Protective Effect Of Mfn2 Protein On Oxidative Apoptosis Of Hippocampal Neurons In Epileptic Rats

Background and objective Epilepsy is a common disease of central nervous system in clinic.Studies at home and abroad have found that seizures can damage mitochondrial structure and function,increase production of reactive oxygen species in respiratory chain,DNA mutation,calcium overload,lipid peroxidation and release of cytochrome C,leading to apoptosis.Therefore,whether we can maintain the normal structure and function of mitochondria in order to find new targets for epilepsy treatment.Mfn2 protein mainly exists in mitochondrial membrane.Mfn2 not only mediates mitochondrial fusion and division,but also plays an important role in mitochondrial transport,endoplasmic reticulum/sarcoplasmic reticulum and mitochondrial interaction,cell metabolism,apoptosis and autophagy.Mfn2 mutations have been extensively studied in degenerative neurological diseases,such as fibula muscular atrophy,alzheimer’s disease and Parkinson’s disease.However,so far,whether Mfn2 is involved in epilepsy injury and what role it plays in the nerve damage of epilepsy has not been reported in domestic and foreign literatures.In this study,rat hippocampal neurons were cultured in vitro,induced epilepsy model without magnesium,lentivirus infection interfered with Mfn2 expression,observed the morphology of hippocampal neurons,detected oxidative stress and apoptosis indicators.To investigate whether the mitochondrial function regulated by Mfn2 plays a protective role in epileptic nerve injury.Materials and Methods Sprague-Dawley rats within 24 hours after birth were decapitated and their brains were taken out quickly.The bilateral hippocampi were stripped into single cell suspension and the primary neurons were cultured in vitro.On the 7th day,the purity of neurons was identified and the epileptic discharge model was established by magnesium-free induction.On the 10 th day of culture,QPCR and westren blot were used to detect the expression of Mfn2 in the control group and magnesium-free treatment for 3h,6h,12 h,24h and 48 h.Mfn2,transfected with lentivirus was divided into five groups: control group(CON),non-magnesium induced epilepsy group(AE),non-magnesium induced epilepsy + empty carrier group(AE+LV),non-magnesium induced epilepsy + interference Mfn2 group(AE+LV-Mfn2-sh RNA)and non-magnesium induced epilepsy + overexpression Mfn2 group(AE+LV-Mfn2).The morphology of hippocampal neurons in the five groups was compared.Oxidative damage was determined by SOD,cell apoptosis was detected by western blot for Bax,and Bcl-2、Cyt-C.Results Cellular morphology After 7 days of culture,hippocampal neurons were observed under an inverted microscope,with plump cell bodies,vacuolation of some nuclei,and obvious corona around the cell body,typical axons and dendrites,and a tight network formed between cells.Neuronal purity determination After 7 days of culture,hippocampal neurons were stained with NSE,and the purity of neurons was more than 90%.The expression of Mfn2 at different time after seizure Compared with CON group,the expression levels of Mfn2 m RNA and Mfn2 protein in the 3h group and 6h group were decreased(P < 0.05).Expression of Mfn2 after lentivirus transfection Compared with CON group,the expressions of Mfn2 m RNA and Mfn2 protein were decreased in AE+LV group and increased in AE+LV-Mfn2-sh RNA group(P<0.05).Compared with the AE group,the expression of Mfn2 m RNA and Mfn2 protein in the AE+LV-Mfn2-sh RNA group decreased,while the expression of Mfn2 m RNA in the AE+LV-Mfn2 group increased,and the difference was statistically significant(P<0.05).The expression of SOD Compared with CON group,SOD expression in AE group,AE+LV group and AE+LV-Mfn2-sh RNA group decreased(P< 0.05).Compared with AE group,SOD expression decreased in AE+LV-Mfn2-sh RNA group and increased in AE+LV-Mfn2 group(P < 0.05).The expression of Bcl-2,Bax and Cyt-C Compared with CON group,the expressions of Bcl-2,Bcl-2 /Bax and mitochondrial Cyt-C in AE group,AE+LV group,and AE+LV-Mfn2-sh RNA group decreased,while the expressions of Bax and cyt-c in cytoplasm increased(P<0.05).Compared with the AE group,the expressions of Bcl-2,Bcl-2 /Bax and mitochondrial Cyt-C in the AE+LV-Mfn2-sh RNA group decreased,while the expressions of Bax and Cyt-C in cytoplasm increased;the expression of Bcl-2,Bcl-2 /Bax and mitochondrial Cyt-C decreased,while the expression of Bax and cyt-c in cytoplasm increased in AE+LV-Mfn2 group(P<0.05).Conclusion 1.In the hippocampal neurons of magnesium-free epileptic rats,the expression of Mfn2 decreased temporarily,SOD decreased,Bcl-2/Bax decreased,and Cyt-C was transferred from mitochondria to cytoplasm.It is suggested that Mfn2 regulated mitochondrial function is involved in the pathological process of epilepsy.Epilepsy can lead to oxidative stress and apoptosis.2.After lentiviral transfection upregulated Mfn2 expression,magnesium-free cells induced a decrease in SOD,down-regulation of Bcl-2/Bax and the inhibition of mitochondrial release of Cyt-C were observed.The down-regulation of Mfn2 expression induced the decrease of SOD,the down-regulation of Bcl-2/Bax and the further aggravation of Cyt-C released by mitochondria.Mfn2 protects hippocampal neurons by reducing mitochondrial oxidative stress and inhibiting apoptosis pathway.3.The regulation of mitochondrial function has potential research value in the treatment of epilepsy.