| Epilepsy is a chronic brain disease that seriously endangers human health and the morbidity is second only to cerebrovascular disease in neurological diseases.About 9 million people in China are affected by epilepsy and 30 percent are diagnosed with refractory epilepsy.Among them,the temporal lobe epilepsy(TLE)is the most common type,and rat epilepsy model caused by lithium chloride-pilocarpine can well simulate the human temporal lobe epilepsy.Salubrinal,many scholars at home think it might be a good neuroprotective drug,selectively inhibits elf2 a to phosphorylate.In the case of cell stress,eelf2 a phosphorylation can keep the cell's internal environment relatively stable.In addition,the elf2 a phosphorylation can reduce the synthesis of intracellular proteins and increase the expression of ATF4.For now,cell apoptosis may have three main signaling pathways:Firstly,the mitochondrial pathway;Secondly,pathway of death receptors;Thirdly,The mitochondrial pathway.The Boycott and other studies[1]have found that salubrinal can effectively inhibit cell apoptosis caused by the stress of endoplasmic reticulum,but the mechanism is not yet clear.Studies have shown that,in many stressor,Parkin can protect cells and inhibit cell death(4,5,6).But so far,the role of salubrinal in neuronal apoptosis is not clear,whether it can reduce the apoptosis of epilepsy rat hippocampal neurons caused by lithium chloride-pilocarpine are not sure.To observe apoptosis of hippocampal neurons after seizures,and examine the phosphorylation of salubrinal and mitochondrial autophagy level mediated Parkin,we seem the epilepsy rat model caused by lithium chloride-pilocarpine as the research object and the salubrinal as the intervention of the model.Finally,clear and definite the apoptosis of rat hippocampal neuron and the protection of salubrinal on apoptosis and its mechanism.This research is divided into two parts.PART ?THE PROTECTIVE EFFECT AND MECHANISM OF SALUBRINAL ON HIPPOCAMPUS APOPTOSIS IN EPILEPTIC RATSObjectiveTo study the protective effect of salubrinal on apoptosis and injury of hippocampal neurons and to investigate the protective mechanism of salubrinal on hippocampal neurons.MethodsSixty adult male wistar rats were divided into control group,PILO group(a rat model of epilepsy induced by lithium pilocarpine),Salubrinal(Sal)+PILO group and Sal group.In the control group and the treatment group,the apoptosis and damage of hippocampal neurons were observed by TUNEL and immunohistochemistry after 24h of the status epilepticus.The expression of P-elf2a,elf2a,LC3 and Parkin were analyzed by western blot.ResultsFirstly,Compared with the control group,the apoptosis of hippocampal neurons were found obviously in PILO group.Secondly,the apoptosis of hippocampal neurons in group Sal+PILO were significantly lower than those in group PILO.Thirdly,compared with PILO group.the expression of P-elf2a in hippocampus of Salubrinal+PILO group was significantly increased(P<0.05).The elf2a expression was not affected while the expression of LC3II and Parkin were significantly higher To investigate the alteration of behavior in lithium-pilocarpine induced seizures in(P<0.05).ConclusionFirstly,there were apoptosis in hippocampal neurons of epileptic rats induced by lithium chloride and pilocarpine.Secondly,salubrinal can reduce the apoptosis of hippocampal neurons in epileptic rats.Salubrinal may effectively reduce the damage and apoptosis of hippocampal neurons in epileptic rats by rising up Parkin level and increasing the level of autophagy.PART ?THE PROTECTIVE EFFECT OF MITOCHONDRIAL AUTOPHAGY MEDIATED BY PARKIN FOR HIPPOCAMPUS APOPTOSIS IN EPILEPTIC RATS INDUCED BY LITHIUM PILOCARPINEObjectiveMake sure the protective effect of mitochondrial autophagy mediated by Parkin for hippocampus apoptosis in epilepsy rats induced by lithium pilocarpine.MethodsThe hippocampal CA1 region were transfected with Parkin overexpression recombinant adenovirus.Establishing temporal lobe epilepsy rats model by intraperitoneal injection of lithium pilocarpine after 7 days.A total of sixty healthy male wistar rats were divided into 4 groups,the wild type control group(Ctr group);epilepsy model group induced by Pilocarpine(PI group);epilepsy model group induced by Pilocarpine and Parkin overexpression adenovirus(PIPO group)and overexpression of Parkin adenovirus group with normal saline(PO group).Observing and comparing the changes of hippocampal neurons apoptosis between Ctr group and the treatment group after 24h status epilepticus.Western blot analysis Parkin and LC3 expression.Compared with the autophagic bodies and apoptotic bodies by transmission electron microscope.ResultsFirstly,compared with the Ctr group,the number of apoptosis in PI group increased significantly while the number of apoptosis in PIPO group was significantly reduced compared with PI group(P<0.05).Secondly,compared with Ctr group,under transmission electron microscope,the hippocampal neuron mitochondrial ultrastructure was damaged,the number of bodies of autophagosomes and apoptosis increased significantly in PI group;compared with PI group,the damage of hippocampal neurons in mitochondrial ultrastructure were decreased and the number of bodies of autophagosomes were increased.However,the number of apoptotic bodies was significantly reduced in PIPO group.Third,western blot experimental analysis showed that compared with Ctr group,the expression level of Parkin decreased while LC3II/LC3I ratio increased in PI group(P<0.05).Compared with PI group,Parkin and LC3II/LC3I ratio were significantly increased in PIPO group(P<0.05).ConclusionFirstly,accompanied by obvious apoptosis and mitochondrial damage,the Parkin expression level was decreased in epilepsy rats induced by lithium pilocarpine.Secondly,Overexpression of Parkin can reduce the apoptosis of epileptic rats and the injury of mitochondria.What's more,mitochondrial autophagy mediated by Parkin has a protective effect on the apoptosis of epileptic rats induced by lithium pilocarpine. |
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