The Effect Of Uncaria Total Alkaloids On The P-gp Gene Expression In The Brain Of Phenytoin-resistant Convulsant Rats

Objective To establish the original direct cortical electrical stimulation phenytoin-resistant convulsant model in rats,investigate the anticonvulsant action of Uncaria total alkaloids(UTA) based on the model.To observe the change of P-glycoprotein(P-gp)gene expression in rats brain injury area,explore the mechanism of UTA on inhibiting the anticonvulsant drug-resistant,and find the relationship of cortex injury,phenytoin(PHT),frequent convulsive acitivity and the P-gp gene(mdr1)expression in the brain of phenytoin-resistant convulsant rats.Methods The rats were implanted with electrodes into the brain cortex to establish the convulsant rat model.They were electrically stimulated after PHT(154mg·kg^-1·d)by intragastric administration(ig)every time for a week,the phenytoin-resistant convulsant rat model was established.Verapamil(Ver)as the positive control drug,we observed the effects of UTA on the change of convulsion threshold and electroencephalogram(EEG)of phenytoin-resistant convulsant rats after both dose of UTA ig.Using reversed transcript polymerase chain reaction (RT-PCR),we detected the gene expression of P-glycoprotein in electrical stimulation impaired brain area of rats,and used statistics method to semiquantitative analyse the information object definition(IOD)ratio(mdr1/GAPDH)of the P-gp gene expression of different groups.Results(1)During establishing the convulsive rats model period,except of 2 rats died after operation and 5 rats which lost their electrodes,all of 35 rats appeared convulsant activity(Ⅰ—Ⅴstages) after cortical electric stimulation.Their EEG were visible spike waves and spike and slow wave complex.The convulsion threshold values of all of convulsive rats were 1042.89±198.14 on the fist day,and stabilized at 631.94±113.59 until 14th day,the achievement ratio of the convulsant model is 83.3%.(2)During establishing the phenytoin-resistant convulsant rats model period,except of 5 rats which lost their electrodes,30 rats were given phenytoin ig.Compared to convulsant model control group(CMCG),the convulsion threshold of rats with phenytoin upgraded conspicuously after PHT ig firstly,there is statistical difference(p＜0.05).Be given continuous PHT ig to the seventh day,their convulsion threshold descended gradually. Compared to CMCG,the convulsion threshold of phenytoin-resistant convulsion control group(PRCG)had no statistical difference;so the phenytoin-resistant convulsant rats model was accomplished.The achievement ratio of the phenytoin-resistant convulsant rats model was 86%. (3)After both dose of UTA(245mg·kg^-1,490 mg·kg^-1)and Ver(38.5mg·kg^-1)ig respectively, the convulsion threshold of phenytoin-resistant convulsant rats increased,compared to themselves,there was statistical differences(p＜0.05),and epileptic discharge lightened.To compare the increase of the convulsion threshold of different groups each other,there was no statistical difference between CMCG and PRCG;to compared with CMCG and PRCG respectively,three groups with drugs were all have statistical differences(p＜0.05).The increase of the convulsion threshold of UTA2 is more than those of UTA1 and verapamil positive control group(VPCG),there were significant statistical difference between them(p＜0.01).There is no statistical difference between VPCG and UTA1.(4)To semiquantitative analyse the IOD ratio (mdr1/GAPDH)of P-gp gene(mdr1)expression of every groups,the CMCG is more than normal group,but there is no statistical difference between them.The mdr1 expression quantity of PRCG is more than CMCG,there was significant statistical difference between them(p＜0.01). Compared to PRCG,the mdr1 expression of UTA1 and UTA2 are all less than them,there are statistical difference(p＜0.01 vs PRCG).Compared to PRCG,the mdr1 expression quantity of VPCG is less too,there is significant statistical difference between PRCG and VPCG(p＜0.01).Conclusions(1)After direct electrical stimulation cortical motor area of rat,rats constantly appeared localized seizure -generalized convulsant activity.The convulsion threshold of rats being permanent on the 14th day,the achievement ratio of the convulsant rats model is 83.3%.After PHT ig,the convulsant rats were electrically stimulated one time a day for 7 days, they become phenytoin-resistant,the achievement ratio of convulsant rats model was 86%.As a original drug-resistant convulsant animal model,it is beneficial for us to investigate the multiple drug resistance(MDR)mechanism of intractable epilepsy and to find new MDR inhibitors.(2) PHT can significantly induce the quantity increase of mdr1 gene expression.The frequent convulsion activity and brain injury of rats maybe have effect on inducing the quantity increase of mdr1 gene expression.It is supposed that the formation mechanism of phenytoin-resistant convulsant rats model has direct relationship with the mdr1 gene expression increased synergistically by PHT induction action,frequent convulsant activity and brain injury of rats.(3) Both dose of UTA or Ver alone all have anticonvulsive action on drug-resistant convulsant animal model,the effect character of UTA are similar as Ver,But the anticonvulsive action of Ver is weaker than UTA2.(4)To use both dose of UTA or Ver alone all can inhibit the P-gp gene expression quantity in brain of rats,so the drug resistant mechanism has relationship with the gene expression of P-gp inhibited,but the effect of UTA are weaker thanVer.(5)As a new kind of P-gp inhibitor,UTA can be used as a utility adjunctive therapy drug for Intractable Epilepsy.