Study Of Adenosine On The Expression Of Phospho-mtor In Pentylenetetrazole-induced Acute Status Epilepticus

Background:Epilepsy is the second most popular disease in the nervous system,which is a chronic and recurring attack,and some are intractable epilepsy.It's causing a lot of physical and mental harm to the patient,and burden to the patient's families,society.Humans have to struggle for many years,up to now,the study of the epileptic mechanism are mainly concentrated in the neurotransmitter,ion channels,glial cells,synapses,genetic and immune abnormalities,abnormal function of the neural network theory.Although we have done a lot of effort on the research and developed some new antiepileptic drug to control the disease,but there are a lot of people with refractory epilepsy and recurrent seizure under the condition.Therefore,we still need to carry out further research on the mechanism of epilepsy to find new treatment points,and develop new therapeutic targets and drugs.Current study found that mTOR and adenosine play an important role in epilepsy occurrence,development and endness.They are the research hotspot in recent years.The study found that the adenosine in the brain increased significantly when seizures occured,and system or local application of adenosine or adenosine kinase inhibitor can effectively control the seizures,and adenosine receptor gene knockout mice can affect the seizures,all these indicated that adenosine played role in the mechanism of epilepsy.Up to now,the anticonvulant effects of adenosine invovled the neurotransmitter release,synaptic plasticity,ion channel and the postsynaptic membrane stability and so on.Adenosine A1 receptor mediated antiepileptic effect,but A2a receptor activation may be opposite,and other receptors were not clearly.At present,although the adenosine has been applied in cardiology,but because of side effects on cardiovascular,respiratory system of the large number of adenosine in systemic medication,it cannot effectively applied to the clinical prevention and treatment of epilepsy.Then a variety of local enhancement adenosine therapy were invented which could enhance local lever of adenosine in the brain and without the side effects of system medication.It is expected to be a new antiepileptic therapy.mTOR was first found in the tumor.It was found that rapamycin,in as mTOR inhibitor,could effectively relieve the seizures of patients with tuberous sclerosis,which opened the door of the research of mTOR involved in seizures.Moreover,rapamycin could reverse the abnormal pathological changes of the central nervous system caused by epilepsy,which indicate that mTOR played an important role in the pathogenesis of epilepsy.It's antiepileptic effects mediated by ion channel,cell autophagy and inflammation etc..It's downstream factors,p70S6 and 4e-Bpl,could control protein translation to affect cell metabolism and apoptosis.The new mTOR inhibitor,ivermox,has been gradually applied to clinical practice,with better tolerance and less side effects than rapamycin,and it is expected to be a new therapeutic drug for epilepsy.In previous studies,adenosine was found to affect mTOR expression through PI3K/Akt in tumor and mTOR may also be involved in energy metabolism.But the there is no research on the relationship of adenosine and mTOR in epilepsy.Through reviewing the previous literature,we found that adenosine could lead to AMP/ATP ratio change,and then to activate AMPK pathway,which was mTOR upstream inhibitory factor.Elevated adenosine or inhibited mTOR could effectively suppress seizures.Based on this,we thought that adenosine in epileptic seizures may affect the expression of mTOR.So,we design experiment to explore the relationship.The study could reveal the pathogenesis of epilepsy,and combines the tedious epilepsy mechanism effectively.Objective:Based on the previous research results,we found that there is theoretical link between adenosine and mTOR.This study examine the relationship of ADO with mTOR in a Wistar rat model of pentylenetetrazole(PTZ)-induced acute status epilepticus and to explore the mechanism in it..Methods:The mature male Wistar rats(200-220g)was divided into blank control group,PTZ induced epilepsy group and ADO group.There were 8 rats in each group.The rats were divided into three groups to study the effect of ADO on mTOR:the vehicle control group,the PTZ group,and the ADO+PTZ group.The rats were intraperitoneally injected with ADO(200 mg/kg,Sigma)or vehicle,followed 30 minutes later by PTZ(55-75 mg/kg,Sigma)or vehicle.Seizure behaviors were graded according to the modified Racine scale:stage 1,mouth or facial movements;stage 2,head nodding;stage 3,unilateral or bilateral forelimb clonus;stage 4,bilateral for limb clonus and rearing;and stage 5,rearing and falling.The initial dose of PTZ to cause seizure(55 mg/kg)was increased by 5 mg per 5 min until sustained generalized motor seizures(stage 4 or 5)were induced.Seizures were terminated by injection with 10%chloral hydrate(3 ml/kg)60 min after the treatment was administered.Eight surviving rats per group that successfully reached stages 4 and 5 were sacrificed by decapitation 3 h after status epileptics(SE)treatment was administered.Their brains were then removed for Western bloffing and immunohistochemistry.Compound C was used to inhibit the expression of AMPK.The application of Compound C in combination with PTZ was lethally toxic to the rats,with the majority(19/20)of rats succumbing to death.We therefore investigated the effects of a combination of ADO and Compound C by intraperitoneally injecting Compound C(20 mg/kg,Selleck)30 min before ADO.In order to further study the relation between the aging effects of intraperitoneal injection of ADO for mTOR,we further respectively detect changes in p-mTOR in rat hippocampus using western technology at 60min,120min,180min and 360min after ADO injection.Similarly,immunohistochemistry detection in hippocampal CA1 area of CA3 District p-mTOR groups,further verify the experimental results.Results:1.Effects of drugs on rat behaviorsPTZ(55-75mg/kg)can lead to acute status epilepticus.The rats appear drowsy(100%)and asthma(23/86)after injection of ADO(200mg/kg)intraperitoneally,which predicted a big side effects of ADO in this dose.We did not detect the effect of ADO on PTZ induced seizure behavior this time.Compound C could induce sustained muscular tremors like wave.Wistar rats after Compound C were extreme intolerance to PTZ-induced seizure with a high mortality rate(19/20).2.Relationship between ADO and mTORThe protein levels of total mTOR,phospho-mTOR,phospho-S6,and p-actin in rat hippocampi of the control,PTZ,and ADO+PTZ groups after SE was induced were detected via Western blot assay.Compared with those in the control group,the ratios of phospho-mTOR to total mTOR and phospho-S6 to ?-actin were increased in the PTZ group.Compared with that in the PTZ group,these ratios were decreased in the ADO+PTZ group.These ratios significantly differed among the groups(P<0.05,n=8 per group;Fig.1).The respective average gray values(average ± SD)of mTOR in the control,PTZ,and ADO+PTZ groups were 109.9 ± 8.8/297.2 ± 25.5,311.7 ±27.3/369.5 ± 32.1,and 129.17 ± 10.1/276.0 ± 23.5 in CA1/CA3 region of the hippocampus,as revealed by immunohistochemistry.Significant differences were observed between the vehicle control group versus the PTZ group and between the PTZ group versus the ADO+PTZ group(P<0.05,n= 8 per group).3.ADO Affects mTOR via the AMPK PathwayExpression levels of total mTOR,phospho-mTOR,phospho-S6,and ?-actin in the control,ADO,and Compound C+ADO groups were determined by Western blot assay.Compared with that in the vehicle control group,the mTOR expression decreased in the ADO group.The introduction of Compound C was found to reverse this effect(P<0.05,n= 8 per group;Fig.3).The respective average gray values of mTOR(average ± SD)in the control,ADO,and Compound C+ADO groups were 112.6 ± 12.6/256.3 ± 21.8,77.7 ± 9.1/132.5 ± 5.5,and 104.1 ± 12.0/197.8 ± 16.2 in the CA1/CA3 region of the hippocampus,as indicated by immunohistochemistry.Significant differences were found between the vehicle control group versus the ADO group and between the ADO group versus the Compound C+ADO group(P<0.05,n= 8 per group;Fig.4).2.1.Subsection4?The aging relationship between adenosine and p-mTOR:In normal rats,adenosine gradually inhibited mTOR expression over time.The inhibitory effect was most obvious at about 3h(p<0.05),and recovered at 6h.Immunohistochemical results were further validated.Conclusions:1?The rats would be drowsy(44/44)and egophony(5/44)after ADO treated and sustained muscular tremors like wave after Compound C treated.2?Adenosine can significantly inhibit p-mtor and p-s6 expression in acute status epilepticus rats induced by pentylenetetrazole;3?In normal wistar rats,adenosine may inhibit mTOR phosphorylation through AM PK pathway;4?In normal wistsar rats,adenosine could significantly inhibit mTOR expression in 3 hours;5?In epileptic seizures,adenosine is associated with mTOR pathway and may inhibit mTOR phosphorylation through AMPK pathway to play the role of antiepileptic effect.