under oxidative stress,we established a neuronal oxidative stress model by H₂O₂stimulation of subsurface primary cortical neurons and neuronal cell line mouse-derived neuroblastoma cells N2a.Methods:1.Primary cortical neurons and neuronal cell line mouse-derived neuroma cells N2a were cultured under H₂O₂gradient concentration for 24 hours to construct a neuronal oxidative stress model,and IC50 concentrations were determined using CCK-8 assay for neuronal viability of primary neurons.2.Gradient concentration of Perampanel was used simultaneously with H₂O₂IC50 concentration,and the effect of Perampanel on oxidative stress was detected using CCK-8.3.Detect neuronal apoptosis after drug treatment using TUNNEL method,detect neuronal ROS level after drug treatment using DCFH-DA probe,detect mitochondrial membrane potential level after drug treatment using JC-1,detect ATP production level using ATP production reagent,detect neuronal LDH-release release level using LDH-release-release method.4.Detection of neuronal calcium signals and mitochondrial calcium signals using live cell calcium imaging5.Western blot was used to detect the expression of mitochondrial division-related protein DRP1 and p-DRP1 s616,autophagy-related protein and mitophagy-related protein P62,LC3II/Ⅰ,Pink1,Parkin,ferroptosis-related protein GPX4,SLC7A11（x CT）,FTH1.6.Detection of intracellular ferrous ion levels using live cell Fe²⁺7.Using Mdivi-1,a mitophagy inhibitor,the effect of different concentrations of Mdivi-1 on neuronal activity was first measured using CCK-8.After determining the concentration of Mdivi-1 used,Mdivi-1 and Peramapnel were added simultaneously,and Western blot was used to detect changes in mitophagy and ferroptosis-related expression after using Mdivi-1,and Ca²⁺and Fe²⁺levels were detected.Conclusion:1.CCK-8 confirmed a significant decrease in neuronal activity in primary mouse cortical neurons and N2a cell lines after 24 hours of treatment with H₂O₂at different IC50 concentrations2.TUNNEL assay demonstrated an increase in neuronal apoptosis,DCFH-DA probe confirmed an increase in cellular ROS,JC-1 confirmed a decrease in cellular membrane potential,LDH-release release assay confirmed an increase in LDH-release enzyme release,and ATP production assay confirmed a decrease in neuronal cellular ATP production.Simultaneous administration of 3μM Perampanel to H₂O₂-treated primary mouse cortical neurons and N2a cell lines for 24 h rescued the oxidative stress,cell death and reduced mitochondrial function caused by H₂O₂.3.Live cell imaging of Fluo-4AM and Rhod 2AM confirmed that cellular and mitochondrial calcium overload occurred under H₂O₂-induced oxidative stress and was reversed with Perampanel.4.Western blot confirmed that LC3BII/Ⅰratio increased,P62 increased,DRP1,p-DRP1 s616 expression increased under H₂O₂stimulation,indicating that autophagy occurred in neurons under oxidative stress,and the expression of ferroptosis related proteins GPX4,SLC7A11（x CT）,FTH1 protein decreased,and the average fluorescence intensity of cellular Fe²⁺increased,suggesting that we H₂O₂can cause cells to undergo ferroptosis.5.The addition of Perampanel treatment revealed further increase in LC3BII/Ⅰratio,decrease in p62 protein level,increase in DRP1,p-DRP1 s616 expression along with significant increase in Pink1 and Parkin proteins,suggesting that Perampanel can promote mitochondrial division,autophagy and mitophagy level.Moreover,theexpressionofferroptosis-relatedproteins GPX4,SLC7A11（x CT）,FTH1 protein increased and the mean fluorescence intensity of cellular Fe²⁺decreased,suggesting that Perampanel treatment could increase neuronal mitophagy and inhibit the occurrence of ferroptosis under oxidative stress conditions.6.Mdivi-1 inhibitor prevented mitophagy,decreased p-DRP1 s616 expression,and increased cellular and mitochondrial calcium mean fluorescence intensity,while Pink1 and Parkin proteins were significantly reduced,ferroptosis-related proteins GPX4,SLC7A11（x CT）,and FTH1 protein expression were decreased,and cellular Fe²⁺mean fluorescence intensity was increased.It was suggested that Mdivi-1 could inhibit neuronal mitophagy and enhance the occurrence of ferroptosis after Perampanel treatment.After H₂O₂treatment,the level of mitophagy of neurons decreased,which increased the level of cell ferroptosis,resulting in neuron death.Using Perampanel increased mitophagy levels and reduced neuronal Ferroptosis,which was inhibited by the mitophagy inhibitor MDIVI-1.