Research On The Effect And Mechanism Of Semaglutide In GABRG2 Gene Defect Mice Upon Genetic Epilepsy With Febrile Seizures Plus

Epilepsy is a common central nervous system（CNS）disease,characterized by long-term recurrent seizure.Recurrent seizure may lead to cognitive,psychological and social disorders,seriously affecting the patient’s quality of life.The etiology of spontaneous seizure is complex,genetic and environmental factors have important influence on it.（Genetic epilepsy with febrile seizures plus,GEFS+）is a special type of epilepsy syndrome.Some patients have spontaneous seizure,which resulting in related complications and bringing huge burden to patients and their families.Seizure can last for years,or even a lifetime.At present,seizure is mainly controlled by antiseizure drugs（ASDs）,but it has little effect on the progression and thechange of epilepsy pathophysiological.Nearly 30 percent of epilepsy resistance to ASDs and are unable to control their seizure effectively.The research of ASDs is necessity for neuroscience.Neuroinflammation refers to the activation of the innate immune system after the internalhomeostasis of the brain is disturbed by the internal and external environment.It is closely related to various CNS disease,such as epilepsy.Neuroinflammation is involved in the development of epilepsy and seizure,resulting in over-excitation of neurons and abnormal connections which lead to seizure.Neuroinflammation is controlled by a variety of glial cells,such as microglia.Microglia is considered to be a key coordinator of neuroinflammation and participate in both pro-inflammatory and anti-inflammatory responses.Neuroinflammation may be important to research into neuroprotection,improvement of cognitive dysfunction,andseizure control.As receptors of innate immunity,inflammasome plays an important role in defending against pathogen and coping with aseptic inflammation,and is an important part of inflammatory signaling pathway.As a familiar inflammatory complex,NLRP3 inflammasome is involved in the occurrence and development of many CNS diseases,including epilepsy,andis a new target for the control of seizure.Glucagon-like peptide-1（GLP-1）was originally thought to be a peptide substance with hypoglycemic effect derived from preglucagon gene cells.However,subsequent studies have found that it is also produced in the CNS,with the function of neuropeptide,with anti-epileptic,anti-inflammatory and neuroprotective functions.Our experimental group constructed genetic epileptic mice with GABRG2 gene defect(Gabrg2^fl/wtCre⁺in hippocampus and neocortex,GABRG2-CKO)with background of C57BL/6J in the early stage,and constructed GEFS+epilepsy model under exogenous heat stimulation.Semaglutide（SEMA）was launched in 2017 as a new weekly GLP-1.Our preliminary trials shown that prophylactic application of 25 nmol/kg SEMA alleviate seizure severity,reduce neuroinflammatory response and improve neuron injury in pentylenetetrazole（PTZ）-kindled mice.However,the underlying mechanism of SEMA’s anti-inflammatory and neuroprotective effects remain unclear.In this study,we used behavioral,electrophysiological,histological and molecular biological methods to investigate the antiepileptic,neuroprotective and anti-inflammatory effects of SEMA in GABRG2-CKO mice upon GEFS+epilepsy model.The effect and mechanism of SEMA on NLRP3inflammasome activation was investigated in Lipopolysaccharide（LPS）and Nigericin-activated mouse microglial cell line（BV2）.The neuroprotective effect and mechanism of SEMA was investigated in the co-cultured activated BV2 cells and HT22 cells.Part 1 The effect of semaglutide on cognitive dysfunction and seizure severity in GABRG2 gene defect mice upon GEFS+epilepsy modelObjective To investigate the effect of SEMA on cognitive dysfunction and seizure severity in GABRG2 gene defect mice upon GEFS+.Methods According to the previous plan of our research group,GABRG2-CKO mice were given exogenous thermal stimulation by electric heating device,and the GABRG2-CKO mice with 3 or higher grade improved Racine convulsions were selected as GEFS+epileptic model mice for subsequent experiments.The experimental animals were randomly divided into5 groups:（1）WT group（wild mice,0.9%physiological sodium chloride injection,0.9%Na Cl injection every other day,intraperitoneal injection,i.p.）;（2）Control-1 group（wild mice,25 nmol/kg SEMA every other day,i.p.）;（3）CKO group（GEFS+mice,0.9%Na Cl injection,every other day,i.p.）;（4）SEMA-1 group（GEFS+mice,25 nmol/kg SEMA every other day,i.p.）;（5）VPA group（GEFS+mice,100 mg/kg Sodium valproate daily,VPA,i.p）.Intellicage test,Field test,modified novel object recognition（NOR）,Shuttle box,Morris water maze（MWM）were performed to evaluate the effect of SEMA on cognitive dysfunction in GEFS+mice.The effect of SEMA on seizure severity in GEFS+mice were evaluated by modified Racine score,Electrocorticography（ECo G）monitoring and Timm staining.Results1.In the Intellicage test,noise test shown no significant difference in the number of visitand noise among all groups（P>0.05）.In the position recognition test,SEMA and VPA reduced the number of visit false in GEFS+mice（P<0.05）.In the reverse position experiment,SEMAand VPA reduced the error visit rate of GEFS+mice（P<0.05）.In the Field test,SEMA and VPA reduced the ratio of edge time to center time in GEFS+mice（P<0.05）.In the modified NOR experiment,SEMA and VPA increased the recognition index of GEFS+mice（P<0.05）.In the shuttle box test,SEMA and VPA increased the active avoidance times of GEFS+mice in active shuttle test（P<0.05）.In the passive shuttle experiment,SEMA and VPA reduced the number of electric shock times in the learning stage of GEFS+mice（P<0.05）,and extended the incubation period of GEFS+mice entering the black box in the test stage（P<0.05）.In the MWM experiment,SEMA and VPA reduced the time for GEFS+mice to find an escape platform（P<0.01）,increased the number of times for GEFS+mice to cross the target location（P<0.05）,and increased the stay time of GEFS+mice in the target quadrant（P<0.05）.2.ECoG monitoring shown that the seizure behavior of GEFS+mice were consistent with ECo G monitoring result.SEMA and VPA both reduced the frequency and amplitude of ECo G during epileptic seizure in GEFS+mice,alleviated the severity of seizure in GEFS+mice（P<0.01）,and reduced the rate of heat-induced seizure in GEFS+mice（CKO:75%;SEMA-1:21.4%;VPA:28.6%）and reduced mortality in GEFS+mice（CKO:20%;SEMA-1:6.7%;The VPA:6.7%）.3.Timm staining shown that SEMAand VPAreduced the germination of mossy fibers in the dentate gyrus（DG）region of GEFS+mice（P<0.01）.Conclusion1.SEMA improves cognitive dysfunction in GEFS+epileptic mice.2.SEMA alleviates seizure severity in GEFS+mice.3.Both SEMA and VPA alleviate seizure severity and improve epilepsy-related cognitive dysfunction in GEFS+mice.SEMA administration may be more beneficial for patients to takelong-term medication.Part 2 The effect of semaglutide on neuronal injury and NLRP3 inflammasome activation in GABRG2 gene defect mice upon GEFS+epilepsy modelObjective To investigate the effect of SEMA on neuroprotection and NLRP3inflammasome activation in GABRG2 gene defect mice upon GEFS+epilepsy model.Methods The grouping of GABRG2-CKO mice and the establishment of GEFS+epilepsy model mice were the same as the part 1.Nissl staining was used to detect the number of nissl staining positive hippocampal neurons.The fluorescence intensity of neuron specific marker（Neu N）and astrocyte specific marker（GFAP）was measured by immunofluorescence double staining,in hippocampal area.The protein expression and average fluorescence intensity of NLRP3,Active caspase-3,Bax,Bcl-2,and Sirtuin 1（SIRT-1）were detected by western blot（WB）and immunofluorescence staining.The protein expression of caspase-1 p20,interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）and NFκB-P65 were detected by WB assay.The percentage of positive cells of Caspase-1p20,IL-1β,TNF-αand NFκB-p65 were detected by immunohistochemical staining.Results1.SEMA and VPA increased the number of nissl staining positive neurons in the hippocampus of GEFS+mice（P<0.05）,enhanced the average fluorescence intensity of Neu N（P<0.05）,and weakened the average fluorescence intensity of GFAP（P<0.05）.Immunofluorescence staining and WB assay also showed that SEMA and VPA decreased the average fluorescence intensity and protein expression of Active caspase-3（P<0.01 and P<0.0001）,and significantly decreased the average fluorescence intensity of Bax（P<0.01）.The mean fluorescence intensity of Bcl-2 was increased（P<0.01）.The ratio of grayvalue of Bcl-2/Bax was increased（P<0.001）.2.SEMA and VPA significantly decreased the average fluorescence intensity（P<0.01）and protein expression（P<0.001）of NLRP3 protein in the hippocampus of GEFS+mice,decreased the percentage of positive cells in immunochemical staining of Caspase-1 p20,IL-1β,TNF-α（P<0.0001）,and decreased the gray value of band in WB experiment（P<0.01）.3.In the GEFS+mice,SEMA and VPA increased the mean fluorescence intensity（P<0.01）and protein expression（P<0.01）of SIRT-1,as well as decreased the percentage of NFκB-p65’s positive cells in immunochemical staining.（P<0.0001）and the gray value of protein band in WB experiment（P<0.01）.Conclusion1.SEMAimprove hippocampal neuron injury in GEFS+epilepsy mice.2.SEMA inhibit the activation of NLRP3 inflammasome in the hippocampus of GEFS+epileptic mice.3.The antiepileptic effect of SEMA may be related to the reduction of NLRP3inflammasome activation and thus neuronal injury.4.SIRT-1 may mediate the anti-inflammatory and neuroprotective effect of SEMA in GEFS+epileptic mice.5.Both SEMA and VPA can improve neuroinflammation and neuron damage caused by epilepsy in mice,and SEMA administration may be more conducive to long-term medication.Part 3 Semaglutide exerts anti-inflammatory and neuroprotective effect through SIRT-1/NLRP3/IL-1βpathwayObjective To research the anti-inflammatory and neuroprotective mechanism of SEMA and provide theoretical support for clinical research.Methods CCK8 kit was used to detect the appropriate concentration of SEMA in BV2 cells.BV2 cells were divided into 5 groups:（1）BV2 group（0.1%dimethyl sulfoxide,DMSO）;（2）Control-2 group（900 n M SEMA）;（3）Model group（1μg/m L LPS+10μM Nigernin）;（4）SEMA-2 group（900 n M SEMA+1μg/m L LPS+10μM Nigernin）（5）EX527（SIRT-1 inhibitor）group（10μM EX527+900 n M SEMA+1μg/m L LPS+10μM Nigernin）.Five groups of BV2 cells and HT22 cells were co-cultured for 24 h by Transwell plate.The m RNA and protein change of NLRP3,Caspase-1 p20 and SIRT-1 in BV2 cells were detected by real-time fluorescence quantitative PCR（RT-q PCR）and WB assay.The contents of inflammatory factors IL-1βand TNA-αin supernatant of BV2 cells were detected by ELISA assay.Immunofluorescence staining and WB assay were used to detect the fluorescence intensity and protein expression of apoptosis related protein Active caspase-3 in co-cultured HT22 cells.Flow cytology was used to detect apoptosis of co-cultured HT22 cells.Results1.900 nM was the appropriate concentration of SEMA in BV2 cells experiment.2.SEMA reduced protein and mRNA concentration of NLRP3 and Caspase-1 p20 in LPS and Nigernin-activated BV2 cells（P<0.0001）,and increased protein and m RNA concentration of SIRT-1（P<0.001）.EX527 partially attenuated the effect of SEMA that decreased protein and m RNA concentration of NLRP3（P<0.05）,as well as decreased protein and m RNA concentration of Caspase-1 p20（P<0.01）.In addition,EX527 partially attenuated the effect of SEMA that increased protein and m RNA concentration of SIRT-1（P<0.05）.3.SEMA reduced the protein expression of IL-1βand TNF-αin the supernatant of LPS and Nigernin activated BV2 cells（P<0.0001）.EX527 partially inhibited the effect of SEMA on IL-1βand TNF-αprotein in supernatant（P<0.01）.4.SEMA attenuated average fluorescence intensity and protein expression of apoptosis-related protein Active caspase-3 in LPS and Nigernin activated co-cultured HT22cells（P<0.0001）,which was partially inhibited by EX527（P<0.01）.5.Flow cytometry results shown that SEMA improved apoptosis in LPS and Nigernin activated co-cultured HT22 cells（P<0.0001）.EX527 partially attenuated the antiapoptotic effect of SEMA in co-cultured HT22 cells（P<0.0001）.Conclusion1.SEMA inhibits NLRP3 inflammasome activation and alleviates downstream inflammatory cytokines release in activated BV2 cells.2.SEMA has neuroprotective effect on co-cultured HT22 cells under the co-culture conditions of activated BV2 cells and HT22 cells.3.The anti-inflammatory and neuroprotective effect of SEMA partly depend on the regulation of SIRT-1/NLRP3/IL-1βpathway.