Sevoflurane Exposure Induces Neuronal Cell Ferroptosis Initiated By ER Stress-mediated ATF3 Activation In The Developing Brain

Background:With the rapid progress and development of anesthesia and surgical techniques,an increasing number of infants and pregnant women are undergoing anesthesia and surgical treatment.The human nervous system experiences a crucial developmental period from the late stages of fetal pregnancy to 2-3 years after infancy.During this developmental phase,the brain is highly susceptible to various factors,including general anesthesia drugs,which may lead to developmental neurotoxic damage.Abundant animal studies and clinical data indicate that exposure to inhaled anesthetics such as sevoflurane and isoflurane during the early stages of life can cause widespread pathological changes in the immature brain of infants,resulting in long-term behavioral abnormalities and impairment of neurocognitive functions.Therefore,investigating the neurodevelopmental toxicity and mechanisms of inhaled anesthetics has become an urgent issue in the field of anesthesia and even in neuroscience.Research indicates that neuronal death is a major characteristic of developmental neurotoxicity induced by inhaled anesthetics,but the specific mechanisms are not clear.Ferroptosis is a form of programmed cell death characterized by an increase in intracellular ferrous iron(Fe²⁺).Accumulated Fe²⁺can react with hydrogen peroxide（H₂O₂）through the Fenton reaction,generating hydroxyl radicals,which then cause lipid peroxidation damage to polyunsaturated fatty acids（PUFA）.Studies have confirmed that ferroptosis not only mediates cell death in organ damage such as the heart,liver,and kidneys but is also associated with cell death in various neurological diseases.The endoplasmic reticulum（ER）is not only the site for protein synthesis,modification,and folding but also a central hub for driving lipid peroxidation.Upon sensing damage or stress from the internal and external environment,ER stress can be triggered.The PERK/ATF4 pathway activation is closely related to the occurrence of ferroptosis.Activating transcription factor 3（ATF3）,as a stress response factor,is rapidly expressed when cells are subjected to various harmful stimuli,regulating the transcriptional activation or inhibition of relevant target genes.Under ER stress,the activation of the PERK/ATF4 pathway can further activate ATF3,playing a crucial role in various forms of cell death,including apoptosis,autophagy,and ferroptosis.Currently,it is unclear whether sevoflurane can induce ferroptosis in developing brain neurons.The role of ER stress-mediated ATF3 activation in the process of sevoflurane-induced ferroptosis in developing brain neurons,the mechanisms regulating the imbalance of intracellular ferrous iron homeostasis,and the pathways through which lipid peroxidation damage is activated are still not fully understood.Objective:（1）Investigating whether ferroptosis was implicated in sevoflurane-induced neuronal cell death in the developing brain.（2）Exploring the potential mechanisms of action of ER stress-mediated ATF3activation in the process of sevoflurane-induced ferroptosis in developing brain neurons.This research aims to provide a potential prevention and treatment strategy for the developmental neurotoxicity of inhaled anesthetics.Methods:（1）HT22 mouse hippocampal neuronal cells and primary hippocampal neurons from rats were subjected to 2%,4%,and 8%sevoflurane exposure for 6,12,and 24 hours.Cell viability was assessed using the MTT assay,and cell death rates were determined through LDH release experiments.Ferrous iron and malondialdehyde（MDA）assay kits were employed to measure the intracellular concentrations of Fe²⁺and MDA in neuronal cells.Changes in cell morphology were observed using an optical microscope,and alterations in mitochondrial morphology were examined using transmission electron microscopy.Protein imprinting was employed to assess the expression of ferrous iron metabolism-related proteins,including transferrin receptor（TFR）,transferrin（TF）,and ferroportin（FPN）,in neuronal cells.Neuronal cells were pretreated with the iron chelator deferoxamine（DFO）（100μmol/L）,antioxidants Fer-1（50μmol/L）,Lip-1（10μmol/L）,Vit E（100μmol/L）,GSH（2.5 mmol/L）,or an equal volume of saline for 1 hour before exposure to 4%or 8%sevoflurane for 24 hours.Subsequently,cell survival rates,death rates,and intracellular concentrations of Fe²⁺and MDA in neuronal cells were measured.（2）HT22 mouse hippocampal neuronal cells and primary hippocampal neurons from rats were subjected to 4%and 8%sevoflurane exposure for 6 to 24 hours.The H₂O₂assay kit was used to measure the intracellular H₂O₂in neuronal cells.Neuronal cells were pretreated with GSH（2.5 mmol/L）or saline for 1 hour before exposure to sevoflurane.Ferrous iron and hydrogen peroxide assay kits were used to measure the intracellular concentrations of Fe²⁺and H₂O₂,and protein imprinting was employed to assess the expression of TFR,TF,and FPN in neuronal cells.Neuronal cells were treated with H₂O₂（500μmol/L）for 24 hours,and the death rate,intracellular Fe²⁺,MDA,and the expression of TFR,TF,and FPN were measured.（3）HT22 mouse hippocampal neuronal cells and primary hippocampal neurons from rats were subjected to 4%and 8%sevoflurane exposure for 6 to 24 hours.The protein imprinting method was used to detect the expression of ATF3 in neuronal cells,and confocal microscopy was employed to observe the aggregation of ATF3 in the cell nucleus.Neuronal cells were treated with si RNA to silence the ATF3 gene,followed by exposure to 8%sevoflurane for 24 hours.The death rate of cells,intracellular Fe²⁺and H₂O₂,MDA,and the expression of TFR,TF,and FPN were measured using ferrous iron,hydrogen peroxide,and MDA assay kits,as well as the protein imprinting method.（4）HT22 mouse hippocampal neuronal cells and primary hippocampal neurons from rats were subjected to 4%and 8%sevoflurane exposure for 6 to 24 hours.The protein imprinting method was used to detect the expression of endoplasmic reticulum stress-related proteins GRP78,PERK,and ATF4 in neuronal cells.Neuronal cells were treated with endoplasmic reticulum stress inhibitors 4-PBA（250μmol/L）,PERK inhibitor GSK2606414（1μmol/L）,or saline for 1 hour before exposure to 8%sevoflurane for 24 hours.Cell death rate,intracellular Fe²⁺,H₂O₂,and MDA concentrations,as well as the expression of GRP78,PERK,ATF4,ATF3,TFR,TF,and FPN,were measured using ferrous iron,hydrogen peroxide,and MDA assay kits,and the protein imprinting method.Neuronal cells were treated with si RNA to silence the ATF4gene,followed by exposure to 8%sevoflurane for 24 hours.The death rate of cells,intracellular Fe²⁺,H₂O₂and MDA content,as well as the expression of ATF4,ATF3,TFR,TF,and FPN were measured.（5）HT22 mouse hippocampal neuronal cells were subjected to 4%and 8%sevoflurane exposure for 24 hours,and the dihydroethidium（DHE）fluorescence probe was used to detect changes in intracellular superoxide levels.Neuronal cells were treated with 4%and 8%sevoflurane for 6 to 24 hours,and the protein imprinting method was used to detect the expression of NADPH oxidase 4（NOX4）,catalase,glutathione peroxidase 4（GPX4）,and cysteine/glutamate transporter light chain（SLC7A11）in neuronal cells.Neuronal cells were treated with NOX4 inhibitor GKT137831（50μmol/L）or saline for 1 hour before exposure to 8%sevoflurane for 24 hours.DHE assay was used to detect changes in intracellular superoxide levels,and ferrous iron,hydrogen peroxide,and MDA assay kits were used to measure intracellular H₂O₂,Fe²⁺and MDA concentrations.The protein imprinting method was used to detect the expression of NOX4,TFR,TF,and FPN in neuronal cells.Neuronal cells were treated with si RNA to silence the ATF3 gene,followed by exposure to 8%sevoflurane for 24 hours.The expression of NOX4,catalase,GPX4,and SLC7A11,as well as the levels of cysteine and GSH in neuronal cells,were measured using the amino acid and GSH assay kits.（6）Newborn C57BL/6J mice and ATF3 gene knockout mice were intraperitoneally injected with GSK2606414（50 mg/kg）,iron chelator deferoxamine（DFP）（75 mg/kg）,or saline 1 hour before anesthesia.Newborn mice were exposed to 3%sevoflurane for 2hours each day on postnatal days 6（P6）,7（P7）,and 8（P8）.Hippocampal tissues were collected 6 hours after three consecutive exposures to sevoflurane,and ferrousc iron,hydrogen peroxide,and MDA assay kits were used to detect intracellular H₂O₂,Fe²⁺,and MDA levels.Protein imprinting was used to detect the expression of PERK,ATF4,ATF3,TFR,TF,NOX4,FPN,catalase,GPX4,and SLC7A11 in the hippocampus.HE staining was performed on the hippocampal tissues obtained 7 days after three consecutive exposures to sevoflurane,and the pathological changes and survival rate of CA1pyramidal neurons in the hippocampus were observed.Morris water maze experiments were conducted on postnatal day 31 to assess spatial learning and memory function,including a place navigation test conducted over 5 days（P31 to P35）and a spatial exploration test on the 6th day（P36）.Results:（1）Sevoflurane exposure can concentration-and time-dependently inhibit the viability of hippocampal neurons and induce neuronal cell death.This leads to a significant increase in intracellular Fe²⁺and MDA,accompanied by upregulation of TFR and TF expression,as well as downregulation of FPN expression.Optical microscopy revealed a decrease in neuronal cell density,reduced cell volume,and a more rounded cell outline after exposure to 8%sevoflurane for 24 hours.Transmission electron microscopy showed mitochondrial shrinkage,increased membrane density,and expanded cristae,consistent with the morphological features of ferroptosis.Treatment with the iron chelator DFO significantly inhibited sevoflurane-induced neuronal cell death and reduced the increase in intracellular Fe²⁺concentration and MDA levels after sevoflurane exposure.Additionally,pre-treatment with lipid inhibitors Fer-1 and Lip-1 markedly suppressed the sevoflurane-induced increase in MDA levels and alleviated the lethal effects of sevoflurane on neuronal cells.（2）Sevoflurane exposure can induce a concentration-and time-dependent increase in intracellular H₂O₂levels in neuronal cells.Pretreatment with GSH reduces H₂O₂generation,effectively suppressing the sevoflurane-induced increase in intracellular Fe²⁺,MDA,and neuronal cell death.Additionally,GSH pretreatment prevents the upregulation of TFR and TF and the downregulation of FPN induced by sevoflurane.Furthermore,exogenous H₂O₂administration induces an increase in intracellular Fe²⁺and MDA,as well as neuronal cell death,promoting the upregulation of TFR and TF and the downregulation of FPN.（3）Sevoflurane exposure can induce a concentration-and time-dependent increase in the expression of ATF3 within neuronal cells,promoting the nuclear translocation of ATF3.Laser confocal microscopy reveals a significant accumulation of ATF3 in the nuclei of neuronal cells after sevoflurane exposure.Silencing the ATF3 gene not only inhibits the sevoflurane-induced increase in intracellular H₂O₂,Fe²⁺,and MDA levels,as well as neuronal cell death but also prevents the upregulation of TFR and TF and the downregulation of FPN induced by sevoflurane.（4）Sevoflurane exposure can induce a concentration-and time-dependent upregulation of endoplasmic reticulum（ER）stress-related proteins,including GRP78,PERK,and ATF4,within neuronal cells.Intervention with the ER stress inhibitor 4-PBA or the PERK inhibitor GSK2606414 not only inhibits the sevoflurane-induced upregulation of GRP78,PERK,ATF4,and neuronal cell death but also suppresses the expression of ATF3 within neuronal cells.Additionally,it reduces the increase in H₂O₂,Fe²⁺,and MDA levels,as well as the upregulation of TFR and TF,and the downregulation of FPN induced by sevoflurane.Furthermore,silencing the ATF4 gene significantly inhibits the expression of ATF3 and neuronal cell death induced by sevoflurane,while also reducing the increase in H₂O₂,Fe²⁺,and MDA levels,as well as the upregulation of TFR and TF and the downregulation of FPN induced by sevoflurane.（5）Sevoflurane exposure can induce a concentration-and time-dependent increase in superoxide anions,upregulation of NOX4 expression,and downregulation of Catalase,GPX4,and SLC7A11 expression within neuronal cells.Administration of GKT137831 to inhibit NOX4 expression significantly reduces the sevoflurane-induced increase in superoxide anions and H₂O₂within neuronal cells,while also inhibiting the increase in Fe²⁺and MDA and the upregulation of TFR and TF,and the downregulation of FPN induced by sevoflurane.Additionally,silencing the ATF3 gene significantly inhibits the upregulation of NOX4 and the downregulation of Catalase,GPX4,and SLC7A11induced by sevoflurane,while also reducing the decrease in cysteine and GSH levels within neuronal cells induced by sevoflurane.（6）Sevoflurane exposure significantly leads to neuronal damage and death in the hippocampal region of newborn mice（CA1 pyramidal neuron survival rate decreases to80%）,accompanied by an increase in H₂O₂,Fe²⁺,MDA,and upregulation of PERK,ATF4,ATF3,TFR,TF,NOX4,and downregulation of FPN,Catalase,GPX4,SLC7A11.Pretreatment with the iron chelator DFP significantly reduces the increase in Fe²⁺and MDA induced by sevoflurane exposure,and markedly increases the survival rate of CA1pyramidal neurons.Administration of GSK2606414 or ATF3 gene knockout not only significantly inhibits the increase in H₂O₂and neuronal death in the hippocampus of newborn mice induced by sevoflurane exposure but also reduces the increase in Fe²⁺and MDA induced by sevoflurane and the upregulation of PERK,ATF4,ATF3,TFR,TF,NOX4,and the downregulation of FPN,Catalase,GPX4,SLC7A11.（7）The results of the Morris Water Maze experiment show that,compared to normal mice,mice exposed to repeated sevoflurane exhibit a significantly prolonged escape latency on the 3th,4th,and 5th days（P<0.05）,and a significantly shortened spatial exploration time on the 6th day（P<0.05）.Compared to the sevoflurane group,mice pre-treated with GSK2606414,DFP,or ATF3 silencing before sevoflurane exposure show a significantly shortened escape latency on the 3th,4th,and 5th days（P<0.05）and a significantly prolonged spatial exploration time on the 6th day（P<0.05）.Conclusion:（1）In vivo and in vitro studies suggest that clinically relevant concentrations of sevoflurane exposure can lead to ferroptosis in developing hippocampal neurons.The mechanism involves an increase in intracellular Fe²⁺concentration and lipid peroxidation.（2）Sevoflurane exposure induces endoplasmic reticulum（ER）stress,leading to the activation of the PERK/ATF4 pathway,resulting in ATF3 activation and nuclear translocation.This,in turn,causes the accumulation of H₂O₂in neuronal cells.Excess H₂O₂directly reacts with Fe²⁺through the Fenton reaction,generating hydroxyl radicals,causing lipid peroxidation damage to cell membrane polyunsaturated fatty acids（PUFA）.Additionally,H₂O₂induces an upregulation of TFR and TF expression while downregulating FPN expression,further increasing intracellular Fe²⁺,leading to sustained lipid peroxidation damage accumulation,initiating the cascade of ferroptosis,ultimately resulting in hippocampal neuronal ferroptosis.（3）The mechanism by which sevoflurane-induced ATF3 activation leads to an increase in intracellular H₂O₂is as follows:ATF3,on the one hand,promotes an increase in intracellular H₂O₂by upregulating NOX4 expression and inhibiting Catalase expression.On the other hand,it inhibits GPX4 and SLC7A11 expression,causing a decrease in intracellular cysteine and GSH,leading to a reduction in the clearance capacity of H₂O₂.（4）Repeated exposure to sevoflurane induces endoplasmic reticulum stress and ATF3 activation in newborn mouse hippocampal neurons,leading to ferroptosis and resulting in spatial learning and memory impairment in newborn mice.