The Effect And Mechanism Of Soddium Valproate On Ferroptosis In Mg²⁺-free Induced Epileptic Hippocampal Neurons

Objective:Epilepsy is a kind of transient brain dysfunction syndrome induced by abnormal discharge of brain area,whose clinical characteristics are chronic and recurrent epileptic seizures.The etiology of epilepsy is complicated and its pathogenesis has not been completely clarified now.At present,programmed cell death has been confirmed to be involved in the pathological mechanisms of epilepsy.Ferroptosis,a regulated cell death pattern,is caused by divalent iron ion-dependent lipid peroxides accumulation,which has been proposed in the last decade and has been confirmed to be involved in the pathology of epilepsy.Sodium valproate（VPA）is a classic first-line antiepileptic drug that exerts neuroprotective effects by inhibiting oxidative stress.In view of this,our study is carried out to decipher whether ferroptosis is coupled with the pathology of epilepsy as well as ferroptosis is involved in antiepileptic mechanisms of VPA in Mg²⁺-free induced epileptic cell model,highlighting the ferroptosis-based mechanisms and elaborating the new perspective therapeutic targets of epilepsy.Methods:Sprague Dawley（SD）embryonic day 19-20（E19-20）rats were decapitated,and then the hippocampal tissue of their pups was isolated for primary hippocampal neurons culture.After 7 days of culture,the cultured hippocampal neurons were divided into 3 groups:blank control group（control group）,epilepsy group(Mg²⁺-free group)and VPA group which were respectively cultured by neuron culture medium,Mg²⁺-free artificial cerebrospinal fluid and Mg²⁺-free artificial cerebrospinal fluid containing 0.5 m M VPA for 3 h for subsequent experiments.Immunofluorescence was used to determine the expression of microtubule-associated protein-2（MAP-2）and 2-（4-Amidinophenyl）-6-indolecarbamidine（DIPA）to identify cultured primary hippocampal neurons.Cell Counting Kit 8（CCK-8）was used to determine the cell viability.Colorimetry was used to determine the level of intracellular iron,glutathione（GSH）and malondialdehyde（MDA）.Western blotting was used to determine the protein expression of solute carrier family 7 member 11（SLC7a11）,long-chain-fatty-acid-Co A ligase 4（ACSL4）and glutathione peroxidase 4（Gpx4）in each group.Results:1.The results of immunofluorescence revealed that the expression of MAP-2was detected in both the neuronal cell bodies and neurites,and the expression of DIPA was detected in the nuclei of the neurons.The primary hippocampal neurons had plump cell bodies and the neurites overlapped and branched freely with each other to form a complex neural network.Primary hippocampal neurons were cultured successfully.2.The effect of sodium valproate on cell viability in Mg²⁺-induced epileptic cell model:（1）The survival rate of primary hippocampal neurons treated by 0.5 m M sodium valproate was significantly elevated compared with epilepsy group（P<0.05）.0.5 m M was the optimal concentration for sodium valproate to treat epilepsy cell models and was selected as sodium valproate group.（2）The cell viability in the epilepsy group was reduced significantly compared with control group（P<0.05）.（3）The cell viability in the sodium valproate group was elevated significantly compared with epilepsy group（P<0.05）.3.The effect of sodium valproate on ferroptosis-related factors1)Sodium valproate reduces iron level in Mg²⁺-induced epileptic cell model:（1）The level of iron was increased significantly in epilepsy group compared with control group（P<0.05）.（2）The iron level in the sodium valproate group was significantly lower than in the epilepsy group（P<0.05）.2)Sodium valproate reduces MDA content in Mg²⁺-induced epileptic cell model:（1）The content of MDA was increased significantly in epilepsy group compared with control group（P<0.05）.（2）The content of MDA was decreased significantly in sodium valproate group compared with epilepsy group（P<0.05）.3)Sodium valproate downregulated the expression of ACSL4 protein in Mg²⁺-induced epileptic cell model:（1）The expression of ACSL4 protein was significantly increased in epilepsy group compared with control group（P<0.0001）.（2）The expression of ACSL4 protein was significantly downregulated in sodium valproate group compared with epilepsy group（P<0.0001）.4)Sodium valproate increased GSH level in Mg²⁺-induced epileptic cell model:（1）The level of GSH was reduced significantly in epilepsy group compared with control group（P<0.05）.（2）The level of GSH was increased significantly in sodium valproate group compared with epilepsy group（P<0.05）.5)Sodium valproate elevated the expression of SLC7a11 and Gpx4 protein in Mg²⁺-induced epileptic cell model:（1）The expression level of SLC7a11 and Gpx4protein was significantly decreased in epilepsy group compared with control group（P<0.0001）.（2）The level of SLC7a11 and Gpx4 protein was significantly upregulated in sodium valproate group compared with epilepsy group（P<0.0001）.Conclusion:Our findings showed that ferroptosis was coupled with the pathology of epilepsy,and the neuroprotective effect of sodium valproate in seizures was closely related to the inhibition of ferroptosis,highlighting the potential therapeutic value for targeting ferroptosis process in patients with seizure-related diseases.