| BackgroundPatients with epilepsy whose seizures do not successfully respond to anti-epileptic drug therapy are considered to have drug-resistant epilepsy(DRE),accounting for one third of all the epilepsy patients.The main course of DRE is the over-expression of the brain P-glycoprotein(P-gp)encoded by Multidrug Resistance gene 1(MDR1).Inhibition P-gp up-expression in the brain will be a new a clinical target in DRE patients.We previously found that epileptic seizures can lead to significant inflammatory responses in brain tissues and the activity of NF-κB,one essential factor in the brain inflammatory response could up-regulate P-gp expression in epilepsy brain.The inhibition of NF-κB activity could reduce the P-gp expression.In addition to frequency epileptic seizures itself,the use of anti-epileptic drugs is another important factor that promote drug-resistant proteins over-expression in the brain,but the detailed mechanism is still unknown.Pregnane X receptor(PXR)is one of the most important nuclear receptors in the metabolism and transporting of drugs and exogenous substances in vivo.The previous study had confirmed that the activity of PXR was closely with the inflammatory NF-κB signaling pathway.However,there have been no studies conducted to determine whether inhibition of PXR activity could reduce P-gp expression in epileptic brain through its inhibition of NF-κB activation,as one of the main purposes of this study.Temporal lobe epilepsy(TLE)is the most common type of DRE,and carbaxnazepine(CBZ)is one of the first-line drugs for the treatment of TLE recommended by most epilepsy treatment guidelines,so the resistance to CBZ is considered as the characteristics of DRE.Thus this subject is also further aimed to explore whether regulating NF-κB/PXR signaling pathways could reduce P-gp expression in brain to reverse the pharmacoresistance of epilepsy,and whether CBZ-induced brain P-gp overexpression is associated with this signaling pathway,especially for the clinical treatment of epilepsy and provide a theoretical basis for DRE.Part Ⅰ Role of PXR/NF-κB signaling pathway in regulation of P-glycoprotein expression at the brain of the acute epileptic seizure ratsObjective:To investigate the effect of PXR/NF-κB signaling pathway on the brain P-glycoprotein(P-gp)expression in the acute epileptie seizures rats and to explore the effect of carbamazepine(CBZ)on the brain P-gp expression in this phase and its relationship with PXR/NF-κB signaling pathway.Methods:One hundred and sixty SD rats were randomly divided into:(1)Sham group,(2)acute epileptic seizure model group(AEP group),(3)the intervention groups of seizure model including three subgroups,①PMA(PXR activity inhibitor)group,②PDTC(NF-κB activity inhibitor)group,③SJW(PXR agonist)group,(4)the CBZ treatment of epilepsy group including three subgroups,①CBZ group,②CBZ+PDTC group,③CBZ+PMA group.The acute epileptic models were induced by the micro-injection of kainic acid(KA)into rats’ hippocampus.Sham group was injected with equal volume of normal saline,and the intervention groups were given PMA(intraperitoneal injection),PDTC(intraperitoneal injection),and SJW(gavage)30 hours before the KA injection respectively.The treatament group was given CBZ orally with or without different intervention agents.We recorded the mortality rate,the success rate of modeling and the latency of epileptic seizures in each group.The protein levels of P-gp,PXR,NF-κBp65 were detected by Western blot and their mRNA(mdr1a,mdr1b,PXR,p65)levels were measured with RT-PCR.Furthermore,the co-expression of P-gp and PXR,or P-gp and NF-κBp65 were measured by immunohistochemistry and immunofluorescence double staining.The levels of IL-1β,IL-6 and TNF-α in blood and brain tissue were measured by ELISA.Results:1.There were no significant differences on the mortality and success rate of each epilepsy group.The seizure susceptibility in the PDTC group(SOT3=90.35±40.39 min&SOT5=105.28±28.26 min)was lower than that in the AEP model group(SOT3=66.31±12.95 min&SOT5=75.69±28.67 min),and the seizure susceptibility in the PMA group(SOT3=47.500±23.21 min&SOT5=60.57±21.10 min)was higher than that in the AEP group.SIW intervention had no significant effect on seizures.2.The proinflammatory cytokines in the brain tissue were increased after epileptic seizures.There was no significant difference on the inflammatory cytokines in the peripheral blood among each group.Compared to the AEP group(IL-1β=49.7±2.6 ng/L,IL-6=28.7+7.2 ng/L,TNF-α=42.8±2.4 ng/L in the brain),the concentrations of the above inflammatory cytokines were significantly decreased in the brain of the PDTC group(IL-1β=31.1±2.3 ng/L,IL-6=16.1+8.2 ng/L,TNF-α=28.2±3.0 ng/L),with a up-expression in PMA group(IL-1β=52.5±8.3 ng/L,IL-6=32.5+6.1 ng/L,TNF-α=45.81=7.2 ng/L in the brain)but with no significant differences.3.Compared to the AEP group,the levels of mdr1a,p65 mRNA and their protein levels were all significantly decreased in the PDTC intervention group and CBZ+PDTC group,with an interestingly high expression trend of PXR protein and its mRNA(but P<0.05).The similar effect was found in the SJW group.There were no significant differences on the above indicators between the other intervention group and CBZ treatment group.There was no significant difference in mdrlb mRNA level between each group.Conclusions:The inflammatory response of brain was promoted in acute epileptic phase with an increase of proinflammatory cytokines level in its brain.In this phase,inhibition of NF-κB activity could attenuate the brain inflammation and decrease the brain expression of mdr1a mRNA and P-gp.Inhibition of PXR activity could not reduce the brain P-gp expression,but elevate the levels of inflammatory cytokines in the epileptic brain.CBZ treatment had no significant effect on inflammatory cytokines levels and P-gp expression in epileptic seizure phase.In conclusion,the brain P-gp expression in acute epileptic seizures may be related to the inflammatory response mediated by NF-κB activation.Part II:Effect of PXR/NF-κB signaling pathway on the P-glycoprotein expression at the brain of the chronic epileptic rats induced by kainic acidObjective:Drug-resistance epilepsy(DRE)is attributed to the brain P-glycoprotein(P-gp)overexpression.We previously reported that nuclear factor-kappa B(NF-κB)played a critical role in regulating P-gp expression at the brain of the acute seizure rats.This study was extended further to investigate the interaction effect of NF-κB and pregnane X receptor(PXR)on P-gp expression at the brain of chronic epileptic rats treated with carbamazepine(CBZ).Methods:One hundred and forty SD rats were randomly divided into:(1)NS group,(2)chronic epileptic(CEP)model group(KA group),(3)the intervention groups of CEP model including two subgroups,①KA+PMA(PXR activity inhibitor)group,②KA+KA+PDTC(NF-κB activity inhibitor)group,(4)the CBZ treatment of epilepsy group including three subgroups,①KA+CBZ group,②KA+CBZ+PMA group,③KA+CBZ+PDTC group.The chronic epileptic models were induced by the micro-injection of kainic acid(KA)into rats’ hippocampus.Subsequently,the successful models were treated with different intervention agents of CBZ,PMA(a non-specific PXR activity inhibitor)or PDTC(a specific NF-κB activity inhibitor)respectively.The mortality rate,the success rate of modeling and the latency of epileptic seizures were recorded by each group.The immunohistochemistry staining of P-gp and PXR was used to detect their distribution in the brain Furthermore,the protein levels of P-gp,PXR and NF-κBp65 were detected by Western blot and their mRNA(mdr1a,mdr1b,PXR,p65)levels were measured with RT-PCR.The levels of IL-1β,IL-6 and TNF-α in serum and brain tissue were measured by ELISA.Results:1.Twenty-six of the 120 epileptic modeling rats died and 14 survived rats did not manifest the unprovoked seizures in the observed time.By compared to the KA group,there were no significant differences on the mortality,failure rate,and epileptic severity in the each epilepsy rats’ group.2.The expression levels of P-gp and its encoded gene mdr1a/b were significantly up-regulated on the brain of KA-induced chronic epilepsy rats or the epilepsy rats treated with CBZ for one week,meanwhile with a high expression of PXR.The treatment of PMA dramatically reduced both PXR and P-gp expressions at the protein and mRNA levels in the chronic epilepsy brain.By compared to the epilepsy model,the P-gp expression was not markedly attenuated by the inhibition of NF-κB activity with PDTC treatment,nevertheless with a decrease of NF-κB expression in this intervention.After PMA intervention,P-gp expression in the brain of CBZ-treated epilepsy rats was decreased(KA+CBZ group V.S.KA+CBZ+PMA group,P<0.05)3.Higher levels of proinflammatory cytokines(IL-1β,IL-6,TNF-α)were found both in the brain tissue and the serum in the epilepsy rats of each.There was a declined trend of the pro-inflammatory cytokines expression of the PDTC treatment but with no statistical significance.Conclusions:This study demonstrates for the first time that P-gp up-regulation is due to increase PXR expression in the chronic phase of epilepsy,differently from that NF-κB signaling may induce the P-gp expression in the acute seizure phase.Our results offer insights into the mechanism underlying the development of DRE using or not using CBZ treatment.Part Ⅲ:Roles of PXR signaling pathway on the tissue diversity of P-glycoprotein expression at the brain and intestine-liver of epileptic ratsObjective:To investigate the tissue diversity of P-glycoprotein(P-gp)expression at the brain and intestine-liver of epileptic rats with different stages(acute phase,clironic phase and drug-resistant phase).Methods:One hundred SD rats were randomly divided into:(1)Sham group,(2)acute epileptic seizure group(AEP group),(3)chronic epilepsy model(CEP)including three subgroups:①the CEP rats treated with NS(NSEP group),②CBZ-responsive CEP group(RCEP group),③CBZ-non-responsive CEP group(NRCEP group).The epileptic models were induced by the micro-injection of kainic add(KA)into rats’ hippocampus.The AEP model was produced at 24 h after KA injection.The CEP model was designed as the appearance of over 2 times of the unprovoked seizures after the acute phase.The CEP rats were given CBZ treatment for one week.The RCEP(CBZ-responsive)group were defined as its total seizure frequency at the reduction of over 50%and the NRCEP(non-CBZ-responsive)group were defined as<50%.The NSEP group rats were treated with NS instead of CBZ.The modeling and intervening reagents were all NS in Sham group.The blood was collected by the amputation of rats’ tails.Then all the rats were sacrificed.Thus the brain liver and jejunum were separated quickly.Formaldehyde were used to fix the tissues above.The P-gp and PXR expression at the intestine,liver and brain were respectively detected by immunohistochemical staining.The levels of IL-1β,TNF-αand IL-6 in serum and brain tissue were measured by ELISA.The differences of the above indicators were compared among the detected tissues.Results:1.In the acute phase of epilepsy,three rats died(3/20).In the chronic phase,nine rats did not reach the seizure criteria;32 rats died(32/60),including 11 cases in the NSEP group,nine cases in the RCEP group and 12 cases in the NRCEP group.The causes of their death included the drilling damage of the acute stage(one case)and the uncontrolled frequent seizures(38 cases).2.The expression of P-gp and PXR protein in brain tissue of chronic epilepsy was significantly higher than that in AEP group(NRCEP group>RCEP group>NSEP group,P<0.05).3.There was no statistical significance on the expression of P-gp and PXR protein in the intestinal tissue among the Sham group,AEP group and NSEP group.Compared with AEP group,the expression of P-gp and PXR protein was significantly increased in the chronic phase epilepsy rats with CBZ(NRCEP group>RCEP group,P<0.05).4.There was no significant difference in P-gp and PXR protein expression between liver tissue between the Sham group and AEP group.Compared with AEP group,the P-gp and PXR protein expression was significantly higher in the liver of the all CEP group(NRCEP group>RCEP group>NSEP group).5.Compared with Sham group,the levels of inflammatory cytokines both in the brain tissue and serum were higher in the KA-induced epilepsy rats(NRCEP group>AEP group>RCEP group>NSEP group,P<0.05).Conclusions:In acute epileptic phase,the over-expression of P-gp and PXR was significantly found in the brain but not in the intestinal tissue and liver.The expression of P-gp and PXR protein was both up-regulated in the brain,intestinal tissue and liver of the chronic epileptic rats.The brain inflammation appeared at both the acute and chronic epileptic phase.The peripheral inflammatory activation was only occurred in phase of drug-resistant chronic epilepsy. |
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