| Objective:Drug-resistant epilepsy(DRE)is a refractory neurological disorder.There is ample evidence that suggest thatγ-aminobutyric acid-a(GABA_A)receptors could be one of the mechanisms responsible for the development of drug resistance in epilepsy.It is also known that the cAMP response element binding protein(CREB)plays a possible key role in the transcriptional regulation of GABA_A.Methods:240 healthy adult male SD rats were included,of which 24 were selected as the normal control group,and the remaining 216 rats were used to establish the epilepsy model.CREB overexpressing lentivirus(n=36),CREB overexpressing control virus(n=36),CREB interfering virus(n=108),and CREB interfering control virus(n=36)were constructed.Lentivirus was used to regulate the expression level of CREB in the hippocampus of rats,and an acute epilepsy model of lithium chlorine-pilocarpine was constructed.The time of gradeⅣand above seizure in rats injected with different groups of lentivirus was recorded,and The Times of additional injection of pilocarpine in rats without gradeⅣand above seizure was recorded.The rats were subjected to 24h continuous video surveillance.Epileptic seizure grade and duration of rats were recorded.After the epileptic seizure of rats entered the chronic phase,two antiepileptic drugs,phenytoin sodium,and phenobarbital,were used to screen drug-resistant epileptic rats.In the overexpression group(n=10),the overexpression control group(n=6),the interference group(n=6),and the interference control group(n=6),the percentage of rats with drug-resistant epilepsy in each group of lentivirus injection was recorded.The drug-resistant epileptic rats injected with lentivirus-resistant lentivirus-resistant epileptic rats were divided into an overexpression group,overexpression control group,interference group,and interference control group.The frequency and duration of seizures were observed during the drug screening process,and the expression levels of CREB and GABA_A subunits in the hippocampus of rats were detected by immunohistochemistry,protein western blotting,and real-time fluorescence quantitative PCR.Results:(1)Compared with the rats in the CREB overexpression group,the rats in the CREB overexpression group had a higher success rate of induction into the acute epilepsy model,a higher possibility of acute epilepsy,and a higher proportion of drug-resistant epilepsy models were screened out.Compared with the rats in the control group,the success rate of acute epilepsy model induced by CREB interference virus group was lower,and it was difficult to screen drug-resistant epilepsy model.(2)When lithium chlorine-pilocarpine method was used to create an acute epilepsy model,the time required for the occurrence of grade 4 or above epileptic seizure after the first injection of pilocarpine in each group was counted,and The Times of additional injection of pilocarpine in rats without gradeⅣor above epileptic seizure were counted.The results showed that the ignition time of rats in the overexpression group was less than that in the overexpression control group.The difference was statistically significant.The number of additional injection of pilocarpine in the overexpression group was less than that in the overexpression control group,but the difference was not statistically significant.The ignition time of rats in the interference group and The Times of additional injection of pilocarpine were longer than those in the interference group,and the difference was statistically significant.(3)Compared with the overexpression control group,the number of grade 3 and above seizures was higher and the duration of seizures was longer in the overexpression group at weeks 5-6,7-8,and the difference was statistically significant(F=14.016,P=0.001,F=29.831,P<0.001;F=30.423,P<0.001,F=60.804,P<0.001);Compared with the interference control group,the number of grade 3 or above seizures was less in the interference group at weeks 5-6,7-8,but the difference was not statistically significant(F=14.016,p=0.09,F=29.831,P=0.15),and the duration of grade 3 or above seizures was shortened in the interference group at weeks 5-6.The difference was statistically significant(F=30.423,P=0.005),and the duration of Grade 3 or above seizures was shortened in the disturbance group at week 7-8,but the difference was not statistically significant(F=60.804,P=0.055).Compared with the overexpression control group,the number of grade 5 seizures in the overexpression group was higher and the duration of seizures was longer at the 5th to 6th week,and the difference was statistically significant(F=17.7937,P=0.013,F=34.158,P<0.001),the number of grade5 seizures was higher in the overexpression group at week 7-8,but the difference was not statistically significant(F=16.094,p=0.054),and the duration of seizures was longer,the difference was statistically significant(F=36.569,P<0.001);Compared with rats in the interference control group,the number of grade 5 seizures was lower in the interference group at 5-6 weeks,the difference was statistically significant(F=17.7937,P=0.03),and the number of Grade 5 seizures was lower in the interference group at weeks 7-8,but the difference was not statistically significant(F=16.094,P=0.364),the duration of class 5 and above seizures was shorter in the disturbance group at 5-6 weeks,but the difference was not statistically significant(F=34.158,P=0.061),and the duration of class 3 and above seizures was shorter in the disturbance group at 7-8 weeks,the difference was statistically significant(F=36.569,P=0.001)(4)Immunohistochemical results showed that the content of CREB protein in the granular layer of hippocampal dentate gyrus of rats in the overexpression group was higher than that in the overexpression control group,the difference was statistically significant(F=2.896,P=0.008),and the expression of GABA_A subunit proteins was lower than that in the control group,the difference was statistically significant(F=2.676,P=0.01,F=2.249,P=0.003,F=10.183,P=0.025),and the content of CREB protein in interference group was lower than that in interference control group,the difference was statistically significant(F=2.896,P=0.021).The protein expression of GABA_A subunit was higher than that of interference control group,and the difference was statistically significant(F=2.676,P=0.001,F=2.249,P<0.001,F=10.183,P=0.003).(5)PCR results showed that the relative expression level of CREB mRNA in the overexpressed group was higher than that in the overexpressed control group,and the difference was statistically significant(F=40.897,P<0.001),the relative expression level of GABA_Aα1 mRNA was lower than that of overexpressed control group,the difference was statistically significant(F=42.145,P<0.001),the relative expression level of CREB mRNA in interference group was lower than that in control group,and the difference was statistically significant(F=40.897,P<0.001),the mRNA relative expression levels of GABA_A receptorα1,β2 andγ2 were higher than those of interference control group,the difference was statistically significant(F=42.145,P<0.001,F=19.334,P<0.001,F=54.767,P<0.001).The mRNA expression levels of GABA_A receptorβ2 andγ2 in overexpression group were lower than those in overexpression control group,but there was no significant difference between groups(F=19.334,p=0.051,F=54.767,P=0.05).(6)The results of WB showed that the protein expression level of CREB in overexpression group was higher than that in overexpression control group(F=4.939,p=0.008),the protein expression levels of GABA_A receptorα1,β2 andγ2 were lower than those of overexpressed control group(F=3.046,P=0.01,F=3.832,P=0.026,F=2.588,P=0.04),the protein expression level of CREB in interference group was lower than that in control group(F=4.939,P=0.04),the protein expression levels of GABA_Areceptorα1,β2 andγ2 were higher than those of interference control group,the difference was significant(F=3.046,P=0.010,F=3.832,P=0.003,F=2.588,P=0.003).Conclusions:Our study shows that enhanced CREB expression promotes the development of DRE and negatively regulates GABA_A receptor levels and that the inhibition of CREB expression may reduce the incidence of DRE. |
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