The Effect Of Glutamate NMDA Receptor Antagonist, MK801, On The Expression Of Multidrug Transporters In The Limbic Lobe Epilepsy Rat Model

Epilepsy is a common clinical syndrome in nervous system, with chronic, accidental,and repeatedly stereotyped as its primary clinical characteristics. Although the therapyfor Epilepsy is advancing, there are still about 30% of all Epilepsy patients, withseizure out of controlled, belonging to refractory epilepsy including complex partialseizures (most seen in adults) and various kinds of epilepsy syndrome (most seen inchildren). Not only they are failed to response to the routine antiepileptic drugs(AEDs) (drug resistance), but also the new AEDs have uncertain effect for them. A lotof studies indicated that the overexpression of Multidrug Transporters (MDTs) on theblood brain barrier (BBB) of epileptogenic brain tissue limited brain uptake AEDs,interfered with AEDs attaining the taget, and developed drug resistance in the end.Seizure could induce the overexpression of MDTs, and it also cause glutamate torelease more. And glutamate could upregulate P-gp's expression by activating NMDAreceptor in the rat brain microvessel endothelial cells (RBMECs), which indicated thatthe overexpression of MDTs during seizure has something to do with the activation ofNMDA receptor. Thus, firstly, we observe the expression of MDTs in hippocampus inthe limbic seizure rat model within 72 h after status epilepticus (SE) was terminated.Secondly, we observe the effects of NMDA receptor antagonist, MK801, on theexpression of MDTs' mRNA and protein respectively at the peak time of their mRNAand protein expression within 72 h after SE was terminated. Thirdly, we explored theeffects of glutamate on Mrp2's mRNA expression and efflux function, and the effectsof MK801's pretreatment on the changes of Mrp2's mRNA expression and effluxfunction of RBMEC caused by glutamate in vitro.PartⅠThe Expression of MDTs after Limbic SeizureAim: To explore the time expression pattern of P-gp, Mrp2, and Bcrp within 72 hafter SE was terminated, and to determine the cell pattern distribution of these MDTs'expression. Methods: Limbic seizure model was induced by lithium chloride andpilocarpine. Realtime fluorescent quantitative RT-PCR (RT-qPCR) was employed todetermine P-gp, Mrp2, and Bcrp mRNA expression in hippocampus at 0h, 3h, 6h,24h, 72h after SE was terminated and of control group. Protein expression of P-gp, Mrp2, and Bcrp in hippocampus was determined by Western blot at 3h, 6h, 24h, 72h after SE was terminated and of control group. Brain slices were double labeled byP-gp, Mrp2, and Bcrp with vWF, MAP2, and GFAP respectively by Immunofluerence.Each group mentioned included 6 rats. Results: mRNA and Protein expression ofthese MDTs increased instantly after SE was terminated, mRNA expression of eachreached peak at 6h, while their protein expression attained peak at 24h. It is at 3hthat pxr mRNA expression reaches its peak. The expression of P-gp, Mrp2, and Bcrplocated on endothelial cells of brain capillary in control group, while their expressionincreased and appeared on a few of neurons and astrocytes at 24 h after SEterminated.Conclusion: Seizure induced transient overexpression of P-gp, Mrp2, and Bcrp. Alsoseizure could induce these MDTs' expression in neurons and astrocytes.PartⅡThe Effects of Glutamate NMDA Receptor Antagonist. MK801, on the Expression of P-gp, Mrp2 and Bcrp after SeizureAim: To observe the effects of NMDA receptor on P-gp, Mrp2, and Bcrp expressionafter limbic seizure, and to explore whether NMDA receptor could regulate theexpression of these MDTs. Methods: Rats were divided into 3 groups: SE group wasrefered to the limbic seizure rat model induced by LiCl-Pilocarpine; MK801 groupwas pretreatmented with MK801 (0.5 mg/kg); control group was given Normal Salineinstead of pilocarpine, and other treatment of MK801 and control group were thesame as SE group. P-gp, Mrp2, and Bcrp's mRNA and protein expression weredetermined by RT-qPCR (6h after SE was terminated) and Western blot (24 h afterSE was terminated) respectively. Immunohistochemistry was employed to determinethe expression distribution of P-gp, Mrp2 and Bcrp. Results: The expression of P-gp,Mrp2 and Bcrp in SE group were significantly higer than control group, while theexpression of these MDTs in MK801 goup were lower than SE group. Exception forthe protein expression of Mrp2 in MK801 group, there was no difference between theexpression of these MDTs in MK801 and control group. Immunhistochemistry resultsshowed that the expression of these MDTs in almost each limbic brain area in SEgroup were much higher than MK801 and control group. Conclusion: NMDAreceptor antagonist, MK801, could reverse the overexpression of P-gp, Mrp2 andBcrp induced by seizure. The activation of NMDA receptor might be involved in theoverexpression of P-gp, Mrp2, and Bcrp during seizure. PartⅢThe Effects of Glutamate, and NMDA Receptor antagonist, MK801, on mRNA expression and efflux function of Mrp2 in RBMECsAim: To observe the effects of glutamate on the mRNA expression and the effluxfunction of Mrp2, and to explore whether NMDA receptor has an effect on thechanges of expression and function of Mrp2 induced by glutamate. Methods: Isolatedmicrovessel segments from the cortex of 2-week-old rats, and rat brain microvesselendothelial cells (RBMECs) primary culture was carried out. After RBMECs becameconfluent, we undertook the following experiments. The cells were divided into 3groups: control, Glu, and MK801 group. The cytotoxicity of different concentration ofglutamate on RBMECs was determined by MTT to identify an appropriateconcentration for glutamate intervention, mRNA expression of mrp2 was determinedby RT-qPCR. And Mrp2 efflux function was determined by substrate effluxexperiment. Results: MTT results showed that glutamate at 300μM or above causedsignificant cytotoxicity, therefore we selected 100μM as the glutamate concentrationfor intervention. RT-qPCR results demonstrated that the mRNA expression of mrp2 inGlu group was significantly higher than MK801 and control group (P＜0.05). And theefflux experiments showed that the ratio of extracellular/intracellular concentration offluorescent substrate was higher in Glu group than MK801 and control group(P＜0.05). Conclusion: Glutamate could induce the upregulation of mrp2 mRNAexpression and the reinforcement of Mrp2's efflux function. Furthermore, NMDAreceptor antagonist, MK801, could reverse these effects of glutamate.Summary1. Seizure induces transient overexpression of P-gp, Mrp2, and Bcrp. Furthermore,seizure could induce these MDTs' expression in neurons and astrocytes.2. NMDA receptor antagonist, MK801, could reverse the overexpression of P-gp,Mrp2 and Bcrp induced by seizure. The activation of NMDA receptor might beinvolved in the overexpression ofP-gp, Mrp2, and Bcrp during seizure.3. Glutamate could induce the upregulation of mrp2 mRNA expression and thereinforcement of Mrp2's efflux function. Furthermore, NMDA receptorantagonist, MK801, could reverse these effects of glutamate.