The Comparative Study Of The Anticonvulsive Effects Of 4-Amino-2-Methyl-Cantharidinimide And Other Three Derivatives And The Influence On GABA And GABA_C Receptor ρ 2mRNA Expression

Objective To compare the anticonvulsive effects, toxicity and pharmacodynamic pa-rameters of AMC, MANC, CANC, and CBNC. The model of the electrical stimulation of sciatic nerve and penicilin (PNC) coordination inducing convulsion in rat was es-tablished to observe the influence of AMC on the convulsion rat's cerebral cortex elec-troencephalogram (EcoG), evoked potential (CEP), the contents ofγ-aminobutyric acid (GABA),ρ2mRNA and GABAC receptor.Methods (1) We used the model of MES with Valproate (VPA) as positive control to determine anti-maximal electroshock seizure effects of 4 different kinds of cantharidin 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 accord-ing to the Bliss's method, then we compared their potency and efficacy. The experiment of sezure induced by Picrotoxin(PTX) was to compare the convulsion seizure latency and anti-convulsion rate of VPA, AMC and other derivatives. (2) The roll club method was adopted to analysis the toxic effect of intragastrical (ig) AMC, VPA, MANC, CANC, and CBNC. Then we calculated TD50 according to the Bliss's method, finally we compared toxic effects of different medicines. (3) Pharmacodynamic parameters of AMC, VPA, MANC, CANC, and CBNC were assayed by means of Pharmacological Effective Method. (4) The model of electricity stimulating sciatic nerve and penicillin (PNC) coordination was used to induce convulsion in rat, with Valproate(VPA) taken as the positive control. The convulsion seizure latency and Racine behavior study gradua-tion was considered as index to evaluate drug efficacy. RM6240C multi-channel biology signal gathered processing system synchronization recorded EcoG and CEP of convul-sion rats, after intragastric AMC(88mg·kg-1,22mg.kg-1), and the anti-convulsion effects, influence on the epileptiform discharge latenperiod, frequency, the highest amplitude and CEP amplitude were investigated. (5) Real time polymerase chain reaction (RT-PCR) was adopted to determine the expression of hippocampus GABAc receptorρ2mRNA. (6) The contents of GABA and the protein expression of GABAc receptor in hippocampus region of epileptic rats were determinated by immunohistochemistry technology.Results (1) AMC, VPA, MANC, CANC, and CBNC could antagonize MES in mice dose-dependently with anti-convulsion rate 100%,100%,90%,90%,100%respectively; The ED50 of them were AMC(22mg·kg-1), VPA (14mg·kg-1), MANC(39mg·kg-1), CANC(49mg·kg-1), CBNC(53mg·kg-1). In the antagonizing PTX experiment, AMC, VPA, MANC, CANC, and CBNC all had aiti-convulsion effects, and the seizure lateny was prolonged as well. MANC, CANC, CBNC, VPA had the significance differ-ence(P＜0.01) compared to PTX model group, and AMC had the significance differ-ence(P＜0.05). The anti-convulsion rate of the drugs were respectively VPA(90%), CANC(80%), MANC(70%), CBNC(60%), AMC(40%). (2) AMC, VPA, MANC, CANC, and CBNC all had CNS toxic effect, and TD50 of them were respectively AMC(545mg·kg-1), VPA(782mg·kg-1), MANC(530mg·kg-1), CANC(402mg·kg-1), CBNC(489mg·kg-1). (3) The results of pharmacodynamic parameters indicated the minimum effect dosage of different medicines from low to high were VPA, AMC, CANC, MANC, CBNC; The peak time of AMC was 12min, while VPA was 3h and other drugs got to their peak time after 50 min. The t1/2 (ED) of AMC was 3h which was the biggest among the drugs, and the persist time of AMC and VPA were similar which were 15h or so; The t1/2 (ED) and persist time of other drugs were all shorter than AMC and VPA. Elimination Constant of AMC were smallest of all. (4) AMC1 group (323.8±19.5),AMC2(296.1±23.0) group and VPA group(345.3±21.4) could inhibit epileptiform behavior, and prolong the seizure latency obviously, compared to the PNC group(161.6±20.7) with the significance difference(P＜0.01). AMC1(88mg·kg-1), AMC2(22mg·kg-1) could prolong the epileptiform discharge latency period, reduce the frequency of epileptiform discharge, and lower the highest wave amplitude and the evoked potential amplitude compared with the PNC group with the significance differ-ence(P＜0.01) after intragastricing. (5) The results of comparsion in GABAC receptor p2 gene in RT-PCR showed that the expression of GABAC receptor in AMC1(1.38±0.99), AMC2(1.21±0.13) and VPA(4.93±0.43) were higher than PNC group(0.65±0.09) with the significance difference(P＜0.01). (6) The influence of AMC and VPA on GABA in the brain of seizure rats in hippocampus spot:GABA expression quantity of PNC group(51320.4±11005.4) was smaller than the normal group(80319.2±15322.3) with the significance difference(P＜0.01); GABA expression quantity of AMC1(100773.1±11853.9), AMC2(98714.3±14028.6) and VPA(120531.0±10950.8) were higher than the normal group and the model group in the value, with statistics significance(P＜0.01); The influence of AMC and VPA on GABAC receptor protein expression in the brain of seizure rats in hippocampus spot:AMC1 group(3772.3±13879.6) AMC2 group (69483.4±10648.2) and VPA group (97316.2±12060.8) had statistics significance(P＜0.01) compared with PNC group (32805.0±13826.6)Conclusions (1) AMC could antagonize MES, and its anticonvulsive potency was similar to VPA and CBNC. The titre intensity of AMC was similar to VPA, and stronger than other three derivates. MANC, CANC, CBNC and VPA had obvious effects to an-tagonize PTX, while AMC showed a little effect of anticonvulsion in this experiment. (2) AMC, VPA, MANC, CANC, and CBNC all had CNS toxicity. The TC50 value of VPA was the biggest among the drugs which indicated its toxicity was the smallest. The TC50 value of AMC was bigger than other three derivates. However, the TC50 value of CANC was the smallest which indicated its toxicity as the biggest. (3) Among the 5 drugs, the peak time of AMC was the fastest. It had the longest T1/2 (ED), the persist time, and the slowest elimination. The lowest effect dose of AMC was more than VPA, but less than other 3 derivates. (4) AMC could inhibit epileptiform behavior, and prolong the seizure latency induced by PNC obviously. It also could prolong the latent period of epilepti-form discharge, and reduce the frequency of epileptiform discharge, the highest wave amplitude and the evoked potential amplitude, which had significant anticonvulsive ef-fective. (5) AMC could enhance the GABAC receptor p2mRNA expression in hippo-campus area. AMC could increase rats GABA content and the GABAC receptor protein content in hippocampus, which indicated AMC could antagonize sezure not only from the genetic level but also protein level.