The Mechanism Study Of Regulating CB2R-PI3K/AKT Signal Pathway To Protect Astrocytes In The Childhood Rat With Epilepsy

ItroductionEpilepsy is a chronic brain disease caused by abnormal discharging of neurons in the brain,which induce a momentary brain dysfunction.The disease will seriously affect the quality of people’s lives,to bring the patient physical and mental torture.According to relevant statistics,5‰-8‰of the population suffers from epilepsy,60%of patients with epilepsy occurs in infants or childhood.The epilepsy is generally considered to being caused by higher excitability of the nervous system,which is closely related to the imbalance of neurotransmitters,ion channels,glial cells,genetic and immunity abnormalities.Studies have shown that the basic pathology of epilepsy has gliosis,glial scar formation.Local glial cells increase and fibrosis can induce the formation of hippocampal sclerosis.But astrocytes may also serve nutrition and supporting to neurons.Now with more in-depth study,astrocytes can mobilize ions and neurotransmitters to act on the neurons,such as the regulation of signaling pathways,glutamate receptors,water and potassium,calcium metabolism,gap junctions and inflammatory immune responses.These effects are closely related to the pathogenesis of epilepsy.In recent years,the role of cannabinoid system in epilepsy has received more and more attention.Cannabinoid system is consist of cannabinoid receptors（cannabinoid receptor,CBR）,cannabinoid（endocannabinoid）and the enzyme which regulate the synthesis and degradation of endocannabinoids.CBR has two subtypes,type 1 cannabinoid receptor（CB1receptor,CB1R）and type 2 cannabinoid receptor（CB2 receptor,CB2R）.Munro discovered and proposed CB1R firstly in 1988.Devane isolated CB2R from human bone marrow cells successfully in 1992.Over the past,CB1R was generally considered existing in the brain tissue,and CB2R was in peripheral immune system in common.But there is more and more researches show CB2R playing an important role in the nervous system.CB2R widely is found distributing in the central nervous system,such as the cerebral cortex,corpus callosum,hippocampus,basal ganglia,cerebellum.Numerous studies display CB2R playing an important role in the nervous system diseases,but there is no report with epilepsy.Nevertheless,cannabinoids has already been proven to inhibit seizures,which alleviates the local brain tissue damage in epilepsy.Based on the above background,this study tests CB2R in temporal lobe epilepsy model rat induced by lithium-pilocarpine in vivo experiments,and it’s effect to the brain tissue and nerve cells.Also we try to find CB2R effect’s mechanism to astrocytes in magnesium-free solution.Materials and Methods1.Preparation of an Animal ModelHealthy male SD rats（18-21 days old）,the experimental animals by intraperitoneal injection of lithium chloride（3mEq/kg,127mg/kg）,then 18h-20h later,intraperitoneal injection of pilocarpine（30mg/kg）.Before pilocarpine intraperitoneal injection 30min,give animals injection of methyl bromide scopolamine（1mg/kg）to antagonize pilocarpine peripheral cholinergic reaction.If the rats don’t get status epilepticus,we gave pilocarpine（10mg/kg）intraperitoneal injection every 30 minutes,until the status epilepticus.Racine（1972）grading standards:aGrade 0:no response;GradeⅠ:ear facial muscle twitching;GradeⅡ:with nod or flick-based muscle twitching;GradeⅢ:unilateral limb clonus and convulsions;GradeⅣ:bilateral limb clonic or generalized tonic-clonic seizures,accompaning the body upright;GradeⅤ:convulsions,continued standing,body tonic dorsiflexion and fell.Based on the above evaluation criteria,level rats in GradeⅣ-Ⅴwas considered successful model.2.The experimental groups of animals（1）Experimental groups of the first paperHealthy male SD rats（18-21 days）,animals were randomly divided into two groups:Group A:control group;Group B:pilocarpine model group,setting up the point after entering the status epilepticus（status epilepsy,SE）at 2h,24h,14d,21d.（2）Experimental groups of the second paperHealthy male SD rats（18-21 days）,the animals were randomly divided into four groups:Group A:normal rat;Group B:pilocarpine model group;Group C:CB2R model agonist treatment group;Group D:pilocarpine model+CB2R agonist（JWH133）+CB2R antagonist（AM630）,setting up the point after entering SE 24h.Young rats were sacrificed rats at this time.All the organizations were divided into two parts,one was paraffin-embedded for immunohistochemical staining and Nissl test,the other one was frozen in liquid nitrogen for Real-time PCR and Western blot detection.3.The culture and groups of astrocytesAstrocyte cell lines in rats,introduced from ATCC.Application of DMEM containing10%fetal bovine serum at 37℃,5%CO₂ conditions of culture,every 2-3d for passage at the ratio of 1:2.（1）Experimental groups of the third paperGroup A:normal rat astrocyte cell monolayer;Group B:rat astrocytes monolayer with magnesium-free solution treatment:with magnesium-free solution treated for 2 hours,then with normal cell culture medium 24 hours;Group C:magnesium-free solution+JWH133:with magnesium-free solution treated for 2 hours,the cell culture medium containing JWH133（4μM）cultivation for 24 hours;D group:magnesium-free solution+JWH133+AM630:with magnesium-free solution treated for 2 hours,then with containing JWH133（4μM）,AM630（1μM）cell culture medium for 24 hours cultivation.（2）Experimental groups of the forth paperGroup A:rat astrocytes monolayer with magnesium-free solution treatment;Group B:magnesium-free solution+JWH133;Group C:magnesium-free solution+Wortmannin:with magnesium-free solution treated for 2 hours,the cell culture medium containing Wortmannin（2μM）cultivationfor24hours;GroupD:magnesium solution+JWH133+Wortmannin:magnesium ion was treated for 2 hours,containing JWH133（4μM）,Wortmannin（2μM）cell culture medium to cultivate for 24 hours.4.The experimental methods and outcome measures（1）Immunohistochemistry:detecting CB2R and GFAP expression in hippocampus of rats in childhood with epilepsy.（2）Nissl staining:detecting hippocampal neurons in rats after CB2R affection.（3）Western blot:detecting GFAP,PI3K/AKT pathway（PI3K,p-AKT,AKT protein）of the hippocampus of epilepsy rats in childhood after CB2R affection;detecting PI3K/AKT pathway（PI3K,p-AKT,AKT,p-mTOR,mTOR protein）of astrocytes with magnesium-free solution and CB2R affection;detecting cell cycle protein CyclinD1,CyclinE,p-Rb protein and anti-apoptotic protein bcl-2;After affecting with PI3K/AKT pathway inhibitor Wortmannin intervention,detecting cyclinD1,p-Rb protein,anti-apoptotic bcl-2 protein.（4）Realtime-PCR:detecting of mRNA expression of CB2R in childhood rats hippocampus suffering from epilepsy.（5）FCM（flow cytometry）:detecting cell cycle changes of astrocytes.5.Statistical methodsAll statistical analyzes were performed using SPSS 12.Software,the value of p less than 0.05 is considered significant difference.Results1.A dynamic expression CB2R in hippocampal tissue of epileptic childhood rats（1）In control group,CB2R content of the hippocampus brain gradually increased with age increasing.When the rats with the age of 35-42 days,CB2R content gradually got stabilized.After status epilepticus for 2h-14d CB2R content of hippocampus is more than the control group,at the point of 21d the control group is more than epileptic group.（2）Immunohistochemistry showed:CB2R positive staining expressed in hippocampal CA1,CA3,DG zone were brown granules.Hippocampus control rats scattered in various districts.hippocampus CB2R positive cells of epilepsy model increased gradually after SE 2 hours to 2 weeks,and after SE 14d the content peaked and gradually showed balance trend.（3）Real-time PCR and Western blot results showed:CB2R content of hippocampus in epileptic group was more than control group at the point after SE 2 hours,24 hours,14days,but the CB2R content of control group was more at 21days.With the changing age of the rats,epileptic hippocampus at different time points difference compared with the control group of young rats are different,the most obvious change is at 24 hours.2.The early effects of CB2R to epileptic rats hippocampus（1）CB2R has protective effect of hippocampal neurons after status epilepticus.Nissl staining:JWH133 can reduce the damage of hippocampal neurons after status epilepticus,while AM630 reduced this protective effect.（2）CB2R can increase the expression of GFAP of hippocampus in epileptic rats after SE.Immunohistochemistry and Western Blot showed JWH133 can increase the expression of GFAP after status epilepticus,while AM630 can reduc this role.（3）CB2R can affect PI3K/AKT pathway in the hippocampus of epileptic rats.Western blot detected the protein of PI3K/AKT pathway（PI3K,p-AKT,AKT）protein changes.We found JWH133 and AM630 can influence the protein（PI3K,p-AKT,AKT）in the hippocampus region.3.CB2R may affect astrocyte proliferation through the PI3K/AKT pathway（1）Western blot analysed cell cycle protein p-Rb,CyclinD1,CyclinE,and anti-apoptotic bcl-2 protein after CB2R agonists JWH133 treatment were increased.But with AM630 operation reduced this change.（2）FCM tested cell cycle changes of each astrocyte group:JWH133 significantly promote the cell from G1 to S transition,but with adding AM630,the activation of this cell cycle reducing.（3）Western blot detected the expression of the protein of PI3K/AKT pathway（PI3K,p-AKT,AKT,p-mTOR,mTOR）changes.JWH133 reduced the inhibition effect of astrocytes with magnesium-free solution,found the expression p-AKT/AKT,p-mTOR/mTOR increasign,but AM630 reduced this effect.4.CB2R can affect astrocyte proliferation by regulating the PI3K/AKT pathwayApplication of PI3K/AKT pathway inhibitor Wortmannin,it blocked the proliferation effect of astrocytes caused by JWH133.Western blot detected the cell cycle protein p-Rb,cyclinD1 and anti-apoptotic bcl-2 are significantly reduced with Wortmannin treatment.Conclusion1.CB2R presented high expression in childhood rat hippocampus suffering from status epilepticus,reached a peak following with prolonged seizures.2.CB2R gives the protection to the childhood rat suffering from pilocarpine-induced epilepsy.3.CB2R may relieve the injury of the neurons and astrocytes in hippocampus after status epilepticus through activation of PI3K/AKT pathway.4.CB2R promotes astrocytes proliferation after"epilepsy microenvironment"treatment by regulating the PI3K/AKT pathway.