The Effect Of TREK-1 On Neurons Viability After Oxygen And Glucose Deprivation In Vitro Of Cocultured Astrocytes And Neurons And Intervention Of Mailuoning Injection

Objective:1.To investigate the effect of TREK-1 on neurons viability after Oxygen and Glucose Deprivation(OGD)in vitro of cocultured astrocytes and neurons and intervention of mailuoning Injection and the influence of glutamate uptake efficacy.Methods: 1.Astrocytes and neurons cultured separately,TREK-1 channel overexpression model was successfully established by using rat recombinant DNA plasmid;2.coculturing astrocytes and neurons after oxygen and glucose deprivation to establish in vitro model of cerebral ischemia.We set a control group,a normal model group,a normal TREK-1 overexpression group,a normal Mailuoning Injection group,a OGD model group,a OGD TREK-1 overexpression group and OGD Mailuoning Injection group seven groups in this study.3.The rates of GLT-1,P2X7,GS,Caspase-3,TREK-1 mRNA were detected by Real-Time PCR.The expression rates of TREK-1,GLT-1,P2X7 protein were detected by Western blot.4.Lactate dehydrogenase(LDH)release was measured using a LDH assay kit.Neuron apoptosis rates were detected by using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)and the Annexin FITC/ Propidium Iodide method.Glutamate uptake rates were detected by Glutamate oxidase Assay Kit.NR2 B and Caspase-3 protein expression were detected by Western blots.Results: 1.TREK-1 overexpression model was established by rat recombinant DNA plasmid.2.TREK-1 overexpression could increase the mRNA expression of TREK-1,GLT-1,GS and glutamate uptake rates,reduce the mRNA content of Caspase-3 whereas they had no significant effect on P2X7 expression under OGD condition.3.Under the condition of OGD,TREK-1 overexpression could increase cell viability of neurons.4.TREK-1 overexpression could decrease NR2 B,Caspase-3 protein expression rates.5.Mailuoning injection had no significant effect on mRNA expression of GLT-1,GS,TREK-1,Caspase-3,P2X7 and glutamate uptake rates in astrocytes.6.Mailuoning injection had no significant effect on protein expression of Caspase-3,NR2 B in neurons.We found that neutron viability increased,NR2 B and Caspase 3 protein expression of neurons decreased significantly while GLT-1 expression and TREK-1 mRNA and protein expression of astrocytes increased significantly in OGD TREK-1 overexpression group.The results indicate that TREK-1 overexpression may increase the astrocytes viability and enhance astrocyte glutamate uptake under OGD conditions,leading to an increase in neutron viability.Our findings will provide new evidences for the treatment of the acute phase of ischemic stroke.Conclusions:1.Using recombinant TREK-1 DNA plasmid could successfully transfect the rat astrocytes.2.TREK-1 overexpression could increase glutamate uptake of astrocytes after oxygen glucose deprivation.3.Astrocytes of TREK-1 overexpression could increase cell viability of neurons.4.Mailuoning injection had no effect on neurons after oxygen glucose deprivation.