GABA-A Receptor-mediated KLF4' Modulatory Effect On Epilepsy

In recent years,perioperative medicine and anesthetic therapy have played an increasingly important role in the entire medical process.Anesthetic therapy and perioperative management of patients with epilepsy are great challenges for anesthesiologists.Epilepsy is a group of neurological disorders,which are characterized by neuronal synchronous discharging-induced recurrent epileptic seizures in specific brain regions.The global prevalence of epilepsy reached 0.5% ~ 1%.There are more than 9 million epileptic patients in China.Patients with epilepsy are often subjected to other cognitive disorders,mood disorders and locomotion disorders.Most patients rely on traditional antiepileptic drug therapy,which may bring heavy economic and psychological burden to the patients and their family.The anaesthesia during surgery for epilepsy is difficult.Hypnotic sedative drugs can affect spontaneous EEG and which share same or similar active sites with AEDs.Therefore,appropriate anesthesia schemes should be selected in clinical epilepsy surgery.It's of significant importance to avoid influence of anesthetics on ECo G and ensure accurate positioning of epileptic foci and avoid intraoperative anesthetic-induced seizures.Both drugs and surgical treatment can only relieve seizures,while cannot prevent development of secondary epilepsy induced by external factors such as cerebral injury.Objective evidence from family and epidemiological investigation suggest that genetic factors are likely to play an essential role in the development of epilepsy.Therefore,it is of great importance to explore the mechanism of epilepsy at the molecular level,and to seek how to prevent or delay the occurrence of chronic epilepsy after initial post-injury(IPI),and to provide new molecular target for perioperative management,anesthetic therapy and cerebral protection in patients with epilepsy.Kruppel-Like Factor 4(KLF4),a member of the Kruppel Like transcription factor protein family,is a zinc finger DNA-binding protein that can regulate gene expression.KLF4 was first found to express in the epithelial cells,including oral epithelium and gastrointestinal epithelium.It was reported that KLF4 plays an important role in regulating the proliferation,differentiation and apoptosis of embryonic cells.In recent years,emerging evidence showed that KLF4 is also expressed in central nervous system(CNS)and is involved in the regulation of the differentiation of stem cells and axons growth,suggesting that KLF4 might affect the occurrence of various neurodevelopmental disorders,such as autism,congenital hydrocephalus and cerebral palsy.However,there is few study that reported the relationship of KLF4 between epilepsy and chronic epileptogenesis.Based on this background,this study will investigate KLF4's modulatory effect on epilepsy.Objective:More and more researchers focus on transcription factor KLF4 and its regulative effect on neuronal differentiation and proliferation during embryonic development.It was well known that traditional anti-epileptic drugs have great limitations in epilepsy therapy.It has become a focus issue in neurological research field to explore the potential mechanism of epileptogenesis and find a novel anti-epileptic therapeutic target,which might benefit the prevention and treatment of epilepsy.Thus,we proposed the questions that whether KLF4 can affect epileptogenesis,and whether KLF4 is involved in the regulation of sleep activity.In this article,we tend to clarify the relationship between the transcription factor KLF4 and epilepsy,and at the same time,discuss the regulative effect of KLF4 on epilepsy and sleep activity preliminarily.KLF4 might be a novel molecular target for the prevention and treatment of epileptic seizures.In this study,we aimed to prevent or delay the occurrence of chronic epilepsy,and also provide a theoretical basis for perioperative management,anesthetic therapy and neuroprotective strategy for clinical patients.GABA-A Receptor-mediated Effect of KLF4 on seizures Methods:1.Sixteen healthy ICR male mice were randomly divided into the control group and the epilepsy model group(PTZ).The expression of mice hippocampal KLF4 was detected by western blotting.2.Twenty-four healthy ICR male mice were selected and randomly divided into PTZ group,PTZ+VPA group and PTZ+CBZ group,with 8 mice in each group.The epileptic behaviors of mice were observed within 20 min after PTZ administration.The expression of hippocampal KLF4 was detected by western blotting.3.Thirty-two healthy ICR male mice were randomly divided into PTZ group,VPA group,Muscimol(Mus)group and Flumazenil(Flu)group.The epileptic behaviors of mice were observed within 20 min after PTZ administration.The expression of hippocampal KLF4 was detected by western blotting.Results:1.Compared with the control group,the expression of hippocampal KLF4 in the PTZ acute administration group was decreased.2.Both the antiepileptic drugs VPA and CBZ can significantly prolong the seizure latency and the seizure latency of VPA group was longer than that of CBZ groups.Both VPA and CBZ can significantly reduce the severity of epileptic seizure in mice.Both VPA and CBZ promoted the expression of hippocampal KLF4 in mice.The expression of KLF4 in the CBZ group was higher than that in the VPA group.3.VPA can significantly prolong the latency of epileptic seizure of mice,while pretreatment with Flu significantly antagonize this effect.The latency of epileptic seizure in Flu group is significantly shorter than that of VPA group.There was no statistical difference for the seizure latency between Mus group and VPA group.In addition,the average seizure level of the Flu group was slightly higher than that of the VPA group,and no significant difference was found between the Mus group and the VPA group.The expression of KLF4 in hippocampus of the Flu group was significantly lower than that of the VPA group,while no significant difference was found between the Mus group and the VPA group.Potential Mechanism of KLF4's Anti-epileptic actions Methods:Thirty-two healthy ICR mice were randomly divided into saline+lv-GFP group,PTZ+lv-GFP group,saline+lv-KLF4 group and PTZ+lv-KLF4 group.The mice were stereotaxic injected with virus and the lentiviral-mediated KLF4 overexpressed mice models were established.After three weeks,the PTZ or saline were administrated based on the group.The epileptic behaviors in mice were observed and recorded within 20 minutes after PTZ administration.The expression of c-fos in the hippocampus and pyriform cortex of mice in each group was detected by immunofluorescence staining.Results:1.Compared with PTZ+lv-GFP group,the epileptic seizure latency of PTZ+lv-KLF4 group was significantly longer,and the average seizure level of mice in the PTZ+lv-KLF4 group was significantly reduced.2.The expression of c-fos in the hippocampus and pyriform cortex of mice was significantly increased after PTZ administration.Comparing the PTZ+lv-KLF4 group with the PTZ+lv-GFP group,we found that the c-fos expression was significantly reduced in the hippocampus and piriform cortex of the over-expressed KLF4 mice.Effect of KLF4 on pentobarbital-induced sleep activity in mice Method:1.Sixteen healthy ICR male mice were selected and divided into control group and pentobarbital(PBBT)group randomly.The expression of hippocampal KLF4 was detected by western blotting.2.Thirty-two healthy ICR male mice were selected and divided into PBBT+lv-GFP group,PBBT+lv-KLF4 group,PBBT+si-GFP group and PBBT+si-KLF4 group randomly.The virus was stereotaxic injected into the hippocampus and the lentiviral-mediated KLF4 overexpression and si RNA-mediated KLF4 interference mice modes were established.After three weeks,PBBT or saline were intraperitoneal injected based on the group and the sleep behavior and activity were recorded.Results:1.Acute PBBT intraperitoneal injection increased the expression of KLF4 in hippocampus significantly.2.The sleep latency and sleep duration of mice in lv-KLF4 group were not statistically different from that of control virus group.The sleep latency of si-KLF4 mice was significantly longer than that of the control virus group,and the sleep duration was significantly shorter than that of the control virus group.Conclusion:1.The antiepileptic effect of VPA and CBZ was related to the promotion of the expression of hippocampal KLF4.2.Flumazenil can antagonize the anti-epileptic effect of VPA via inhibiting the promotion of hippocampal KLF4 expression.3.KLF4 may exert its anti-epileptic effect by inhibiting PTZ-induced c-fos expression in the hippocampus and piriform cortex.4.KLF4 can regulate sleep activity.