| IntroductionEpilepsy is a common disease in the nervous system caused by the sudden abnormal discharge of neurons in the brain.The pathogenesis of the disease is complex and the causes are numerous.Repeated seizures can lead to damage of neurons in the brain.Previous studies have proved that the formation of epileptic diseases needs to go through the acute period,silent period and spontaneous seizures period,in which the acute period of SE model is an important period in the formation of epileptic diseases.It is believed that the formation of spontaneous seizures are resulted from the damages generated in the acute period of SE model accompanied by some pathophysiological changes such as neuronal apoptosis,mossy fiber germination,synaptic circuit re-formation,ectopic cell proliferation,and glial cell activation.Arachidonic acid(AA)is an unsaturated fatty acid widely present in the human body.Previous studies have shown that AA will accumulate in the brain after SE,and then promote the increase of glutamate release in the synaptic,leading to the further increase of excitability of the neural network.However,the role of AA metabolism in the development of epilepsy need to be further identified.Arachidonate lipoxygenase 3(ALOXE3)is the main catalytic enzyme in the lipoxygenase pathway of AA.We have recently shown that ALOXE3 colocates with neurons in the mouse hippocampus,and that local overexpression of ALOXE3 in the mouse hippocampus can reduce the severity of pilocarpine-induced epilepsy.However,the role of ALOXE3 overexpression on the whole animal level in the occurrence and development of epilepsy has not been studied.In another of our studies,we found that a mutation of a locus in the ALOXE3 promoter region in a patient with Dravet syndrome resulted in a decrease in ALOXE3 enzyme activity and an increase in AA content through this mutation.In conclusion,ALOXE3 may play a certain role in the occurrence and development of epilepsy by participating in the metabolism of AA in the brain,but whether ALOXE3 plays a promoting or inhibitory role still needs to be further discussed.ObjectiveTo explore the effect of Aloxe3 overexpression on the AA level in the hippocampus of pilocarpine-induced SE model and the seizure severity during the acute phase,and the neuroprotective effect on the occurrence and development of epilepsy.This study will help to understand the pathogenesis of epilepsy and provide a potential strategy for clinical therapy.Methods(1)Using Piggy Bac transposable enzyme system,Thy1-aloxe3-3x Flag overexpression transgenic mouse model was established,and the primary positive mice were identified by PCR.Western blot and RT-q PCR were used to verify the expression levels of ALOXE3 protein and m RNA expression levels in the hippocampus of the offspring of the primary positive mice.(2)Observation and weighing methods were used to compare the differences in appearance,body length and body weight between Aloxe3 overexpressed transgenic mice and WT mice.(3)Western blot was used to analyze the protein expression level of ALOXE3 in WT and transgenic mice during the acute phase of epilepsy.ELISA was used to analyze the content of AA in the hippocampus of pilocarpine epilepsy model of WT mice at the acute phase,pilocarpine overexpressed transgenic mice at the acute phase,WT mouse control group and Aloxe3 overexpressed transgenic mice control group.(4)Epilepsy susceptibility of 8-week-old WT mice and Aloxe3 overexpressed transgenic mice in the acute phase of mouse epilepsy was detected by behavioral observation method.(5)Western blot was used to analyze the protein expression level of ALOXE3 in the hippocampus of WT and transgenic mice during the seizure-silent phase(10 d).(6)FJC and was used to measure the hippocampus neuron damage in CA1 and CA3 of WT mice at pilocarpine-induced epilepsy model seizure-silent phase,overexpressed transgenic mice at pilocarpine-induced epileptic model seizure-silent phase,WT mouse control group and Aloxe3 overexpressed transgenic mice control group.Electron microscope was used to measure the hippocampus neuron state and the morphological changes of mitochondria in the above 4 groups.(7)The spontaneous-seizure severity of pilocarpine-induced epileptic model in 8-week-old WT mice and Aloxe3 overexpressed mice during spontaneous-seizure phase were detected by behavioral observation method.Results The establishment of the tool mice:(1)In order to study the relationship between Aloxe3 and epilepsy,a transgenic mouse model of Aloxe3 overexpression was established.The primary positive mice were verified by PCR and the effect of ALOXE3 overexpression in the hippocampus of their offspring was verified by Western blot and RT-q PCR.(2)No difference in appearance,body length and body weight between the transgenic mice overexpressed with Aloxe3 and WT mice of the same week of age was observed.These results indicated that the overexpression of Aloxe3 transgenic mice had no effect on phenotype.The role of ALOXE3 overexpression during the acute phase:(1)Western blot showed that the expression of ALOXE3 protein in the hippocampus of mice in the acute epilepsy stage(12 h)was decreased(**P<0.01),and ELISA test showed that the overexpression of ALOXE3 could reduce the content of AA in the hippocampus of mice in the acute epilepsy stage(**P<0.01).These results suggested that ALOXE3 overexpression could reverse the decrease of ALOXE3 protein expression caused by SE,and regulate AA content in the hippocampus.(2)Behavioral observation of the severity of seizure in the acute phase of epilepsy in WT mice and Aloxe3 overexpression mice showed that,compared with WT mice,Aloxe3 overexpression mice had significantly longer initial myoclonic twitch,tonic hind limb extension,latency of death and clonic convulsion lasting more than 5 s.The mortality rate was decreased(*P < 0.05).These results indicate that Aloxe3 overexpression can reduce the severity of acute epileptic seizures in mice.The role of ALOXE3 overexpression during the seizure-silent phase:(1)Western blot showed that Aloxe3 overexpression could up-regulate the protein expression of ALOXE3 in pilocarpine-induced epilepsy model during the epileptic silent period(10 d)(*P < 0.05).These results suggest that Aloxe3 overexpression can reverse the decrease of ALOXE3 protein expression in mice due to entering the seizure-silent phase.(2)The FJC data showed that Aloxe3 overexpression transgenic mice significantly reduced the apoptotic neurons of CA1 and CA3 in the hippocampus during seizure-silent phase(*P<0.05),suggesting that Aloxe3 overexpression has a protective effect on hippocampal neurons.(3)Electron microscopy images showed that the morphology of neurons and mitochondria in the seizure-silent phase of hippocampus of Aloxe3 overexpressed transgenic mice was slightly abnormal,and the neurons and mitochondria in the seizure-silent phase of hippocampus of WT mice was severely abnormal,which suggesting that Aloxe3 overexpression had a protective effect on neurons and mitochondria in the hippocampus.The role of ALOXE3 overexpression during the spontaneous-seizure phase:(1)The behavioral results of spontaneous epilepsy in WT mice and Aloxe3 overexpressed transgenic mice showed that the frequency and duration of seizures in Aloxe3 overexpressed transgenic mice were significantly decreased compared with WT mice(*P < 0.05).These results suggest that ALOXE3 overexpression can reduce the severity of spontaneous seizures in the pilocarpine-induced epileptic model.ConclusionThese results demonstrate that the overexpression of ALOXE3 can reduce the seizure severity during the acute phase of epilepsy by reducing the content of AA in the hippocampus of the pilocarpine-induced epileptic model,play a protective role in the occurrence and development of epilepsy disease,and also alleviate the seizure severity of spontaneous epilepsy.In this study,the neuroprotective role of ALOXE3 overexpression was verified in the pilocarpine-induced epileptic model,providing data for the search of potential therapeutic targets for epilepsy. |
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