CB2Agonist Pretreatment Induced Neuroprotction Through Activating Microglia Excitatory Amino Acid Transporter

Cerebral ischemia/reperfusion injury seriously threatens people’s lives andhealth, and how to prevent and reduce ischemic brain injury effectively hasbecome one of the priorities of medical research. Glutamate is the mostimportant excitatory amino acid, but it can also lead to brain damage. Inphysiological conditions, the concentration of glutamate out of neurons is indynamic equilibrium, but in pathological conditions, glutamate would beproduced too much, lead to accumulation in synaptic cleft and resulting inneuron damage. Glutamate transporters expression in the glial cells surface cantransport glutamate into glial cells, and transform it into glutamine withoutneurotoxicity. Therefore, maintain normal levels of extracellular glutamateconcentration by glutamate transporter, in CNS plays an important role inprevention and mitigation of the neuron damage.Our laboratory first reported that “Baihui” electro-acupuncture stimulationinduced brain ischemic tolerance in rats, with acupoint specificity (Baihui) andoptimal stimulation parameters (2/15Hz,1mA,30min/d,5d) throughincreasing the endogenous cannabinoid ligand (2-AG and AEA) synthesis inbrain. Further studies demonstrated that cannabinoid receptors CB1and CB2 play an important role in the fast phase and delayed phase of neuroprotectionrespectively. But the mechanism is not clear. As well known, CB2mainlyexpressed in microglia which play key role in cell damage following cerebralischemia by activating inflammation and/or modulating EAA concentrationthrough EAAT. According to these data, we develop our hypothesis that CB2induced delayed phase neuroprotection by activating EAAT on microglia.Therefore, in this study we use cell culture methods to observe the effect of CB2agonist pretreatment against glutamate toxicity by acting on microglia excitatoryamino acid transporter (EAAT).Part1AM1241pretreatment protection on microglialinjury induced by glutamateMetheds1. To build glutamate injury model.Microglia were assigned into6groups: Control and5injury groups of bydifferent glutamate concentrations (1、3、5、10and20mmol/L). Cell suvival wasdetermined by MTT after24hours.2. To find the effective concentration of AM1241pretreatment.Microglia were assigned into6groups pretreated with different AM1241concentrations (0、0.5、1、2、3and5μmol/L) for2hours, then cells were culturedwith normal medium for2hours, after that the medium was changed intoIMDM containing10mmol/L glutamate for24hours, cell suvival wasdetermined by MTT. The group with the highest cell suvival and lower AM1241concentration has be choosen to use in next steps. 3. To investigate the effects of AM1241pretreatment on microglia injuryinduced by glutamate.Cells were assigned to6groups, Control group、AM1241group、AM630group、Glu group、AM1241+Glu group and AM1241+AM630+Glu group. Themouse N9microglial cells were respectively pretreated with2μmol/L AM1241and2μmol/L AM1241+6μmol/L AM630in AM1241+Glu group andAM1241+AM630+Glu group. They were then exposed to10mmol/L glutamatefor24h at2h after pretreatment. The cell suvival was measured by MTT assay;the LDH release was measured by Reagent Kit; the shapes of microglia wereobserved by microscope.Results1. The cell suvival after glutamate explosure for24h.Compared with control group, cell suvival of the cells exposed to differentconcentrations of glutamate decreased markedly(P＜0.05).10mmol/Lglutamate can decrease cell suvival to about50%compared with the controlgroup.2. The variation on cell suvival by different concentrations of AM1241pretreatment.Compared with the Glu group, AM1241pretreatment reduced theglutamate-induced decrease of cell suvival (P＜0.05).2μmol/L of AM1241waschosen according to the protective effects and drug concentrations. 3. The influence of AM1241pretreatment in microglial injury induced byglutamate.(1) The cell suvivalCompared with Glu group, cell suvival of AM1241+Glu group increasedmarkedly(P＜0.05). Compared with AM1241+Glu group, cell suvival ofAM1241+AM630+Glu group markedly decreased(P＜0.05).(2) The LDH releaseCompared with Glu group, LDH release in AM1241+Glu group decreasedsignificantly(P＜0.05). Compared with AM1241+Glu group, LDH release inAM1241+AM630+Glu group significantly increased (P＜0.05).(3) The shape of microgliaCompared with Glu group, the injury level of AM1241+Glu groupdecreased significantly, that of AM1241+AM630+Glu group, however, showsno apparent difference with Glu group.Part2CB2agonists reduce neuron injury induced by glutamatethrough regulating EAAT of microglialMetheds1. The effecte of AM1241pretreatment on neuron injury induced byglutamate.Cells were assigned to7groups: Control group、AM1241group、AM630group、 Glu group、 AM1241+Glu group、 AM630+Glu group andAM1241+AM630+Glu group. The mouse N9microglial cells were respectivelypretreated with2μmol/L AM1241、6μmol/L AM630and2μmol/L AM1241+6μmol/L AM630in AM1241+Glu group、AM630+Glu group andAM1241+AM630+Glu group. They were then exposed to10mmol/L glutamatefor24h after pretreatment, then the medium was collected and used to cultureneuron for24h. Neuron suvival was assessed by MTT assay.2. The effect of AM1241pretreatment on microglial EAAT expression invitro.Cells were assigned to4groups: Glu group、AM1241group、AM630group and TBOA group. N9microglial cells were respectively pretreated with2μmol/L AM1241、2μmol/L AM1241+6μmol/L AM630and2μmol/LAM1241+100μmol/L TBOA in AM1241group、AM630group and TBOA group.They were then exposed to10mmol/L glutamate for24h after pretreatment,Then the expression of EAAT2were detected.3. The effect of EAAT antagonists on neuroprotection induced by AM1241pretreatment.Cells were assigned to7groups: Control group、AM1241group、TBOAgroup、 Glu group、 AM1241+Glu group、 TBOA+Glu group andAM1241+TBOA+Glu group. The mouse N9microglial cells were respectivelypretreated with2μmol/L AM1241、100μmol/L TBOA and2μmol/LAM1241+100μmol/L TBOA in AM1241+Glu group、TBOA+Glu group andAM1241+TBOA+Glu group. They were then exposed to10mmol/L glutamatefor24h after pretreatment, then the medium was collected and used to cultureneuron for24h. Neuron suvival was assessed by MTT assay.Results1. The effect of AM1241pretreatment on neuron injury induced byglutamate.Compared with Glu group, neuron viability of AM1241+Glu group increased significantly(P＜0.05). Compared with AM1241+Glu group, neuronviability of AM1241+AM630+Glu group significantly decreased(P＜0.05).2. The effect of AM1241pretreatment on microglial EAAT expression.2.1Western blotThe analysis of western blot shows that the expression of EAAT2markedlyincreased in AM1241group compared with that in Glu group (P<0.05). Theexpression of EAAT2protein significantly decreased in AM630and TBOAgroup compared with that in AM1241group (P<0.05).2.2ImmunocellulerchemistryImmunocellulerchemistry shows that the expression of EAAT2proteinincreased in AM1241group compared with that in Glu group.The expression ofEAAT2protein decreased in AM630and TBOA group compared with that inAM1241.3. The effect of EAAT antagonists on neuroprotection induced by AM1241pretreatment.Compared with Glu group, neuron viability of AM1241+Glu groupincreased significantly(P＜0.05). Compared with AM1241+Glu group, neuronviability of AM1241+TBOA+Glu group significantly decreased (P＜0.05).Summary1. CB2receptor agonist AM1241pretreatment reduced the microglial damageinduced by glutamate, which can be reversed by the CB2receptor antagonistAM630, suggesting that microglial CB2receptor activation can reduceglutamate damage.2. Pretreatment of microglia with CB2receptor agonists can reduceglutamate-induced neuronal damage, and it can be reversed by the CB2receptorantagonist AM630, suggesting that microglia CB2activation is a way to eliminate the cytotoxic effects of glutamate in the medium. CB2receptoragonist pretreatment enhanced microglia EAAT2protein expression, whileEAAT antagonist TBOA can also reverse the protective effect of CB2receptoragonist pretreatment, suggesting that microglial EAAT is involved in the CB2receptor agonist pretreatment neuroprotection.