The Study Of Protective Mechanism Of 5-HT6 Receptor Antagonist SB271046 On Hippocampal Neurons Of Epileptic Rats

Objects we went to observe the changes of autophagy level in different period after epileptic seizures with lithium chloride – Pilocarpine-induced epileptic rat model.;we investigate the effects of 5-HT6 receptor antagonist SB271046 on JNK pathway,and observe the changes of autophagy and apoptosis of hippocampal neurous and neuronal degeneration.We aimed to explore protective mechanism of 5-HT6 receptor antagonist on hippocampal neurons of epileptic rats.Methods 1. Experimental animals and groups:(1)First group:30 male adult SpragueDawly rats were divided into 2 groups,vehicle control group(n=6) and pilocarpine(PILO) treated group(n=24). The success SE of PILO group then was divided into 5 subgroups according to the different time after seizures(2h、6h、12h、24h).(2)Second group: 40 male adult Sprague-Dawly rats were divided into 4 groups, vehicle control group(n=10),epilepsy group(n=10), epileptic rats administrated DMSO(n=10)and epileptic rats treated with SB271046(n=10). 2. Establish epileptic model: intraperitoneal injection PILO( 30mg/kg) after 16-20 h Li Cl( 127 mg/kg) was administered, observed behavior changes continuously 30 min to decide the success of status epilepticus(SE), according to Racine classify(seizures up to IV and continue more then 30 min was supposed SE). 3.Method of administration:use brain solid positioner,the recording electrode was inserted in right frontal lobe cortex and SB-271046 or DMSO was microinjected into right LV in 1h. 4. JNK signaling pathway related proteins including c-jun 、 JNK 、p-JNK,in hippocampus were detected by western blot; Then observe the changes of autophagy and apoptosis by detected LC3II/LC3 I 、 beclin1 、 bax and bcl-2 with western blot.the m RNA level of beclin1、bax and bcl-2 were tested by Real-time PCR. 5. Through the Fluoro- Jade C(FLC) dyeing neuron degeneration after seizures(24h).Results 1.The level of LC3II/LC3 I increased gradually after seizures(2h、6h、12h、24h) in hippocampus of rats. 2.Epilepsy group compared with vehicle group,the expression of c-jun and p JNK/JNK protein was obvious increased,the antagonist of 5-HT6 R SB271046 decrease c-jun and p JNK/JNK protein expression after administating SB-271046. 3.Autophagy indicators: epilepsy group compared with vehicle group,the expression of LC3II/LC3 I and beclin1 protein was obvious increased,the antagonist of 5-HT6 SB-271046 decreased LC3II/LC3 I and beclin1 protein expression, Detection of Beclin1 m RNA and protein had same change trend. 4.Apoptosis indicators: Compared with vehicle group,the expression of bax protein in epileptic group was obvious increased, but the expression of bcl-2 protein was significant decreased.the antagonist of 5-HT6 R SB271046 decrease bax protein expression after administating SB-271046. Detection of bax m RNA and protein had same change trend. 5. Neuron degeneration:The FLC dyeing found group degeneration of neurons in hippocampal CA1 area epilepsy significantly higher than normal group. 5- HT6 receptor antagonist group no significant change in the epilepsy group.Conclusions 1.Status epilepticus occurred within 24 hours of autophagy level increased gradually in the hippocampus 2.5- HT6 R antagonists SB-271046 may through the JNK/ c jun signaling pathways, it have protective effect on hippocampal neurons by inhibit autophagy and apoptosis in the hippocampus of epileptic rats. 4. 5- HT6 R antagonists SB- 271046 may not have obvious effect on epilepsy degeneration of rat hippocampal neurons.