Effects Of NLRC4 Inflammasome On Status Epilepticus And The Hippocampal Injury Post-status Epilepticus In Mice

Objective:At present,around 50 million people worldwide have epilepsy,which is mainly clinically treated with drugs.However,about 30%of patients still do not have effective seizure control,and a considerable part of them develop intractable epilepsy.It is a clinical problem that needs to be solved urgently to explore its occurrence mechanism and find new treatments.More and more studies have found that the increased susceptibility to seizures and the formation of temporal lobe epilepsy are closely related to neuroinflammation.As an inflammatory complex,the role of NLRC4 inflammasome in the pathogenesis of nervous system diseases is receiving increasing attention.In this study,a model of status epilepticus(SE)in mice induced by kainic acid(KA)was established to investigate the effect of NLRC4 inflammasome on the seizures of SE and the hippocampal injury after SE,which provided theoretical and experimental evidence for the treatment of temporal lobe epilepsy after SE with immune intervention as the core.Methods:One.Expression of NLRC4 and its downstream signaling molecules in hippocampus of mice after SE1.Establishment of KA-induced SE ModelA total of 95 healthy 8-week-old male C57BL/6 mice were randomly divided into two groups:the modeling group(n=80)and the blank control group(n=15).The mice in the modeling group were injected intraperitoneally with the corresponding dose of KA(20 mg/kg)according to their body weight,and the behavioral seizures of the mice were observed.The mice successfully modeled were randomly assigned to each subgroup at time points 3,6,12 and 24 hours after SE.In the CON group,the same amount of normal saline(NS)was injected intraperitoneally at the same time.2.Expression of NLRC4,cleaved-caspase-1 and cleaved-IL-1? in the hippocampus of mice after SEThe expressions of NLRC4,cleaved-caspase-1 and cleaved-IL-1? in the hippocampus of mice after SE were detected by immunoblotting.Immunofluorescence was used to further verify the expression of NLRC4 in the hippocampus of mice after SE.3.Cell localization of NLRC4 in hippocampus of SE miceCo-expression of NLRC4 with astrocyte markers glial fibrillary acidic protein(GFAP),microglial marker ionized calcium-binding adapter molecule 1(IBA1)and neuron marker microtubule associated protein 2(MAP2)were observed by immunofluorescence double-labeling method.Two.Effect of knockdown of NLRC4 expression in hippocampus on status epilepticus and the hippocampal injury post-status epilepticus in mice1.Detection of LV-NLRC4-RNAi interference efficiency on NLRC4 expressionLV-NLRC4-RNAi or negative control lentivirus(LV-CON)was injected into the hippocampus of mice using stereotactic technology.After 14 days,the interference efficiency of LV-NLRC4-RNAi was detected by immunoblotting and immunofluorescence.2.Effect of knockdown of NLRC4 expression in hippocampus on seizuresA total of 72 healthy male C57BL/6 mice at 6 weeks of age were randomly divided into four groups(n=18 in each group).LV-NLRC4-RNAi/LV-CON was injected into the hippocampus,and KA/NS was injected intraperitoneally 14 days later.(1)LV-NLRC4-RNAi+SE group,(2)LV-NLRC4-RNAi+NS group,(3)LV-CON+SE group,(4)LV-CON+NS group.Stereotactic technology was used to inject lentivirus into the hippocampus of mice.After 14 days,a mouse SE Model was established,and the seizure latent period and seizure levels of SE mice were observed.3.Effect of knockdown of NLRC4 expression in hippocampus on NLRC4-caspase1-IL-1? axis in hippocampus after SEThe expressions of NLRC4,cleaved-caspase-1 and cleaved-IL-1? in hippocampus 12 hours after SE were detected by immunoblotting.4.Effect of knockdown of NLRC4 expression in hippocampus on neuron loss in hippocampus after SEAt 14 days after SE,the number and morphology of neurons in the hippocampal CA1 and CA3 regions of each group were compared by Nissl staining.5.Effect of knockdown of NLRC4 expression in hippocampus on astrogliosis in hippocampus after SEAt 14 days after SE,the expression of GFAP in the hippocampal CA1,CA3 and dentate gyrus(DG)regions of each group was analyzed by immunohistochemistry.Results:One.Expression of NLRC4 and its downstream signaling molecules in hippocampus of mice after SE1.Establishment of KA-induced SE ModelAfter intraperitoneal injection of KA in the model group,a total of 73 mice developed grade 4 or more persistent seizures,so the success rate was 91%(73/80).Five mice died during the modeling process,so a total of 68 mice could be included in the modeling group.These 68 mice were randomly divided into each subgroup of the modeling group:the SE 3h group,the SE 6h group,the SE 12h group,and the SE 24h group,with 17 mice in each group.No seizures occurred in the control group after intraperitoneal injection of the same amount of NS,and no mice died.2.Expression of NLRC4,cleaved-caspase-1 and cleaved-IL-1 ? in the hippocampus of mice after SE(1)Immunoblotting results showed that at 3,6,12 and 24 hours after SE,the expression level of NLRC4 protein in mouse hippocampus was significantly higher than that of the control group(P<0.05),and reached the highest at 12 hours after SE.At 3,6,12 and 24 hours after SE,cleaved-caspase-1 and cleaved-IL-1? also increased correspondingly,which was significantly different from the control group(P<0.05),and showed the same change trend as NLRC4.(2)Immunofluorescence results showed that the NLRC4 fluorescence intensity at 12 hours after SE was significantly increased compared with the control group(P<0.05).3.Cell localization of NLRC4 in hippocampus of SE miceImmunofluorescence results showed that NLRC4 co-expressed with GFAP and IBA1 in the hippocampus of SE mice,and the co-expression with GFAP was more obvious,but no co-expression with MAP2 was seen.Two.Effect of knockdown of NLRC4 expression in hippocampus on status epilepticus and the hippocampal injury post-status epilepticus in mice1.Interference efficiency of LV-NLRC4-RNAi on NLRC4 expressionOn the 14th day after lentivirus injection into the hippocampus of mice,(1)Immunofluorescence results showed that the mouse cerebral cortex and hippocampus had green fluorescence distribution.The CA1 and DG regions were the most obvious in the hippocampus,indicating that stereotactic injection of LV-NLRC4-RNAi into the hippocampus successfully infected the hippocampus of mice.(2)Western blot results showed that the expression of NLRC4 in the hippocampus of the LV-NLRC4-RNAi group was significantly lower than that of LV-CON group(P<0.05),indicating that stereotactic injection of LV-NLRC4-RNAi in hippocampus effectively knocked down the expression of NLRC4 in hippocampus.2.Effect of knockdown of NLRC4 expression in hippocampus on seizuresThe results of behavioral seizures studies showed that:(1)After intraperitoneal injection of KA into mice,three mice in the LV-NLRC4-RNAi+SE group did not reach a level 4 or more persistent seizures,and one mouse died,so this group eventually 14 mice remained.Three mice in the LV-CON+SE group did not reach a convulsive seizure of grade 4 or above,and two mice died,so there were 13 remaining mice in this group.No seizures occurred in the control group(LV-CON+NS group and LV-NLRC4-RNAi+NS group)after intraperitoneal injection of the same amount of NS,and no mice died,so 18 mice remained in both groups.(2)The seizure latent period of mice in the LV-NLRC4-RNAi+SE group was 42.64±7.32 min,while in the LV-CON+SE group was 35.77±4.21 min,the former was significantly prolonged(P<0.05).It is suggested that knocking down of NLRC4 expression in hippocampus can reduce the susceptibility of SE in mice.(3)The level of seizures in the LV-NLRC4-RNAi+SE group was significantly lower than that in the LV-CON+SE group,and the differences were statistically significant at 30,40 and 50 minutes after intraperitoneal injection of KA(P<0.05).It is suggested that knocking down of NLRC4 expression in hippocampus can reduce the severity of SE in mice.3.Effect of knockdown of NLRC4 expression in hippocampus on NLRC4-caspase1-IL-1? axis in hippocampus after SEImmunoblotting results showed that at 12 hours after SE,the expression of NLRC4 in the hippocampus of LV-NLRC4-RNAi+SE group was significantly lower than that of LV-CON+SE group(P<0.05),and the expressions of cleaved-caspase-1 and cleaved-IL-1? were also significantly lower than those in the LV-CON+SE group(P<0.05).4.Effect of knockdown of NLRC4 expression in hippocampus on neuron loss in hippocampus after SEThe results of Nissl staining showed that at 14 days after SE,the number of neurons in hippocampal CA1 area and CA3 area of LV-CON+SE group was significantly lower than that of LV-CON+NS group(P<0.05),and the neurons were arranged disorderly and loosely.The number of neurons in the hippocampal CA1 and CA3 regions of the LV-NLRC4-RNAi+SE group was significantly higher than that of the LV-CON+SE group(P<0.05),and there was no significant difference from the LV-NLRC4-RNAi+NS group and the LV-CON+NS group(P>0.05),the neuron structure tended to be normal.It is suggested that neuron loss and structural damage can occur in the hippocampus 14 days after SE,and knocking down the expression of NLRC4 in the hippocampus can reduce the degree of neuron damage in the hippocampus after SE.5.Effect of knockdown of NLRC4 expression in hippocampus on astrogliosis in hippocampus after SEImmunohistochemical results showed that at 14 days after SE,the expression of GFAP in hippocampal CA1,CA3 and DG regions of LV-CON+SE group was significantly higher than that of LV-CON+NS group(P<0.05).The expression of GFAP in the hippocampal CA1,CA3 and DG regions of the LV-NLRC4-RNAi+SE group was significantly lower than that of the LV-CON+SE group(P<0.05),and there was no significant difference from the LV-NLRC4-RNAi+NS group and the LV-CON+NS group(P>0.05).It is suggested that astrogliosis can occur in the hippocampus 14 days after SE,and knocking down the expression of NLRC4 in the hippocampus can reduce the degree of astrogliosis in the hippocampus after SE.Conclusions:1.The NLRC4 inflammasome is activated in hippocampus of KA-induced SE mice,and is strongly expressed in astrocytes.Activation of the NLRC4 inflammasome is associated with neuroinflammatory responses in the hippocampus after SE.2.Knocking down the expression of NLRC4 in hippocampus not only reduces the susceptibility and severity of convulsive seizures induced by KA,but also protects hippocampal damage after SE.