Gentiopicroside Play A Potential Role In Prevention Of Epileptogenesis And Neuroprotective In Immature Rats Via Inflammatory Pathway

Objective This study aimed to elucidate whether Gentiopicroside(Gent)affects epileptogenesis in immature rats by Li Cl-Pilocarpine(Li Cl-PILO)and to explore its relationship with P2X7R/NLRP3/Caspase-1 signaling pathway.Methods 1.Effects of Gent on seizure susceptibility and cognitive dysfunction in epileptic immature rats by Li Cl-PILO.Male SD immature rats were randomly divided into sham+vehicle group,sham+Gent(800mg/kg),SE group(model group),SE+VPA(200mg/kg),SE+Gent(200,400,and 800 mg/kg)group.The animal model was established by Li Cl-PILO.Immature rats were firstly intraperitoneally injected(i.p.)with Li Cl(125 mg/kg)and PILO(50 mg/kg,Sigma)24h later.The Racine scale,Electroencephalogram,Morris water maze test and New-Object-Recognition were used to detect the effects of Gent on seizure,learning and memory,and cognitive functions in epileptic immature rats.2.Effects of Gent on the histopathological changes and inflammatory cytokines in epileptic immature rats hippocampus.The dosing and grouping were the same as before.Nissl,FJB,and TUNEL staining were performed to evaluate the hippocampus damage.Their inflammatory factors(IL-1?,IL-18,and TNF-?)were also determined using ELISA.Immunofluorescence,and Western blot were used to detect Iba-1 expression.The HAPI cells were randomly divided into sham+vehicle group,LPS(500ng/ml)+ATP(1m M),LPS+ATP +Gent(50,100,and 200?M)group.The cell viability of HAPI was dected by CCK8,and the level of IL-1?,IL-18,and TNF-? in cell culture supernatants were determined using ELISA.3.Gent affects epileptogenesis in epileptic immature rats involved in P2X7R/NLRP3/Caspase-1 signaling pathway.The dosing and grouping were the same as before.Nissl,FJB,and TUNEL staining were performed to evaluate the hippocampus damage.Immunofluorescence was used to detect P2X7 R and NLRP3 expression in epileptic immature rats hippocampus.Wersten blot was used to detect P2X7 R,NLRP3,ASC,and caspase-1 expression in epileptic immature rats hippocampus.The HAPI cells were randomly divide into sham+vehicle group,LPS(500ng/ml)+ATP(1 m M),LPS+ATP+Gent(50,100,and 200?M)group,LPS+ATP+A839977(P2X7R inhibitor,50 n M).Wersten blot was used to detect P2X7 R,NLRP3,ASC,and Caspase-1 expression in epileptic immature rats hippocampus.Wersten blot was used to detect P2X7 R,NLRP3,ASC,and Caspase-1 expression in HAPI cells.q PCR(Quantitative Real-time)was used to detect P2X7 R,NLRP3,ASC,and Caspase-1 m RNA expression in HAPI cells.Results 1.Effects of Gent on seizure susceptibility and cognitive dysfunction in epileptic immature rats by Li Cl-PILO.Gent administration inhibited epileptogenesis as evidenced by decreased the seizure score from 4.45 ± 0.34 to 2.93 ± 0.17(P<0.01),reduced the incidence of seizures from 68.75% to 50% in Li Cl-PILO induced SE immature rats,increased the latency of seizures(P<0.01),attenuated the duration of seizures(P<0.01)and mortality rate(P<0.001),and reduced the total power(P<0.01)and wave amplitude(P<0.01)of epileptic EEG activity.Moreover,Gent treatment also significantly decreased the the escape latency(P<0.01)and escape distance(P<0.01)to find the hidden platform,increased the number of target quadrant crossings(P<0.05)and target quadrant residence time,(P<0.01)and enhanced the total time exploring the novel object(P<0.01).2.Effects of Gent on the histopathological changes and pro-inflammatory cytokines in epileptic immature rats hippocampus.In vivo:Pretreatment with Gent remarkably decreased neuronal injury,and down-regulated the pro-inflammatory cytokines expression of IL-1?(P<0.05),IL-18(P<0.01),TNF-?(P < 0.05),In epileptic immature rats hippocampus.Compared with the SE+vehicle model group,Gent administration reduced the fluorescence expression and protein level of Iba-1 in epileptic immature rats hippocampus(P<0.01).In vitro: compared with the LPS+ATP model group,Gent administration decreased the release of IL-1?,IL-18,TNF-? in cell cuture supernatants of HAPI cells(P<0.01).3.Gent affects epileptogenesis in epileptic immature rats involved in P2X7R/NLRP3/Caspase-1 signaling pathway.In vivo: Gent treatment remarkably reduced the protein expression of P2X7R(P<0.01),NLRP3(P<0.01),ASC(P<0.05),Caspase-1(P<0.01)in epileptic immature rats hippocampus.Gent administration also reduced the fluorescence expression of P2X7R(P<0.01),NLRP3(P<0.01)in epileptic immature rats hippocampus.In vitro: Compared with the LPS+ATP model group,Gent administration decreased the protein level of P2X7R(P<0.01),NLRP3(P<0.01),ASC(P<0.001),Caspase-1(P<0.01),and also reduce the m RNA expression of P2X7R(P<0.05),NLRP3(P<0.05),ASC(P<0.01),Caspase-1(P<0.05)in the HAPI cells with pre-treatment LPS+ATP.Conclusions 1.Gentiopicroside reduces the severity of epilepsy seizures and improve the cognitive function of epileptic immature rats.2.Gentiopicroside decreases hippocampal neuron damage and apoptosis,reduces the inflammation,and inhibits the activition of microglia in epileptic immature rats hippocampus.3.Gentiopicroside attenuates the expression of P2X7 R and the activation of NLRP3 inflammasome in epileptic immature rats hippocampus and HAPI micrglia.