Research On The Role Of CREB In Regulating The Expression Of GABA_A Receptor In Hippocampus In Drug Resistant Epilepsy

Objective:Epilepsy is a common neurological disease.The c AMP response element binding protein(CREB) and mossy fiber sprouting(MFS) may play an important role in the pathogenesis,seizure,drug resistance and cognitive impairment of epilepsy.But the detailed mechanism of their participation in the pathological process of epilepsy is not clear.The purpose of this study is to investigate the effect of CREB on the expression of gamma-Aminobutyric acid-A receptor(GABA_A receptor) and seizures,and the correlations among MFS,seizures,drug resistance and cognitive impairment in cell and rat model of epilepsy.Methods:Part Ⅰ:CREB overexpression,CREB shRNA and control lentiviruses were constructed.Primary hippocampal neurons were isolated from suckling SD rats.After 10 days of culture and immunofluorescence identification of NeuN antibody,the primary hippocampal neurons were divided into control group,overexpression group,overexpression control group,interference group and interference control group.CREB overexpression lentivirus,CREB interference lentivirus and their control lentivirus were used to transfect and change the expression level of CREB in primary hippocampal neurons in the corresponding group.Seven days later,the expression levels of CREB,p-CREB and GABA_A receptor α1,β2 and γ2 subunits proteins in primary hippocampal neurons in different groups(n=3) were detected and compared by Western blot.At the same time,the m RNA levels of CREB and GABA_A receptor α1,β2 and γ2 subunits in primary hippocampal neurons in different groups(n=3) were detected and compared by fluorescence quantitative PCR.Part Ⅱ:Except for 10 normal controls,190 SD rats were used to establish lithium-pilocarpine induced epilepsy model.Phenytoin and phenobarbital were then used for drug resistance screening.According to the results of drug resistance screening,SD rats were divided into control group,drug-sensitivity(DS) overexpression group,DS overexpression control group,DS interference group,drug resistance(DR) overexpression group,DR overexpression control group,DR interference group and DR interference control group(n=10).CREB overexpression lentivirus,CREB interference lentivirus and their control lentiviruses were injected into the bilateral hippocampus of rats in the corresponding group.The frequency and duration of seizures before and after transfection were monitored by video.The electroencephalogram(EEG) of right hippocampus was recorded with a deep brain electrode.The protein levels of CREB,p-CREB and GABA_A receptor α1,β2 andγ2 subunits in hippocampus of rats in each group were detected by Western blot.The protein levels of CREB and GABA_A receptor α1 subunit in the granular layer of dentate gyrus of rats in each group were detected by immunohistochemistry.Timm staining was used to detect the MFS of dentate gyrus in each group.The seizure frequency,seizure duration,EEG frequency,EEG amplitude,protein expression level and Timm score in different groups were statistically analyzed.Part Ⅲ:Except for 12 rats as control group,38 SD rats were used to establish lithium-pilocarpine epilepsy model.After drug resistance screening using phenytoin and phenobarbital,epileptic rats were divided into DS group(n=18) and DR group(n=8).The seizure frequency of rats in each group was monitored by video.Novel object recognition test and Morris water maze test were used to detect their cognitive impairment.The MFS in dentate gyrus of rats was detected by Timm staining.The seizure frequency and the levels of object memory,spatial memory and MFS were compared among the groups.Spearman rank correlation test was used to analyze the correlations among these factors.Results:Part Ⅰ:Compared with the overexpression control group,the levels of CREB protein,CREB mRNA and p-CREB protein were significantly higher in primary hippocampal neurons in the overexpression group,and the protein and mRNA levels of GABA_A receptor α1,β2 and γ2 subunits were significantly decreased in the overexpression group.Compared with the interference control group,the levels of CREB protein,CREB mRNA and p-CREB protein were significantly lower in primary hippocampal neurons in the interference group,and the protein and mRNA levels of GABA_A receptor α1,β2 and γ2 subunits were significantly increased in the interference group.Part Ⅱ:In rats with DS and DR epilepsy,the frequency and duration of seizures after injection of overexpressed lentivirus were significantly higher than those before injection,while the frequency and duration of seizures after injection of interfering lentivirus were significantly lower than those before injection.Compared with the corresponding control group,the rats in the DS and DR overexpression group had higher EEG frequency and amplitude,higher CREB and p-CREB protein levels and lower GABA_A receptor α1,β2 and γ2 subunit protein levels.Compared with the corresponding control group,the rats in the DS and DR interference group had lower EEG frequency and amplitude,lower CREB and p-CREB protein levels and higher GABA_A receptor α1,β2 and γ2 subunit protein levels.Part Ⅲ:The abilities of object memory and spatial memory learning of rats with lithium-pilocarpine induced epilepsy were significantly lower than those of normal rats.Compared with DS epileptic rats,DR epileptic rats have more frequent seizures,more obvious MFS and more severe impairment of spatial memory and learning ability.There may be a correlation between seizure frequency and spatial memory and learning ability in epileptic rats.Conclusion:Changing the expression of CREB protein in rat hippocampal neurons can change the level of p-CREB protein and negatively regulate the expression and transcription level of GABA_A receptor α1,β2 and γ2 subunits.Overexpression of CREB protein can increase the frequency and intensity of seizures in rats of epilepsy model,while interfering with the expression of CREB protein can decrease the frequency and intensity of seizures.MFS may be related to drug resistance of epilepsy.There may be correlations among seizure frequency,drug resistance and spatial memory impairment in epilepsy.