Research On The Relationship And Influence Factors Between Organic Anion Transporting Polypeptide 2 And Anti-Epileptic Drugs

PARTⅠEXPRESSION FEATURE AND AGE CHARACTERISTICS OF OATP2 IN RAT【Objective】To explore(1)the expression of Oatp2 in the brain and liver of Wistar rat;(2)wether there are age differences in rat Oatp2 expression.【Methods】Wistar rats were divided into 3 groups according to age: adult group, infant group(20d after birth)and newborn group(3d after birth),there were 12 rats in every group, and the number of male and female rats were equal. Expression of Oatp2 protein was detected by immunohistochemistry and mRNA using RT-PCR. Levels of Oatp2 of different groups were compared in brain and liver .【Results】(1)In brain, Oatp2 immunopositive cells were observed in brain capillary endothelium,choroid plexus epithelium and neurons of cerebellum;(2) In liver, Oatp2 was expressed at the membrane of hepatocytes,predominantly around vena portae hepatis region;(3)In brain,expression of Oatp2 decreased with age,whereas,hepatic expression of Oatp2 gradually increased after birth.【Conclusion】(1) Oatp2 were expressed in rat brain and liver ,suggested that the transporter may contribute to the chemical disposition of living systems; (2) The age-denpendent feature of Oatp2 in rat bran and liver will aid in understanding the pharmacokinetics and toxicokinetics of drugs and other chemicals.PARTⅡINFLUENCE OF SIZURE ACTIVITY AND AEDS THERAPY ON EXPRESSION OF OATP2 IN RAT BRAIN【Objective】To investigate the expression of Oatp2 during epileptogenesis and AEDs therapy in rat brain.【Methods】Status convulsion lasting for 60min were induced by intraperitoneal injection with lithium chloride and pilocarpine(.1)Rats were sacrificed at 6 different time points from 6th hour to 8th week after SC termination.Expression of Oatp2 and P-gp in brain and liver were detected by immunohistochemistry ;Dynamic change of Oatp2 in brain and liver were investigated by Western blot and RT-PCR; Levels of Oatp2 and P-gp in brain and liver were compared of chronic epileptic and control rats.(2)Chronic spontaneous epilepsy rats ( 4th week after 60'SC ) were treated with therapeutic dose of AEDs (VPA 187.5mg/kg,CBZ 125mg/kg,PHT 62.5mg/kg ) for 4 weeks.All the rats were killed after AEDs administration.Expression of Oatp2 protein were detected by immunohistochemistry and western blot analysis and mRNA using RT-PCR.【Results】(1)In brain, P-gp immunopositive cells were mainly detected in capillary endothelium and choroid plexus epithelium;(2)Compared with normal control rats,there were no difference in the location of Oatp2 and P-gp after sizure and AEDs therapy; (3) Decreased expression of Oatp2 was observed in acute,latent and chronic periods of epileptogenesis, particularly in 8th week after 60'SC(P<0.01);(4) Compared to control rats, Oatp2 protein expression in brain was significantly decreased while P-gp was obviously increased in chronic epileptic rats(8th week and 4th week after 60'SC),and there was a positive correlation between the change degree of the two transporter and the occurrence of SRSs;(5)Compared to control group,Oatp2 protein expression was significantly down-regulated(P<0.05)while P-gp was up-regulated(P<0.05) in chronic epileptic rat treated with AEDs;(6)Oatp2- mRNA in SC group,chronic rat treated with AEDs group did not have the similar change to Oatp2 protein.【Conclusion】(1) Seizure activity and AEDs down-regulated cerebral expression of Oatp2 ,however up-regulated the brain expression of P-gp, these data suggested that Oatp2 might be play a different role as P-gp in the development of pharmacoresistance. (2)Both seizure activity and AEDs might be responsible for the expression and post-transcriptional control of Oatp2 gene.PARTⅢIN VITRO STUDY ON THE RELATIONSHIP BETWEEN OATP2 AND AEDS【objective】Cultured rat brain microvascular endothelial cells(BMECs) were used as the in vitro model of BBB to observe (1) the transport of valproate (VPA) which was mediated by Oatp2 ;(2) the effects of antiepileptic drugs(AEDs) on the expression of Oatp2 .【Methods】(1)Rat brain microvascular endothelial cells(BMECs) were cultured with brain segment sticking mass method.The BMECs were identified by observing under phase-contrast microscope, and by using immunocytochemical methods with anti-rat factorⅧantibody and CD34.The expression of organic anion transporting polypeptide2 (Oatp2) and P-glycoprotein (P-gp) were detected by immunocytochemical methods, western blot and RT-PCR analysis;(2)VPA uptake over time was recorded by incubation of the cell in a medium containing VPA,the steady-state uptake of VPA by BMECs was tested for different VPA concentrations;(3) Compared the uptake of VPA by cells expressed Oatp2(BMECs)with cells without Oatp2(fibroblasts);(4)The effects of digoxin(inhibitor of Oatp2)on the uptake of VPA were also studied; (5 )Different concentrations of VPA,phenytoin(PHT),carbamazepine(CBZ) were added to the culture media for 7,28 days. Expression of Oatp2 and P-gp in BMECs after AEDs therapy were detected by Western blot.【Results】(1)The cultured BMECs showed regular cobblestone and polygonal morphology and positive forⅧF-Ag and CD34.The purity of positive stained cells was 90%. Both Oatp2 and P-gp expressed in BMECs.( 2 ) The uptake of VPA by BMECs was time- and concentration-dependent.;(3)Compared to fibroblasts,the uptake of VPA by BMECs was significangtly higher(P<0.01);(4)The co-administration of digoxin significangtly decreased the uptake of VPA by the BMECs (P<0.01);(5)Compared to control group,Oatp2 protein expression was significantly down-regulated,while P-gp was up-regulated in BMECs treated with AEDs.【Conclusion】(1) The rat BMECs were successfully cultured in vitro by brain segment sticking mass methods.The primary BMECs expressed the Oatp2 and P-gp. This experiment provide a possible pathway for studying transporters of the Blood-Brain Barrier;(2)The transport of VPA at the BBB is mediated by transpoters,Oatp2 may be involved in the transport of VPA;(3) AEDs may induce the expression of Oatp2 and P-gp in BMECs,which is correlated with the dose.