The Effect Of Scorpion Alcoholic Extraction On The Pgp-mdr1 Gene And Protein Expression In The Brain Of Phenytoin-resistant Convulsive Rats

Objective To establish the phenytoin(PHT)-resistant convulsive rats model induced by di-rectly cortical electrical stimulation and study the anticonvulsive action of Scorpion alcoholic extraction(SAE)based on the model. To investigate the effect of SAE on the expression of mdr1(multidrug resistance gene 1)and its product Pgp(P-glucoprotein)in phenytoin-resistant convulsive rats brain injury area, and explore the antagonizing drug-resistant mechanism of SAE from the levels of gene and protein.In addition,to find the relationship between PHT in-duction,frequently convulsive activity and expression of mdr1 and Pgp.Methods Using the method of implanting microelectrodes in the cortical motor area of the brains of rats and stimulating the area with monophase clivus ladder-shaped dipolar square pulse which increased from 0 to 1000μA within 15s until kindling convulsion seizure so that estab-lished the convulsive rats models. Then,ig PHT(0.154g·kg-1·d )for 7 days and stimulating the cortex of rats in the same parameter as above-mentioned after each administration PHT,established phenytoin-resistant convulsive rats model. Total 6 groups were seted up in the experiment:normal control group(Normal),convulsion model control group(CMCG), pheny-toin-resistant convulsion control group(PRCG),verapamil positive control group (VPCG, 0.0385g·kg-1),Scorpion alcohol extraction(SAE1,6.5g·kg-1)and Scorpion alcohol extraction (SAE2,13.0g·kg-1).After both doses of SAE ig,observed the effects of SAE on the changes of convulsion threshold and electroencephalogram(EEG) in phenytoin-resistant convulsive rats. The method of RT-polymerase chain reaction(RT-PCR) was used to detect the changes of mdr1 gene expression;the ratio of mdr1 IOD and gapdh IOD was considered as the expression quan-tity of mdr1 mRNA in rats brain. The method of immunohistochemistry (SABC) was adopted to determine the changes of Pgp expression;under light microscope,pick up 6 fields in each his-tological section and calculated Pgp-positive cell number in each field,the average Pgp-positive cell number of 6 fields was recognized as the expression quantity of Pgp in rats brain. By statis-tically analyzing the effect of SAE on the expression quantity of mdr1 mRNA and Pgp in phenytoin-resistant rats brain,investigate the mechanism of antagonizing drug-resistance of SAE.Results (1)The preparation of rats convulsive model:Among 42 rats,except of 6 as Nor-mal,2 dying after operation and 4 losting their electrodes,the rest 30 rats all appeared convulsive activity(Ⅰ-Ⅴstages) after cortical electric stimulation.Their EEG showed visible sharp waves,spike wives and sharp (spike) slow resultant wave. On the first day of stimulating,the convulsant threshold of all rats was 1191.00±181.98μАon average. In the following days, the value continuously declined until the 14th day when it stabilized at the level of 513.50±26.16μА.The rats convulsive model was established successfully.(2)The preparation of PHT-resistant rats convulsive model:Among the 30 convulsive rats,except of 6 as CMCG,the rest 24 rats were ig administrated PHT(154mg·kg-1·d).After ig PHT for the first time, the convulsant threshold of all rats with PHT-treated significantly increased,with statistical difference (P<0.01) compared to CMCG.Administrating PHT once a day and then stimulating in the next couple days, the value declined again and stabilized at 480.38±88.48μАin the 7th day.At this time, the value of convulsant threshold had no statistical difference compared with that of before PHT given(515.34±23.511μА) and that of CMCG(478.17±15.16μА)(P>0.05 respec-tively).PHT-resistant rats convulsive model was established successfully. (3) Anticon- vulsive action of SAE on PHT-resistant convulsive rats:Both doses of SAE and verapamil(Ver)all raised the convulsant threshold of PHT-resistant rats, there was statistical difference(P<0.05)compared with before drug-treated respectively. The group comparison after drug demonstrated:there was no statistical difference between CMCG and PRCG(P>0.05);The convulsant threshold in three drug-treated groups were all higher than CMCG and PRCG,there were statistical differ-ence(P<0.05)respectively. In the same time,epileptiform discharges were all weakened. From higher to lower, the convulsant threshold were:SAE2> VPCG> SAE1,but there were no statis-tical differences between them.(4)The results of RT-PCR:After stimulating the cortex of rats brain,the mdr1 mRNA expression in CMCG was higher than Normal,but with no statistical dif-ference between them(P>0.05);PHT was administrated, the mdr1 mRNA expression in PRCG was much higher than CMCG,and there was significantly statistical difference(P<0.01); After both doses of SAE and Ver administrated by ig respectively,the mdr1 mRNA expression de-creased,with statistical difference(P<0.01)compared with PRCG;VPCG had less mdr1 mRNA expression than SAE1,but more than SAE2,there was no statistical difference between them respectively(P>0.05).(5)The results of immunohistochemistry: Although CMCG had more Pgp expression than Normal,but there was no statistical difference between them(P>0.05);Pgp ex-pression in PRCG was much higher than CMCG,and there was significantly statistical differ-ence between them(P<0.01);Both doses of SAE and Ver all had less Pgp expression than PRCG,with P<0.01 respectively;Pgp expression in VPCG had no statistical difference com-pared with SAE1 and SAE2 respectively. The trend of changes of Pgp expression was similar with that of mdr1 mRNA.Conclusions (1)After direct electrical stimulation cortical motor area of rat for 14days, rats constantly appeared localized seizure-generalized convulsant activity (ⅳ~ⅴdegree)and typical epileptiform discharge.PHT administrated by ig for 7 days,convulsive rats become phenytoin-resistant, which showed that phenytoin-resistant convulsive rats models was estab-lished successfully.(2)PHT could significantly induce the expression of mdr1 gene mRNA and Pgp. The frequent convulsion activity also have effect on their expression on some degree. It is suppose that the formation mechanism of phenytoin-resistant convulsive rats model has direct relationship with the increasing of mdr1 mRNA and Pgp expression which were resulted in by the co-induction of PHT and frequent convulsion activity.(3)Both doses of SAE and Ver all raised the convulsant threshold of PHT-resistant rats, weakened epileptiform discharge and produced antagonistic action on this model.In addition,the nature of antagonizing PHT-resistant convulsion of SAE was similar with Ver. The above all showed SAE could cure refractory epi-lepsy.(4)Both doses of SAE and Ver all could inhibit the expression of mdr1 mRNA in pheny-toin-resistant convulsive rats brain,and decrease the Pgp content,which showed that the mecha-nism of antagonizing drug-resistance of SAE and Ver may be related with inhibiting the expres-sion of mdr1 mRNA and decreasing Pgp content in phenytoin-resistant convulsive rats brain. Because the trend of changes of Pgp expression in SAE and VPCG was similar with that of mdr1 mRNA,we presumed the inhibiting action of SAE on Pgp expression was directly resulted in by the inhibiting action of SAE on mdr1mRNA expression,also the nature of inhibiting ac-tion is similar with Ver.(5)Ver,as the first generation Pgp inhibitor received by international-ity,could cure partial seizures of refractory epilepsy clinically as additives in the method of doses administrated increasingly by degrees,with therapeutic effect certain and safe.SAE had the similar acting nature with Ver,and as a new Pgp inhibitor,is hoped to be an effective adjunc-tive therapeutic drug of refractory epilepsy.