Ghrelin Protects Against Cell Death Of Hippocampal Neurons In Pilocarpine-Induced Seizures In Rats

Epilepsy is a serious neurological disorder in human beings.At present,there are about 9 million patients with epilepsy in china.About 25%of patients suffer with intractable epilepsy.Temporal lobe epilepsy(TLE) presents the most prevalent refractory epilepsy and its pathogenesy still remains obscure.It is difficult to gain significant curative effects through administration of antiepileptic drugs(AEDs) and operating.The alteration of behavior,electroencephalogram(EEG) and the hippocampal neuronal injury in pilocarpine-induced seizures in rats is similar to that in TLE patients,so pilocarpine-induced seizures have been one of the most frequently used models to research SE and TLE.As is known,seizures can result in the hippocampal neuronal injury,but the molecular mechanisms of neuronal injury are still far from clear.Since the etiopathogenisis of epilepsy is very complicated,it is very important to explore the new AED and the potential antiepileptic mechanism.Ghrelin,a new 28-amino-acid peptide,is mainly secreted by the stomach.It acts on the pituitary and the hypothalamus to stimulate the release of growth hormone and promote adiposity and appetite via its fully functional receptor,GHSR-1α.Besides,it was suggested that ghrelin inhibited apoptosis through multiple mechanisms in some cell types.Recently,the neuroprotective effects of ghrelin were researched deeply.It was found that ghrelin could inhibit the neuronal damage induced by glucose-oxygen deprivation in hypothalamus and protect neurons in the hippocampus and cortex against cerebral ischemia/reperfusion.Cumulative data showed that the anti-apoptotic effects of ghrelin might be related to activation of not only the PI3K/Akt signaling pathway,but also the mitochondrial pathway. Ghrelin is involved in the anti-apoptotic and neuroprotective effects,but it is little known whether ghrelin exerts biological effects on hippocampal neuronal injury in pilocarpine-induced seizures.In this study,we extensively studied the molecular biological mechanisms of the neuronal injury after pilocarpine-induced seizures,and explored the antiepileptic effects of ghrelin and its potential mechanisms.PARTⅠA STUDY OF ELECTROENCEPHALOGRAM AND PATHOBIOLOGY IN LITHIUM-PILOCARPINE-INDUCED SEIZURES IN RATSObjectiveTo investigate the alteration of behavior,electrophysiology and pathology in lithium-pilocarpine-induced seizures in rats,and to explore the neuronal injury of hippocampus.MethodsAdult male Wistar rats were given lithium-pilocarpine intraperitoneally to induce SE.The change of behavior in rats was observed.Seizures were allowed to last for 60 min and then were terminated by administration of diazepam.EEG was recorded at 2 h after seizures.The hippocampal neuronal damage was observed at 72 h after pilocarpine-induced seizures by using HE and Nissl staining.Results1.93.3%of the rats were induced to develop SE after administration of lithium and pilocarpine(according to Racine,the rats showing stage 4 or 5 convulsive seizures were considered to develop SE successfully ),and the death rate in acute phase was 6.7%.2.EEG in cortex and hippocampus of rats showed accumulated spike waves at 2 h after seizures(in acute phase).3.HE and Nissl staining showed the neuronal damage in hippocampal CA1 and CA3 regions at 72 h after pilocarpine-induced seizures.The surviving neurons showed round and palely stained nuclei,meanwhile,the dead neurons in hippocampus showed pyknotic nuclei and shrunken plasma body.ConclusionsLithium-pilocarpine could induce acute seizures(SE) according to the alteration of behavior and EEG.Seizures induced by lithium-pilocarpine caused hippocampal neuronal damage in rats.PARTⅡALTERATION OF THE PI3K/Akt SIGNALING PATHWAY AND APOPTOSIS-RELATED PROTEIN LEVELS OF HIPPOCAMPUS IN PILOCARPINE-INDUCED SEIZURES IN RATSObjectiveTo detect the alteration of the PI3K/Akt signaling pathway and apoptosis-related protein(Bax,Bcl-2 and activated caspase-3) levels of hippocampus in pilocarpine-induced seizures in rats,so to explore the molecular mechanisms of hippocampal neuronal damage in seizures.MethodsAdult male Wistar rats were divided randomly into control group,2 h,8 h,16 h, 24 h and 72 h after seizures groups.The levels of the phosphorylated phosphatidylinositol-3-kinase(phospho-PI3K p85),the phosphorylated Akt (phospho-Akt),Bax,Bcl-2 and the activated caspase-3 in hippocampus in each group were detected by using western blot.Results1.Immunoreactivity of phospho-PI3K p85 and phospho-Akt was significantly higher at 2 h and 8 h after seizures(p＜0.05),and lower 8 h later than that in control(p＜0.05).2.Bax showed a significantly higher expression in pilocarpine groups than in control (p＜0.05).In contrast,immunoreactivity of Bcl-2 in the hippocampus was decreased after seizures(p＜0.05).Moreover,the active cleavage product of caspase-3 appeared at 24 h after seizures,and increased persistently until 72 h(p＜0.05).ConclusionsOur results showed that pilocarpine-induced seizures inhibited the activation of the PI3K/Akt signaling pathway and regulated the mitochondrial apoptosis pathway. Caspase-3 was activated finally to lead to neuronal death.We verified the molecular mechanisms of hippocampal neuronal injury induced by seizures,which provided theoretical evidence for the neuroprotective therapy in epilepsy.PARTⅢGHRELIN PROTECTS AGAINST CELL DEATH OF HIPPOCAMPAL NEURONS IN PILOCARPINE-INDUCED SEIZURES IN RATSObjectiveTo study the protective effects of exogenous ghrelin on hippocampal neurons after seizures induced by pilocarpine,and to investigate the effect of ghrelin on the PI3K/Akt signaling pathway and the mitochondrial apoptosis-related protein(Bax and Bcl-2) and activated caspase-3,so to explore the underlying mechanisms of neuroprotective effects of ghrelin in pilocarpine-induced seizures in rats.MethodsAdult male Wistar rats were randomly divided into 4 groups for treatment:control, pilocarpine,pilocarpine + saline and pilocarpine + ghrelin.We performed Nissl staining at 72 h after seizures to examine the number of surviving neurons in hippocampal CA1 and CA3 regions in rats.The expression of GHSR-1αmRNA and protein at 24 h after seizures in rat hippocampus was detected respectively by real-time quantitative-PCR and Western blot.The levels of phospho-PI3K p85, phospho-Akt,Bax,Bcl-2 and the activated caspase-3 in hippocampus were detected by western blot,and the expression of Bax and Bcl-2 at 24 h after seizures in hippocampus was also detected by immunohistochemistry. Results1.Nissl staining showed the neuronal damage in hippocampal CA1 and CA3 regions at 72 h after pilocarpine-induced seizures.The surviving neurons showed round and palely stained nuclei,meanwhile,the dead neurons in hippocampus showed pyknotic nuclei and shrunken plasma body.Few cells showed the characters of dead neuron after ghrelin adminiatration.2.The number of surviving neurons at 72h after seizures(105.8±10.8;116.5±12.9) was decreased significantly as compared with control(241.5±24.5;265.5±24.3) (p＜0.05),moreover,ghrelin treatment significantly attenuated the neuronal loss induced by seizures(201.3±17.7;217.5±20.0)(p＜0.05).There was no significant difference in neuronal loss between the pilocarpine and pilocarpine +saline groups (103.75±9.8;112.7±10.3).3.GHSR-1αmRNA and protein levels showed no significant changes at 24 h after seizures as compared with control and ghrelin treatment had no effect on the expression(p＞0.05).4.After ghrelin administration,the decrease in phospho-PI3K p85,phospho-Akt and Bcl-2 levels induced by seizures was reversed significantly(p＜0.05),and ghrelin reversed the increased Bax and activated caspase-3 levels at 24 h after pilocarpine treatment.There was no significant difference between pilocarpine group and pilocarpine + saline group.5.Immtmohistochemistry:Bcl-2 staining in hippocampal CA3 region was decreased at 24 h after pilocarpine treatment(p＜0.05),in contrast,Bax staining was increased (p＜0.05).Ghrelin pretreatment reversed the decreased Bcl-2 staining and the increased Bax staining in hippocampal CA3 region(p＜0.05),with no effect by saline.ConclusionsOur results implied that ghrelin had neuroprotective effects which were exerted through promoting the PI3K/Akt signaling pathway and inhibiting the mitochondrial apoptosis pathway.Ghrelin could be a potential benefit treatment for the hippocampal neuron demise caused by pilocarpine-induced prolonged seizures.The study of the neuroprotective effects of ghrelin may provide novel insights into the therapy of epilepsy. SignificanceThe study deeply researched the effects of the PI3K/Akt signaling pathway and the mitochondrial apoptotic pathway on hippocampal neuronal injury in pilocarpine-induced seizures in rats.We verified that ghrelin exerted its neuroprotective effects in seizures through promoting the PI3K/Akt signaling pathway and inhibiting the mitochondrial apoptosis pathway at the first time.Although we did not find that ghrelin could lessen the degree of pilocarpine-induced seizures,the results illuminated the molecular biological mechanisms of hippocampal neuronal injury induced by seizures and the neuroprotective mechanisms of ghrelin,which provided the novel clue for the neuroprotective therapy in epilepsy.