Effects And Mechanism Of Sirt3 On Kainite Induced Seizures

Epilepsy is a common and serious neurological disease that often has a huge impact on daily life of patients.After acute injuries in the brain,reactive oxygen sprcies and neuroinflammation were rapidly induced and accumulated that enhance the risk for epilepsy.The occurrence of epilepsy further aggravates oxidative stress,and thus become a vicious circle.Sirt3 is a class Ⅲ deacetylase that depends on nicotinamide adenine dinucleotide.It stabilizes mitochondrial function integrity by deacetylating related mitochondrial proteins.However,the role of Sirt3 in the development of epilepsy remains unknown.This project intends to explore the effect and mechanism of Sirt3 in the occurrence of seizures.OBJECTIVE: To explore the effect of Sirt3 on kainic acid(KA)induced epilepsy,and to clarify the underlying mechanism.METHODS: Two kinds of mice,Sirt3 knockout(KO)and C57BL/6 wild type(WT)were treated with KA(22mg/kg)as epilepsy model.In addition to recording the latency of seizure,the seizure scores was calculated.The protein expression was evaluated by western blot analysis and RT-PCR;Fluoro-Jade B(FJB)was used to stain degenerating neurons in brain sections;DCFH-DA probe was applied to detect the ROS activity in the brain;Morris water maze and open filed test were used to calculate the learning and memory as well as anxiety in mice.In vitro,SiRNA were transfected to N2 A cell using Lipo3000 to knock down relative gene.The cell viability was measured by MTT assay.RESULTS: In animal epilepsy model,the latency of seizure of Sirt3 KO mice were longer than that of WT mice,the seizure scores of Sirt3 KO mice were lower than that of WT mice.The ROS activity in the brain of Sirt3 deficiency mice was significantly decreased compared with WT mice.The expression of acetylated SOD was reduced in Sirt3 deficiency mice,suggesting the ability for eliminate ROS was enhanced when Sirt3 was knocked.Meanwhile,Behavioral test shows that knockout of Sirt3 gene could alleviate learning disabilities and anxiety induced by epilepsy.The results of FJB indicate that the neuron death caused by epilepsy was reduced in Sirt3 KO mice.All these results suggest that the protective role of Sirt3 deficiency in the occurrence of epilepsy may be related to the decrease of acetylated SOD and ROS in the brain.The further results showed that the expression of Sirt4,a member of Sirtuins family located in mitochondria,was significantly increased in KO mice compared with WT mice.In order to find out whether the protective effect of Sirt3 deficiency was related to the increase expression of Sirt4,N2 A cell was transfected to silence Sirt3 and Sirt4.ROS assay and MTT assay showed that the protective effects of Sirt3 deficiency disappeared when Sirt3 and Sirt4 were knocked down at the same time.CONCLUSIONS: Sirt3 deficiency could relieve the occurrence of KA induced epilepsy.And the protective effect of that may be related to the increase in expression of the Sirtuins family member Sirt4 when Sirt3 was knocked out.