| BackgroundEpilepsy(EP)is defined as the abnormal synchronous neuronal activity in the brain that causes transient clinical symptoms or signs.It is often accompanied by diseases such as stigma and comorbid affective disorders.In recent decades,experts and scholars have solved part of the burden of intractable epilepsy(IE)by developing new antiepileptic drugs(AEDs)and developing surgical techniques.However,the percentage of IE patients did not change substantially.The pathogenesis of epilepsy is very complicated,mainly including family inheritance,head injury,infection,metabolism and immunity.Transient receptor potential melatonin type 2 channel(TRPM2)is a non selective cation channel through Ca2+,which is widely expressed in the brain.The TRPM2 channel participates in the development of a variety of central nervous system diseases by mediating oxidative stress and inflammation,including traumatic brain injury(TBI),alzheimer’s disease(AD).In bipolar disorder(BP),the use of TRPM2 knockout mice has observed that the activity of glycogen synthase kinase-3(GSK-3)in the mice is lost.In hypoxic-ischemic brain injury,blocking the TRPM2 channel can increase neuroprotection and reduce brain damage caused by hypoxic-ischemia,and this effect is achieved by regulating GSK-3β.It can be concluded that the phosphorylation of GSK-3β depends on the TRPM2 channel,but the specific mechanism is still unclear.Especially in epilepsy,no relevant reports have been found.Studies have shown that TRPM2 can be activated or inhibited by many factors,among which N-(p-amylcinnamoyl)anthranilic acid(ACA)is a broad-spectrum TRP channel blocker.Based on the above,we hypothesized that TRPM2,as a non-selective cationic channel,is activated after epileptic seizure.It causes calcium ion influx and activates calcineurin,which activates GSK-3β and nuclear factor kappa-B(NF-κB)to induce an inflammatory response,thereby promoting the release of interleukin-1β(IL-1β)and causing epilepsy.We attempted to investigate the mechanism of TRPM2-GSK-3β-NF-κB-IL-1β in epilepsy and provide a new idea for the treatment of epilepsy.ObjectiveWe examined the expression of TRPM2,GSK-3β,NF-κB and IL-1β in the brain tissue of patients and the hippocampus tissue of an animal model of chronic temporal lobe epilepsy.It is clarified that the mechanism of GSK-3β induced inflammatory response mediated by TRPM2 channels in refractory epilepsy,to demonstrate the therapeutic effect of TRPM2 intervention on refractory epilepsy.Methods1.Collect the brain tissue of 10 patients with refractory epilepsy who underwent temporal lobe cortex resection as the epilepsy group,and the brain tissue of 10 patients with traumatic brain injury who underwent intracranial decompression and resection of the temporal lobe cortex as the control group.Western blot and qPCR were used to study the expression changes of TRPM2,GSK-3β,NF-κB,and IL-1β in the temporal lobe cortex.2.Group of experimental rats:the epilepsy model group(group E,n=20),namely intraperitoneal injection of LiCl,18-24h after injection of PILO for epilepsy modeling;ACA intervention group(group E+A,n=20),namely intraperitoneal injection of ACA 4h after successful modeling;control group(group C,n=20),namely the same amount of intraperitoneal injection normal saline.The expression of TRPM2,GSK-3β,NF-κB,and IL-1β in rat hippocampus was studied by qPCR and western blot.The expression of TRPM2 and GSK-3β in rat brain tissue sections were detected by immunohistochemistry and immunofluorescence methods.Results1.After injection of LiCI-PILO,36 temporal lobe epilepsy model rats survived.The success rate of model building is 90%.There was no death in the ACA injection group and the normal saline group after the successful model building.2.The results of qPCR showed that the levels of TRPM2(p<0.01),GSK-3β(p<0.01),NF-κB p65(p<0.001),and IL-1β(p<0.01)in the brain tissue of patients in the epilepsy group were higher than control group.In animal models,the levels of TRPM2(p<0.001),GSK-3β(p<0.001),NF-κB p65(p<0.01),and IL-1β(p<0.001)in the group E were higher than those of group C.The levels of TRPM2(p<0.001),GSK-3β(p<0.05),NF-κB p65(p<0.001),and IL-1β(p<0.001)in the group E+A were lower than those in the group E.3.The results of western blot showed that the expression level of TRPM2(p<0.01),GSK-3β(p<0.01),GSK-3β(Y216)/GSK-3β(p<0.05),NF-κB p65(p<0.01),IL-1β(p<0.05)in the epilepsy group were higher than that of the control group,while the expression level of GSK-3β(S9)/GSK-3β(p<0.05)was lower than that of the control group.The expression level of TRPM2(p<0.01),GSK-3β(p<0.05),GSK-3β(Y216)/GSK-3β(p<0.05),NF-κB p65(p<0.01),IL-1β(p<0.05)in the hippocampus of group E rats was higher than that of group C,while the expression levels of TRPM2(p<0.05),GSK-3β(p<0.05),GSK-3β(Y216)/GSK-3β(p<0.05),NF-κB p65(p<0.05),and IL-1β(p<0.05)in the hippocampus of group E+A were lower than those of group E.The expression level of GSK-3β(S9)/GSK-3β(p<0.01)in the group E was lower than that of group C,while the level of GSK-3β(S9)/GSK-3β(p<0.05)in the group E+A was higher than that of group E.4.The results of immunohistochemistry showed that the level of TRPM2(p<0.01)and GSK-3β(Y216)(p<0.001)in the brain tissue slices of rats in the group E was higher than that in the group C,while the level of TRPM2(p<0.01)and GSK-3β(Y216)(p<0.01)in group E+A was lower than that of group E.The level of GSK-3β(S9)(p<0.05)in the hippocampus of group E was lower than that of group C,while the level of GSK-3β(S9)(p<0.001)in group E+A was higher than that in group E.5.Immunofluorescence results showed that TRPM2 and GSK-3β co-localized in the CA1 area of the rat hippocampus.Conclusion1.In the brain tissue of patients and the hippocampus tissue of the animal model of chronic temporal lobe epilepsy,the levels of TRPM2,GSK-3 β,GSK-3β(Y216)/GSK-3β,NF-κB p65 and IL-1β in the epilepsy group were higher than those in the control group,while the levels of GSK-3β(S9)/GSK-3β is lower than the control group.2.After the intervention of TRPM2 inhibitors,TRPM2,GSK-3β,GSK-3β(Y216)/GSK-3β,NF-κB p65,IL-1β were lower than those in the E group,while GSK-3β(S9)/GSK-3β was higher than that in the E group.3.In rat brain tissue slices,immunofluorescence results suggest that TRPM2 and GSK-3β are co-localized in hippocampal CA1 area,indicating that TRPM2 may be involved in the onset of epilepsy through GSK-3β regulation.This study provides a certain experimental basis for further exploring the mechanism of TRPM2-GSK-3β-NF-κB-IL-1β in refractory epilepsy. |
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