| Objects To explore the potential mechanism of 5-HT6 receptor antagonist in epilepsy, the spontaneous recurrent seizures of pilocarpine-induced chronic epileptic rats were observed after SB-271046 intervention, the expression of Glutaminase and glutamic acid decarboxylase was detected via WB and RT-PCR and the level of glutamate and ?-aminobutyric acid wad determinated by HPLC.Methods 1. Experimental animals and groups: 50 male adult Sprague-Dawly rats were divided into 2 groups, vehicle group(n=10) and pilocarpine(PILO) treated group(n=40).Those succeed in SE then were divided into 5 groups:epileptic group(EP), control group(DMSO, injected to lateral ventricle(LV)with DMSO, 4ul), 10ug-SB271046 group(10ug-SB, injected to LV with SB-271046, 4ul), 20ug-SB271046 group(20ug-SB,injected to LV with SB-271046, 4ul), 30ug-SB271046 group(30ug-SB, injected to LV with SB-271046, 4ul). 2.Pilocarpine-induced epileptic model:PILO(30mg/kg) was intraperitoneally injected after Li Cl(127mg/kg) administered,then behavioral changes were continuously observed and those succeed in status epilepticus were included to further experiment.. Rats' spontaneous recurrent seizures(SSRs) were observed according to Veliskova and Goffin's criteria. 3.Intervention: SB-271046 or DMSO was microinjected into right LV in chronic phsae,with the help of brain solid positioner and watson ?rat brain stereotaxic atlas? 4. 4 weeks after SE,the hippocampus of epileptic rats were collected to detect the expression of Glutaminase, GAD65?GAD67 via western blot and RT-PCR. 5. Determination of glutamate and GABA in hippocampus via HPLC.Results 1.Successful rate and survival rate of pilocarpine-induced epileptic model: In the first part experiment, the successful rate and the survival rate were87.5% and 65%. In the second part experiment, the successful rate and the survival rate were 92.5% and 60%. In the third part experiment, the successful rate and the survival rate were 77.5% and 40%. 2. SSRs in chronic phase: all groups were observed in chronic phase. After the injection of SB-271046,both the frequences and stage of SSRs were decreased. 3.Western Blot:(1)The level of GAD65 decrease significantly in EP and EP+DMSO(p <0.05,Vehicle versus EP or EP+DMSO). After the injection of SB-271046,the level of GAD65 was increased when compared with EP group(p <0.05 EP vs 20 ug SB, 30 ug SB),while there was no significant change in 10ug-SB(p >0.05);(2)When compared with vehicle group, the level of GAD67 decrease significantly in EP and EP+DMSO(p <0.05),the level of GAD67 was increased after SB-271046 intervention(p <0.05 EP versus 10 ug SB, 20 ug SB, 30 ug SB), while there was no significant change between EP and EP+DMSO(p >0.05);(3)When compared with vehicle group, the level of GLS increase significantly in EP and EP+DMSO(p <0.05),after SB-271046 intervention it was decreased,especially in 30 ug SB group(p <0.05 EP versus 30 ug SB). 4.RT-PCR:(1)The level of GAD65 m RNA and GAD67 m RNA decrease significantly in EP and EP+DMSO when compared with vehicle group(p <0.05)after SB271046 intervention both GAD65 and GAD67 increase(p <0.05)(2) The level of GLS m RNA increase significantly in EP and EP+DMSO when compared with vehicle group(p <0.05)after SB271046 intervention it was decrease,especially in 20ug-SB and 30ug-SB group. 5.HPLC: Compared to the vehicle, the ratio of GLU/GABA significantly increased in EP group. With the injection of SB271046, the ratio of GLU/GABA decrease, but without statistical significance.conclusion1. 5-HT6 receptor antagonists SB-271046 can alleviate spontaneous seizures of pilocarpine-induced chronic epileptic rats. 2. 5-HT6 receptor antagonists may modulate the imbalance of GLU and GABA via regulate glutamic acid decarboxylase(GAD65, GAD67), glutaminase(GLS). |
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