Neuroprotective Effect And Mechanism Of GLP-1 Analogue Liraglutide In Scn1a Gene Knockout Epilepsy Mice Model

ObjectiveDravet Syndrome(Dravet Syndrome,DS)is a type of refractory epilepsy usually caused by a heterozygous mutation in the Scn1 a gene,which encodes the voltage-gated sodium channel Nav1.1.Glucagon-like peptide-1(GLP-1)analogs are effective therapeutic agents for the treatment of diabetes.In recent years,they have been found to show neuroprotective effects in neurological-related diseases.However,whether GLP-1 analogs have neuroprotective effects on DS is unknown.This study aimed to investigate the neuroprotective effects and mechanisms of liraglutide in Scn1 a knockout-induced epilepsy mouse models and cell models.MethodsIn the Scn1 a knockout epilepsy(Scn1a KO)mouse model,The experiment was divided into four groups:(I)saline control WT mouse group(WT + NS group),(II)liraglutide-treated WT mouse group(WT + Lira group),(III)saline control Scn1 a KO Mouse group(KO + NS group),(IV)Liraglutide treated the Scn1 a KO mouse group(KO + Lira group).DS was evaluated using electroencephalography(EGG),TSE integrated intelligent behavior analysis system(IntelliCage),and open field experiment(OFT).Mice have seizure susceptibility,behavioral changes,and the severity of seizures.Hematoxylin & eosin(HE)and Nissl staining were used to observe the morphological changes of neurons in brain tissue.Immunofluorescence and Western Blotting were used to detect the expression of apoptosis-related proteins(Cleaved Caspase-3,BAX,BCL-2)and mammalian rapamycin target proteins(p-mTOR,mTOR).In the Scn1 a knockout HT22 cell line experiment and divided into four groups:(I)HT22 cell control group(HT22 Control group),(II)HT22 cell liraglutide treatment group(HT22 + Lira group),(III)Scn1a KO HT22 cell control group(Scn1a KO Control Group),(IV)Scn1a KO HT22 cell liraglutide treatment group(Scn1a KO + Lira group).The CCK-8(Cell Counting Kit 8)method was used to detect the proliferation of the Scn1 a knockout cell model,the morphological changes of the cells were observed with an inverted microscope,the number of cells was counted with a cell counter,Annexin V-FITC/PI flow cytometry was used to detect the effect of liraglutide on the apoptosis level,and Western blotting was used to detect the expression of apoptosis-related proteins and the protein of the mTOR signaling pathway.Results1.EEG results showed that compared to the Scn1 a KO + NS group,the Scn1 a KO + Lira group can significantly reduce the epileptic EEG seizures(P < 0.001).2.IntelliCage results showed that compared to the Scn1 a KO + NS group,the Scn1 a KO + Lira group can improve the learning and memory ability(P < 0.001).3.Open field results showed that compared to the Scn1 a KO + NS group,the Scn1 a KO + Lira group could alleviate anxiety symptoms in epilepsy mice(P < 0.001).4.The results of HE and Nissl showed that compared to the Scn1 a KO + NS group,the Scn1 a KO + Lira group could reduce neuronal damage in seizures(P < 0.001);5.CCK-8 results showed that compared to the Scn1 a KO in HT22 Control group,the Scn1 a KO+Lira group could promote cell proliferation(P < 0.001).6.Annexin V-FITC / PI flow cytometry results showed that compared to the Scn1 a KO in HT22 Control group,the Scn1 a KO+Lira group could reduce cellular apoptosis(P < 0.001);7.Immunofluorescence and Western Blotting experiments: Compared to Scn1 a KO group,liraglutide increased the expression of SCN1 A protein(P < 0.001);down-regulated the expression of mTOR(P < 0.001);up-regulated the expression of BCL-2(P < 0.001),down-regulated the expression of caspase-3(P < 0.001),and down-regulated the expression of BAX(P < 0.001).Conclusion1.Liraglutide can reduce seizure susceptibility and cognitive dysfunction in a mouse model of Dravet syndrome.2.Liraglutide may have neuroprotective effects by inhibiting the mTOR signaling pathway to reduce neuronal apoptosis.