The Effect And Mechanism Of The Micro RNA-16 Regulation On The SERT In Chronic Epileptic Rats Induced By Pilocarpine

ObjectsTo explore the potential mechanism that of SERT and mi RNA-16 in epilepsy, we observe the seizures, the change of the proteins related to the neuron proliferation and apoptosis, and the expression of the SERT and micro RNA-16 in pilocarpine-induced chronic epileptic rats.Methods1. Experimental animals and groups: 30 male adult Sprague-Dawly rats were divided into 2 groups,vehicle control group(n=6) and pilocarpine(PILO) treated group(n=30). The success SE of PILO group then was divided into 2 groups:SE+Paroxetine group(Paroxetin 5mg/kg intraperitoneal injection) and SE group(NS 5mg/kg i.p.). 2. Establish epileptic model: intraperitoneal injection PILO( 30mg/kg) after 16-18 h LICL( 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), if seizures not to the success standard,can add PILO(15mg/kg) per 15 minute. 3. Drug intervention: intraperitoneal injection Paroxetin( 5mg/kg) for 4 weeks after 4 weeks in the SE. Rats’ spontaneous recurrent seizures(SSRs) were observed according to Veliskova and Goffin’s criteria in the 5 to 8 weeks. 4. Materials and target detection: Collect rats brain specimen after the 8th week by heart perfusion, One part of the hippocampal tissue was use to TUNEL/HRP to observe the apoptotic cells, another part of the hippocampus and raphe nucleus tissue was use to observe SERT、bcl-2、BDNF and mi RNA-16 by Western-blot and RT-PCR.Results1. Behavioral results:(1) Successful rate and survival rate of pilocarpine-induced epileptic model: the successful rate and the survival rate were 77.78% and 35.71%.(2) SSRs in chronic period : The rats seizure of the SE and SE+Paroxetine groups were observed between 5-8 weeks, most attack stage are between Racin I-IV. After intraperitoneal injection Paroxetin the attack frequence and stage were obviously reduced. 2. TUNEL/HRP: ①To campare with Vehicle group,the numbers of the apoptotic cells in Hippocampus were increased and the intensity of colour was enhanced, After injection PILO.②After intraperitoneal injection Paroxetin, the numbers of the apoptotic cells were reduced and the intensity of colour was weakened,compared to the SE group. 3. Western blot : ①To compare with Vehicle group, in the SE groups, the expression of SERT protein had no changes in the raphe nucleus,but was decreased in the hippocampus; To compare with SE group, in the SE+Paroxetine groups, the expression of SERT protein was decreased in the raphe nucleus,but was increased in the hippocampus; ②To compare with Vehicle group, in the SE groups, the expression of BDNF and Bcl-2 protein was decreased in the hippocampus; After injection Paroxetin injection, the expression of Bcl-2 protein were increased, but the BDNF had no changes. 4. RT-PCR : There are different expression changes of Mi RNA-16 in the chronic epileptic rats before and after Paroxetine treatment. ①To compare with Vehicle group, in the SE group, The mi RNA-16 level decreased obviously in Raphe Nucleus, and increased obviously in hippocampus. ② To compare with SE group, in the SE+Paroxetine group, The mi RNA-16 level there is no change of statistical significance in Raphe Nucleus, but decreased after paroxetine treatment.Conclusions1. The seizure of the chronic epileptic rats can be alleviated by Paroxetine treatment. 2. In chronic epileptic rats,the numbers of the apoptotic cells in the hippocampus were increased,may be related to the decrease of Bcl-2 and BDNF; the SSRI can alleviatedthe apoptotic of hippocampus cells by increase the Bcl-2, and may promote the hippocampus neurogenesis by increase the BDNF. 3. In chronic epileptic rats,the change of SERT in hippocampus and raphe nucleus are different,and SSRI can increase the 5-HT in the hippocampus SERT by special regulation to SERT. 4. There are different expression changes of Mi RNA-16 in the chronic epileptic rats before and after Paroxetine treatment, it may participate in the regulation of SERT, the Specific mechanism needs further research.