| Objective To compare the anticonvulsive effects, toxicity and pharmacodynamic parameters of AMOC,NMOB,NMPB,NDMB.To investigate the effect of AMOC on the expression of mdr1(multidrug resistance gene 1)and its product Pgp(P-glucoprotein)in Valproate -resistant convulsive rats brain, and explore its anti-drug-resistant mechanism on the level of gene and protein.Methods (1) We used the model of convulsion seizure induced by Picrotoxin (PTX) with NS as control and Valproate(VPA)as positive control,then compare the convulsion seizure latency and anti- convulsion rate of AMOC,NMOB,NMPB,NDMB.(2)We used the model of MES with VPA as positive control to determine anti-maximal electroshock seizure effects of four cantharidinimide derivatives and analysis the dose-effect and the time-effect relationship of different doses of the drugs in intragastrical (ig) mice. ED50 of anti-MES was calculated according to the Bliss's method, then we compared their potency and efficacy.(3)The roll club method was adopted to analysis the toxic effect of ig AMOC,NMOB,NMPB,NDMB. Then we calculated TD50 according to the Bliss's method;Therapeutic index(TI) was calculated by LD50 / ED50.Finally , we compared toxic effects of different medicines.(4) Pharmacodynamic parameters of AMOC,NMOB,NMPB,NDMB and VPA were assayed by means of Pharmacological Effective Method.(5)The PNC-chronic kindling convulsive rat model was established by ip PNC(3×106U·kg-1) for 13 days. Valproate -resistant model was set up by Valproate (VPA,250mg·kg-1·d) ig for 21 days. The convulsive latency and Racine behavior classification was used to evaluate the anticonvulsive effects after ig AMOC(175mg·kg-1,87.5mg·kg-1) and flunarizine (FLU, 87.5mg·kg-1, positive control). Reverse transcription polymerase chain reaction (RT-PCR) technology was used to detect the effect of AMOC and FLU on the mdr1 gene expression; Immuno histochemistry (SABC method) was assayed to detect the changes of Pgp expression in the brain of drug-resistant convulsive rat.Results (1) In the antagonizing PTX experiment, AMOC,NMOB,NMPB,NDMB and VPA all had aiti-convulsion effects, the convulsive latency was prolonged as well,and they had the significance difference(P<0.05) compared to NS control. The anti- convulsion rate of AMOC and NMOB are the same as VPA,but NMPB and NDMB are less than VPA. (2) VPA,AMOC,NMOB,NMPB and NDMB could dose-dependently antagonize MES in mice, anticonvulsive rates were respectively VPA100%,AMOC 100%,NMOB100%,NMPB 100%, NDMB90%; The ED50 from low to high as follows: VPA AMOC>NMPB> NMOB>NDMB,and TI of them were respectively VPA(55.9),AMOC(11.5),NMPB(8.1),NMOB(8.4),NDMB(6.1). (4) The results of pharmacodynamic parameters indicated the minimum effect dosage of different medicines from low to high were VPAAMOC>NDMB> NMOB> NMPB. The elimination rate of AMOC (0.2) were smaller than the other two drugs; (5) Preparation of valproate-resistant rat model: with 40 rats, one of them died,six of them were divided in normal control, others were given penicillin (PNC,3×106U·kg-1) by ip for 13 days,all of the 33 rats were appearance Racine IV-V-class attack more than 6-8 times. Six of the 33 were divided in convulsion control group,and the others were given Valproate (VPA,250mg·kg-1·d )by ig for 21days.Until lastly,all of the 39 rats were selected by PNC(3×106U·kg-1,ip) after 24 h . There is no statistical significance (P> 0.05) between the convulsion latency of valproate -resistant(26.11±7.80min) and convulsion control(19.26±10.55min).So the model of valproate–resistant was accomplished and the achievement rate was 100﹪.(6) AMOC(175mg·kg-1,87.5mg·kg-1) and FLU which is respectively given by ig could inhibit epileptiform behavior and prolong the convulsion latency in valproate–resistant rats(P<0.01). (7)The results of RT-PCR and Immuno histochemistry (SABC method): The level of mdr1 mRNA and Pgp in convulsion control group are higher than normal control,and there is statistical significance(P<0.05) between them;the expression of mdr1 mRNA and Pgp in valproate–resistant are more than convulsion control and there is statistical significance(P<0.01) between them;the content of mdr1 mRNA and Pgp in AMOC1, AMOC2, FLU are respectively less than valproate–resistant and there is statistical significance(P<0.01) between them; the level of mdr1 mRNA and Pgp in AMOC1, AMOC2, FLU from low to high as follows: AMOC2 |
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