Study On The Correlation Between CNN3 And Epilepsy Susceptibility And Treatment

Objectives:In recent years,studies have found that astrocytes can regulate neuronal excitability and even participate in epilepsy.The calponin-3 protein encoded by the CNN3 gene is a calcium/calmodulin binding protein and is expressed in neurons and astrocytes.Previous studies have shown that the expression of calponin-3 is abnormally increased in the brain tissue of patients with drug-resistant epilepsy and the brain of chronic epilepsy mice,and may affect the susceptibility to epilepsy,but the specific link is not clear.Therefore,it is proposed to distinguish whether calponin-3 of different nerve cell types is different from epilepsy susceptibility.It is clear whether the existing broad-spectrum antiepileptic drugs can change the expression of calponin-3 and its downstream CAMK ? protein in the brain of epilepsy mice.The treatment of drug-induced epilepsy provides experimental basis.Methods:The syn-CNN3fl+/+and Gfap-cre+/-transgenic mice were used for propagation,and their progeny mice were genetically identified by PCR technology,and syn-CNN3fl+/fl+and Gfap-cre+/-genotype mice were selected.Under the guidance of the stereotaxic instrument,syn-CNN3fl+/fl+transgenic mouse hippocampus multi-sites were loaded with syn-cre virus to selectively knock down the CNN3 gene in hippocampal neurons;to Gfap-cre+/-transgenic mouse hippocampus The site was loaded with DIO virus(HBAAV2/9-CMV-DIO-MIR30-M-CNN3-GFP)to selectively knock down the CNN3 gene in hippocampal astrocytes.Twenty-one days after injection of the virus,the expression of calponin-3 protein in neurons and astrocytes was detected by immunofluorescence triple staining and Western Blotting(WB)technology.After confirming the knockdown efficiency of the CNN3 gene in the experimental mice,pilocarpine was intraperitoneally injected to build a model of status epilepticus,and the latency of Racine IV or higher behavioral neurons in selective knockdown neurons/astroglia cells and control mice was recorded,And compare.Choose healthy C57 mice,ignite pilocarpine by intraperitoneal injection,and randomly divide the experimental mice that survived 48 hours and still have seizures into normal saline(NS)group,valproic acid(VAP)group and left Piracetam(LEV)group.At 8 o'clock in the morning and evening every day,normal saline,450 mg/kg of VAP and 450 mg/kg LEV were intragastrically administered;14 C57 mice were set up without pilocarpine injection,and only normal saline was intragastrically administered as a blank(KB)control group.At the same time,the behavioral changes of the experimental mice in each group were observed every day,and the seizures were graded by Rancine score.After 1 week,2 weeks,3 weeks,and 4 weeks of initiation of gavage,3 mice were randomly selected from each group to decapitate the hippocampus.The expression levels of calponin-3 and CAMK? were detected by WB experiment,and calponin-3 was observed by immunofluorescence staining.The distribution and expression of CAMK? in two kinds of nerve cells.Results:On agarose gel electrophoresis,the sizes of mouse PCR products of syn-CNN3 fl+/fl+and Gfap-cre+/-genotypes are consistent with the expected primers before further experiments.After 21 days of multi-point injection of AAV virus into hippocampus,WB and immunofluorescence showed selective knockdown of neurons/astroglia of CNN3 gene mice calponin-3 expression in hippocampal neurons/astroglia The control group decreased significantly.After injecting pilocarpine in each experimental rat,the seizure latency of CNN3 genome of selective knockdown of hippocampal neurons was longer than that of control group;while the epileptic latency of CNN3 genome of selective knockdown of hippocampal astrocytes was not significantly different from that of control group Sexual differences.Valproic acid began to produce antiepileptic effects in the second week after intragastric administration of epilepsy mice,while levetiracetam had significant antiepileptic effects in the first week after intragastric administration of epilepsy mice.Compared with the non-gastric antiepileptic drugs and the unlit KB group,the calponin-3 expression in the NS group,VAP group,and LEV group increased from the first week after intragastric administration,and the second week was higher,but the third week It began to decline until the fourth week.However,the total level of calponin-3 expression in these four weeks was still significantly higher than that in the KB group;the expression of CAMK ? in the three groups was different,and the expression of CAMK? in the NS group slightly increased in the first week.The second week began to continue to decline,and was lower than that with the KB group;the VAP group CAMK? expression continued to decline in the first week,and slightly increased in the third week,but the overall expression level was lower than the KB group;the LEV group CAMK? expression level in the first week It fell,and continued to rise in the second week,and was higher than the KB group in the third and fourth weeks.In NS group,the expression level of calponin-3 and CAMK? expression had the opposite trend in 1-3 weeks.In the fourth week,the expression level of calponin-3 began to decrease,and the degree of decrease in CAMK ? expression slowed down.The expression level of calponin-3 in the VAP group and LEV group did not change with the rebound of the expression level of CAMK?.Conclusions:The AAV virus specifically knocked down the CNN3 gene in astrocytes and neurons of mouse hippocampus;calponin-3 affects the susceptibility to epilepsy through the action of neurons,but has no obvious relationship with astrocytes.The broad-spectrum antiepileptic drugs valproic acid and levetiracetam did not significantly change the expression of calponin-3 in the hippocampus of epilepsy within 4 weeks.The expression level of CAMK? continuously decreases with repeated seizures and rebounds with the reduction of the severity of epilepsy.VAP may not affect the expression of CAMK ?,but LEV can significantly increase the level of CAMK?.The change in the expression of CAMK? does not affect calponin.-3 expression level.This study found a possible new target involved in the onset of epilepsy,broadening the understanding of the antiepileptic effect of LEV.