Effect Of CREB On Temporal Lobe Epilepsy Cognitive Impairment Mediated By The Oxidative Stress Of Mitochondria

Background:Patients with epilepsy are easily to be associated with other psychiatric or neurological disorders,which are called epileptic co-morbidity.Epileptic co-morbidity which is very common in epileptic patients usually involves 30-50 percent of epileptic patients.And the treatment of epilepsy in modern time is not confined to the control of epileptic seizures.In recent years,the treatment and prevention of epileptic co-morbidity have become the focus in the field of epilepsy.Temporal lobe epilepsy(TLE),originated from the hippocampus,parahippocampal gyrus,amygdaloid nucleus and lateral temporal neocortex,is the most common epilepsy syndrome.Hippocampal sclerosis is the most common etiology and pathological change in TLE.Cognitive impairment which is epileptic co-morbidity in TLE can seriously affect patients' quality of life and its mechanism is unknown.The previous research has been shown that neurons activated by c AMP response element binding protein(CREB)preferentially participate in memory formation.Oxidative stress and mitochondrial dysfunction are thought to be the main pathogenesis of many degenerative nervous system diseases,such as Alzheimer's disease,Parkinson's disease,vascular dementia and so on.This research attempts to reveal the role of CREB in TLE cognitive impairment mediated by mitochondrial oxidative stress.Objective:This research is to explore the role of CREB in mitochondrial oxidative stress as well as the effect of CREB on TLE cognitive impairment mediated by the mitochondrial oxidative stress,and to provide perspectives and directions of updated therapeutic targets for TLE cognitive impairment.Methods:(1)Cell Aspect:Hippocampal neurons of neonatal C57 mice within 24 hours of birth were isolated for primary culture and identified by immunofluorescence.The constructed CREB empty expression vector,empty interference vector,expression vector and interference vector were transfected into the cultured neurons.There were six groups in the cell experiment.They were control group,empty expression vector group,empty interference vector group,expression vector group and interference vector group and Mito Q group.ELISA was used to detect the level of SOD and MDA in neurons.PKA,Ca MKIV,CREB,p CREB,arc and c-fos protein was detected by Western blot.The expression m RNA of Ca MKIV,CREB,arc and c-fos was detected by q RT-PCR.Apoptosis rate of neurons was detected by mitochondrial membrane potential.The level of reactive oxygen species(ROS)was detected by flow cytometry.(2)Animal Aspect:The mice of epileptic model induced by pilocarpine were established.There were five groups in the animal experiment.They were control group,sham group,model+saline group,model+CREB gene inhibitor 666-15 group and model+Mito Q group.All mice in each group were tested by water maze behavior test.ELISA was used to detect the level of CA1 SOD and MDA.Western blot was used to detect the protein expression of Ca MKIV,CREB,arc,c-fos,PKA,p CREB in CA1.q RT-PCR was used to detect m RNA expression of Ca MKIV,CREB,arc,c-fos in CA1.The expression of Ca MKIV,CREB,arc,c-fos,PKA,p CREB protein in CA1 was detected by immunohistochemistry.Apoptosis rate of CA1 neurons was detected by mitochondrial membrane potential.Results:CA1 neurons were successfully isolated,cultured and identified.The CREB expression vector and interference vector were successfully constructed.ELISA showed the SOD level of the expression vector group was not different from that of the control group.The level of SOD in the interference vector group was significantly lower than that in the control group and the expression vector group(P<0.05).The level of MDA in the interference vector group was higher than that in the control group(P<0.05).Western blot showed that CREB interference vector could suppress protein expression of PKA,Ca MKIV,CREB,p CREB,arc and c-fos.q RT-PCR showed that CREB interference vector could suppress m RNA expression of Ca MKIV,CREB,arc,c-fos.Mitochondrial membrane potential test showed that the expression vector group could inhibit the neuronal apoptosis compared with the control group(P<0.05),and the interference vector group could promote the neuronal apoptosis compared with the control group(P<0.05).Flow cytometry showed that the level of ROS increased in the interference vector group compared with the control group and expression vector group.The level of ROS decreased in the expression vector group compared with the control group(P<0.05).Animal model of TLE was successfully constructed.In the water maze experiment,the latency of the place navigation test in model+666-15 group was prolonged compared with the control group and the model+saline group(P<0.05).The residence time around the original platform in the spatial probe test was decreased in the model+666-15 group compared with and the control group,the model+saline group and model+Mito Q group(P<0.05).TLE groups showed learning and memory impairment compared with the control group and sham group(P<0.05),ELISA showed that the SOD level of the model+666-15 group in CA1 was lower than that of the control group(P<0.05),and that of the model+666-15 group was lower than that of the model+saline group(P<0.05).The level of SOD in model+saline group was lower than that in model Mito Q group(P<0.05).The level of MDA of model+saline group and model+666-15 group in CA1 was higher than that of control group(P<0.05),and the model+666-15 group was higher than that of model+saline group.The level of MDA in model+saline group was higher than that in model+Mito Q group(P<0.05).Immunohistochemical staining showed that the expression of Ca MKIV?CREB?arc?c-fos?PKA?p CREB protein in CA1 was significantly decreased in model+666-15 group.Western blot showed that the CA1 protein expression of Ca MKIV,CREB,arc,c-fos,PKA,p CREB in model+Mito Q group was significantly higher than that in model+666-15 group.q RT-PCR showed that the CA1 m RNA expression of Ca MKIV,CREB,arc,c-fos in model+Mito Q group was significantly higher than that in model+666-15 group.The mitochondrial membrane potential test showed that the neuronal apoptosis rate of model+saline group and model+666-15 group was higher than that of control group,and that of model+666-15 group was higher than that of model+saline group(P<0.05).The neuronal apoptosis rate in the model+saline group was higher than that in the model+Mito Q group(P<0.05).Conclusions:(1)Down-regulating CREB expression could induce apoptosis of neurons and increase ROS level.The antioxidant Mito Q could inhibit the neuronal apoptosis and ROS level.Up-regulating CREB expression could inhibit neuronal apoptosis and decrease ROS level.CREB was involved in the pathophysiological process of mitochondrial oxidative stress in neurons.(2)In the TLE mice model,mitochondrial oxidative stress was involved in TLE cognitive impairment,and Ca MKIV-CREB signaling pathway was impaired.Inhibition of CREB increased the level of oxidative stress,the apoptosis rate and aggravated cognitive impairment.CREB was involved in the cognitive impairment of temporal lobe epilepsy induced by mitochondrial oxidative stress.