| Background: Epilepsy is one of children’s most common central nervous system diseases,characterized by brain dysfunction caused by abnormal neuronal discharge induced by various factors.The etiology of epilepsy is complex,and the current mainstream view is that it is mainly related to the imbalance of excitatory and inhibitory neurotransmitters secretion between synapses of the brain,that is,the imbalance of γ-aminobutyric acid(GABA)and glutamate metabolism.At the same time,with the continuous symptoms of epilepsy,the oxidative stress regulation system within the body becomes disrupted,leading to the massive death of hippocampal neurons.Although oral antiepileptic drugs can control the vast majority of epilepsy,there are still about 20% of epilepsy cases that are drug-resistant,and its treatment has always been a clinical challenge.In recent years,exploring effective measures to regulate the human body’s oxidative stress system to protect neurons has become a hot research topic.It has provided potential targets for treating various central nervous system diseases,including epilepsy.Ferroptosis,a new form of oxidative stress system disorder-induced cell death,is characterized by the massive accumulation of iron-dependent lipid peroxides.Increasingly,cutting-edge research suggests that ferroptosis,especially the Nrf2-mediated ferroptosis pathway closely related to central nervous system diseases,can be a potential target for regulating neuronal cell death and treating central nervous system diseases,including epilepsy.With further research into the regulation mechanism of ferroptosis,the natural,non-toxic bioactive substance,quercetin has been shown to inhibit ferroptosis through its good silencing information regulator 1(SIRT1)activation effect.Studies have suggested that quercetin can treat diseases such as acute lung injury,diabetes,and Alzheimer’s disease by inhibiting ferroptosis.However,current research on using quercetin to inhibit the ferroptosis pathway in hippocampal neurons to protect neurons and treat epilepsy is incomplete.The relevant regulatory mechanisms have yet to be fully elucidated.Objective: This paper mainly studies and discusses the potential association between ferroptosis and epilepsy,the feasibility of quercetin inhibiting ferroptosis in hippocampal neurons as a treatment for epilepsy,and its possible molecular mechanisms.Methods: This study first analyzed the high-throughput transcriptome sequencing data of 10 temporal lobe epilepsy brain tissue samples and their surrounding normal brain tissue samples obtained from the Gene Expression Omnibus(GEO)database.Subsequently,gene expression analysis was performed on peripheral blood samples collected from 20 normal children and 20 pediatric patients with newly diagnosed epilepsy,matched in age to the control group.The classic model of kainic-induced epilepsy in mice was employed to investigate whether quercetin can alleviate epilepsy symptoms and regulate neuronal ferroptosis.The glutamate-induced excitotoxicity model was used to explore whether quercetin can protect neurons by regulating neuronal ferroptosis.Cell transcriptomics experiments were conducted to investigate whether neuronal ferroptosis is one of the main pathways regulated by quercetin.Results: Ⅰ.The occurrence of epilepsy is associated with the Nrf2-mediated ferroptosis pathway: Firstly,we downloaded high-throughput transcriptome sequencing data of 10 temporal lobe epilepsy brain tissue samples and their surrounding normal brain tissue from the US public gene database(GEO).The analysis showed that the expression of Nrf2-mediated ferroptosis pathway-related genes was statistically different between epileptic brain tissue and surrounding normal brain tissue.Compared with normal brain tissue,the expression of Nrf2-mediated ferroptosis pathway-related genes in epileptic brain tissue was significantly decreased.We further analyzed the genes expression in peripheral blood samples collected from 20 normal children and 20 newly diagnosed pediatric epilepsy patients of corresponding age.The results showed that compared with the peripheral blood of normal children,the expression of Nrf2-mediated ferroptosis pathway-related genes in the peripheral blood of epilepsy patients was significantly decreased,indicating a statistically significant difference.In addition,in patients with epilepsy,the expression of Nrf2,SLC7A11,and GPX4 showed a positive correlation trend,with Pearson correlation coefficients of 0.496 and 0.481,respectively.The above results suggest that the Nrf2-mediated ferroptosis pathway may be associated with epilepsy.Ⅱ.Quercetin relieves symptoms and hippocampal neuronal death induced by kainic-acid in epileptic mice by inhibiting Nrf2-mediated ferroptosis:(1)In animal experiments,no significant abnormalities were observed in the body weight of mice in the quercetin pre-treatment group compared to the control group;(2)In the behavioral observation experiment,compared to the epileptic group of mice,the Racine behavioral score,the average number of epileptic seizures in the 4-hour observation period,and the average duration of epileptic seizures in the quercetin pre-treatment group of epileptic mice were all significantly decreased,and the same phenomenon was observed in the ferroptosis inhibitor pre-treatment group;(3)In the water maze experiment,the cognitive dysfunction of epileptic mice in the quercetin pre-treatment group was significantly improved compared to the epileptic group of mice,and the same phenomenon was observed in the ferroptosis inhibition group;(4)In the Nissl staining experiment,the loss of neurons in the epileptic mice in the quercetin pre-treatment group and the ferroptosis inhibitor pre-treatment group was significantly improved compared to the epileptic group of mice.Transmission electron microscopy showed that the characteristic mitochondrial shrinkage of ferroptosis was observed in the hippocampal tissue of epileptic mice in the epileptic group.At the same time,this phenomenon was improved in epileptic mice pre-treated with quercetin and ferroptosis inhibitors.(5)Compared to the control group,the contents of 4HNE,MDA,and PTGS2 mRNA in the hippocampal tissue of epileptic mice in the epileptic group were significantly increased.The GSH content was significantly decreased,while this phenomenon was reversed in the hippocampal tissue of mice pre-treated with quercetin.Similar phenomena were observed in the ferroptosis inhibitor pre-treatment group.(6)Western-blot results showed that compared to the epileptic group of mice,the expression of SIRT1,Nrf2,SLC7A11,and GPX4 in the quercetin treatment group was significantly increased.The above results indicate that the natural substance quercetin has no obvious toxicity and has protective effects on hippocampal neurons,improvement of epilepsy symptoms,and cognitive dysfunction,which may be closely related to its inhibition of Nrf2-mediated ferroptosis.Ⅲ.Quercetin inhibits neuronal ferroptosis via the SIRT1/Nrf2/SLC7A11/GPX4 pathway:(1)In cell experiments,the CCK-8 experiment showed that the viability of HT22 cells in the glutamate treatment group decreased to about 50% of the control group.In comparison,the viability of HT22 cells in the group treated with quercetin or ferroptosis inhibitors was restored to about 90% of the control group.If inhibitors of SIRT1,Nrf2,and GPX4 were also added,the cell viability would decrease again to about 50% of the control group.(2)Biological transmission electron microscopy suggested that HT22 cells treated with glutamate exhibited characteristic mitochondrial shrinkage of ferroptosis.In contrast,mitochondrial shrinkage in HT22 cells improved after treatment with quercetin or ferroptosis inhibitors.(3)In cell experiments,compared with the control group,the 4HNE,MDA,and PTGS2 mRNA content of HT22 cells treated with glutamate was significantly increased,while the GSH content was significantly decreased.This phenomenon was reversed in the group treated with quercetin and in the group pre-treated with ferroptosis inhibitors.(4)Western-blot results showed that the expression of SIRT1,Nrf2,SLC7A11,and GPX4 in the group treated with quercetin was significantly higher than that in the epilepsy group of mice.(5)Cell immunofluorescence showed that the GPX4 in the group treated with quercetin was significantly increased compared with the group treated with glutamate.These results suggest quercetin may regulate neuronal ferroptosis through the SIRT1/Nrf2/SLC7A11/GPX4 pathway to protect neurons.Ⅳ.The Nrf2-mediated ferroptosis pathway is the main regulatory pathway of quercetin in regulating neuronal ferroptosis:(1)Heat maps and volcano plots of differentially expressed genes in the group treated with quercetin and the group treated with glutamate suggest that quercetin promotes the gene expression of SIRT1,Nrf2,SLC7A11,and GPX4 in hippocampal neurons.(2)Enrichment analysis of differentially expressed genes in the group treated with quercetin and the group treated with glutamate based on the GO and KEGG databases showed that programmatic cell death plays an important role in epilepsy.Further,KEGG database enrichment analysis confirmed that ferroptosis is the main cell programmatic death pathway.(3)The protein-protein interaction(PPI)network of upregulated genes and the Venn diagram combining FerrDB data suggest that SIRT1,Nrf2,SLC7A11,and GPX4 are key genes in quercetin-regulated neuronal ferroptosis.In addition,the bioinformatics Cytohubba plugin also suggests that SIRT1,Nrf2,SLC7A11,and GPX4 are key genes in quercetin-regulated neuronal ferroptosis.Conclusion: this study explored and found a potential association between the Nrf2-dependent ferroptosis pathway and the occurrence of epilepsy.At the same time,the natural and non-toxic substance quercetin can regulate the Nrf2-dependent ferroptosis pathway and achieve the therapeutic effect of treating epilepsy and protecting cognitive function through the SIRT1/Nrf2/SLC7A11/GPX4 pathway.This study disclosed the potential relationship between ferroptosis and epilepsy,clarified the Nrf2-dependent ferroptosis pathway as a potential target for the treatment of epilepsy,and provided a theoretical basis for developing new therapeutic methods for treating epilepsy in the future. |
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