Protective Effects And Mechanisms Of DPP-Ⅳ Inhibitor On The Inflammatory Response In Repetitive Febrile Seizures Rats

Background:Febrile seizures(FS)are the most common seizure disorders in children aged from 6 months to 5 years.Children suffering from complex FS have a high risk of developing subsequent temporal lobe epilepsy(TLE).Neuroinflammation is involved in the pathogenesis of FS although the mechanism remains unknown.Increasing evidence supports the involvement of immune and inflammatory processes in the pathogenesis of seizures.Reactive gliosis and inflammatory cytokines contribute to neuronal excitotoxicity and epileptogenesis.We previously reported two new strains of rats that were identified as hyperthermia-prone(HP,lower seizure threshold)and hyperthermia-resistant(HR,higher seizure threshold).Our results identified 1140 genes that were significantly different in these two strains of rats using the Whole Rat Genome Oligo Microarray.Among these genes,DPP4 is a gene closely linked to IL-6 in the network map,which played a key role in the gene network analysis.Those studies suggested the important role of DPP4 in FS.This study addressed the potential anti-inflammatory properties of DPP4 inhibitor during FS.Objective:(1)To observe the expression of DPP4 in the cortical region of rats with febrile seizures and the expression in primary astrocytes.(2)To investigate the anti-inflammatory effects of DPP4 inhibitor sitagliptin on astrocyte activation and the expression of inflammatory cytokines.(3)To explore the molecular mechanism of sitagliptin in FS protection.Methods:(1)We established a rat model of complex febrile seizure by using hot water bath,and the expression of DPP4 in primary cultured astrocytes were investigated by immunofluorescence staining.The RNA and protein levels of DPP4 in cultured astrocytes under hypertherima were measured with Q-PCR and Western Blot.(2)The DPP4 inhibitor sitagliptin was injected into the lateral ventricle 15 min before the FS induction.The density of GFAP in hippocampal astrocytes was detected by immunohistochemistry.The expression of IL-1β,IL-6,TNF-a and IL-10 was detected by ELISA and Q-PCR.(3)ELIS A was used to measure the IL-1β、IL-6、TNF-α、EL-10 concentration of the supernatant of astrocytes.(4)The effect of DPP4 inhibitor sitagliptin on NF-κB p65 subunit phosphorylation in astrocytes of febrile seizures rats was detected by immunofluorescence double labeling.The NF-κB signal pathway was blocked by NF-κB inhibitor PDTC,and the phosphorylated level of NF-κB p65 subunit was detected using Western blot with sitagliptin or PDTC treatment.Results:(1)DPP4 expression was significantly increased at both the protein and mRNA levels after hyperthermia induction.Immunofluorescence staining with antibodies recognizing DPP4 and GFAP was conducted.We observed that DPP4 is extensively expressed in astrocytes in the cortex of rats.(2)Sitagliptin,a specific enzyme inhibitor of DPP4,remarkably attenuated hyperthermia-induced astrocytosis was suppressed after DPP4 inhibition.The number of GFAP-positive cells in the hippocampus and cortex sections was significantly higher in the FS group than in the control group,and sitagliptin pretreatment decreased the population of astrocytes.Inhibition of DPP4 blunted the increase of IL-1β,TNF-a,and IL-6 but not IL-10 in the sitagliptin-treated group compared with the hyperthermia-induced group.Similar to the ELISA results,the mRNA levels of IL-1β,TNF-α,IL-6 and IL-10 in the FS group were significantly higher than those levels in the control group.(3)The expression of IL-1β,TNF-a,IL-6 and IL-10 in primary cultured astrocytes were increased after LPS induction.Similar to the in vivo results,levels of the pro-inflammatory cytokines IL-1β,TNF-a and IL-6 generated from sitagliptin-treated astrocytes were lower than the levels in the LPS-induced group.However,IL-10 secretion remained unchanged after sitagliptin treatment in LPS-induced astrocytes.Similarly,IL-1β,TNF-a,IL-6 and IL-10 mRNA levels increased in astrocytes exposed to LPS 5 and the mRNA expression levels of IL-1β,TNF-a,IL-6,but not IL-10 were significantly reduced by sitagliptin.(4)In FS animals,p-p65 NF-κB level apparently increased in astrocytes whereas the level was reduced in the sitagliptin-pretreated group.Western blot analysis also revealed a significant decrease of p-p65 NF-κB in the sitagliptin-pretreated group of FS rats and primary astrocytes.This effect of sitagliptin was similar to the effect of PDTC,an NF-κB inhibitor.Conclusions:Our findings demonstrate that DPP4 functions as a critical regulator of neuroinflammation in hyperthermia-induced seizures and the DPP4 inhibitor may be a viable option for FS therapeutics.