Anticonvulsant Effect And Mechanisms Of DPP4 Inhibitor Sitagliptin

Background:Epilepsy is a common disease of the central nervous system(CNS)characterized by spontaneous and recurrent seizures.Risk factors for epilepsy include febrile seizures and traumatic brain injury.Our previous study demonstrated that dipeptidyl peptidase IV(DPP4)was associated with febrile seizures.Moreover,febrile seizures are closely associated with the development of epilepsy.The most common and important treatment of epilepsy is still drug.Despite the increasing numbers of antiepileptic drugs(AEDs),one-third of individuals with epilepsy are refractory to AEDs.Therefore,it is vital to discover anticonvulsant medicines with better efficacy and safety profiles.RAGE-JAK2/STAT3 pathway and CXCL4/CXCR3 axis are expressed in the brain with low basal expression,but they are rapidly upregulated in response to CNS injury.Therefore,our present study was designed to investigate whether DPP4 inhibitor sitagliptin has anticonvulsant effects and its mechanism.Our study will provide theoretic and experimental basis on the treatment of epilepsy.Objective:To determine the anticonvulsant effect of DPP4 inhibitor sitagliptin.And further identify the anticonvulsant mechanisms of sitagliptin.Methods:(1)Male Sprague Dawley(SD)rats(200 ? 20 g body weight)were randomly divided into the control group(Cnt),the sitagliptin group(Sita),the pentylenetetrazol(PTZ)group,the sitagliptin treatment group(PTZ + Sita).The PTZ group rats received PTZ(35mg/kg/d),which was used to induce seizures,for 5 days.The control and sitagliptin groups rats received saline and sitagliptin(5mg/kg/d)for 5 days,respectively.The PTZ + sitagliptin group rats were first given sitagliptin,followed by injection of PTZ 20 min later for 5 days.The behavioral changes were observed and recorded after injection.The seizure susceptibility of rats in each group was analyzed by electroencephalogram(EEG).(2)Primary cultured hippocampal neurons were randomly divided into three groups:The control group(Cnt)neurons,the model group(Mg2+ free group,Mg2+ free),the sitagliptin treatment group(Mg2+ free + Sita).The Mg2+ free group neurons were incubated with Mg2+ medium for 3 h and then cultured with Neurobasal medium.The Mg2+ free + sitagliptin group neurons were incubated with Mg2+ free medium containing sitagliptin(100 ?M)for 3 h then cultured with Neurobasal medium.Twenty-four hours later the action potential of hippocampal neurons was recorded by whole-cell patch-clamp method.(3)Twenty-four hours after the 5th day of animal behavior experiments,brain tissue was obtained under anaesthesia.The neuronal loss of CA1,CA3 and dentate gyrus(DG)neurons in the hippocampus of rats in each group were observed by Nissl staining.(4)The mRNA and protein levels of RAGE,phosphorylated JAK2(p-JAK2)and phosphorylated STAT3(p-STAT3)in hippocampal tissues of rats and cultured neurons in each group were detected by RT-qPCR and Western blot,respectively.(5)The mRNA and protein levels of CXCL4 and CXCR3 in hippocampal tissues of rats and cultured neurons in each group were detected by RT-qPCR and Western blot,respectively.(6)Hippocampal neurons were transfected with siRNA-CXCR3 plasmid,Western blot detect the levels of CXCR3,RAGE,p-JAK2 and p-STAT3 after transfection.(7)After hippocampal neurons were transfected with siRNA-CXCR3 plasmid,the extracellular medium was replaced with Mg2+ free extracellular medium.Immunofluorescence staining with RAGE antibody was conducted.Results:(1)Pretreatment with sitagliptin increased the latency to the GTCs,shortened the GTCs seizure duration and reduced the level of seizure score compared to the PTZ group.The EEG traces showed that sitagliptin pretreatment decreased the higher amplitude and frequency compared to the PTZgroup.In the control group and the sitagliptin group,the behavior and the EEG of the rats was normal.(2)A significant decrease in action potential burst frequency was observed in the Mg2+ free + sitagliptin group compared to the Mg2+ free group.(3)Hippocampal neurons in the PTZ group exhibited irregular distribution,cytoplasmic shrinkage and triangulated pyknotic nuclei in hippocampal CA1,CA3 and DG regions.The number of surviving neurons were significantly reduced compared to the control group.However,rats in the PTZ + sitagliptin group showed a reduction of neuronal loss in CA1 CA3 and DG regions.In the control and sitagliptin groups,no neuronal loss was found.(4)RT-qPCR and Western blot indicated that both the mRNA and protein levels of RAGE and the protein levels of p-JAK2 and p-STAT3 were significantly increased in the PTZ and Mg2+ free group.However,the expression levels were markedly decreased when pre-treated with sitagliptin.(5)RT-qPCR and Western blot indicated that both the mRNA and protein levels of CXCR3 and the protein levels of CXCL4 were significantly increased in the PTZ and Mg2+ free group.However,the expression levels were markedly decreased when pre-treated with sitagliptin.(6)Western blot showed that the levels of CXCR3,RAGE,p-JAK2 and p-STAT3 were decreased when the neurons were transfected with siCXCR3.(7)Immunofluorescence staining showed that the expression level of RAGE in the siCXCR3 group was lower than the control and negative control groups.Conclusions:In conclusion,this study provides evidence that DPP4 inhibitor sitagliptin provides anticonvulsant effects via suppression of the CXCL4/CXCR3 axis,which in turn inhibits the activation of the RAGE-JAK2/STAT3 pathway.This study provides new insight into the mechanisms of epilepsy and therapeutic approaches,pointing to DPP4 inhibitors as viable options for new anticonvulsant drugs.