Inhibitory Effect And The Mechanism Of Apelin-13on The Oxygen-glucose Deprivation-induced Apoptosis In The Vitro Culture Of Neonatal Rats’ Cortical Neurons

PurposeHere the purpose of the present study is to observe the validity of apoptosis modelinduced by oxygen-glucose deprivation in vitro cultured cortical neurons. To study theinhibitory effect of apelin-13on apoptosis induced by oxygen-glucose deprivation, theneuroprotective effect, and its influence on the activity of cAMP/PKA/CREB signalingpathway. And to investigate the possible inhibitory mechanism of apelin-13on apoptosisand to attempt to offer experimental methods and proofs for new therapeutic target forischemic stroke.Methods1.Primary cortical neurons of neonatal rats were cultured and purified, then identifiedthe neurons and calculated the purity by immunocytochemistry of Neuronspecific Enolase.2.Establishment of the oxygen-glucose deprivation model of primary neurons andconformation its validity: The newborn rats’ cortical neurons cultured for7days, and thewhole cell culture medium was replaced with Earle’s solution without glucose. Theglucose-free Earle’s solution was sealed with steriled paraffin liquid. Neurons wereobserved under inverted microscope. Then MAP-2fluorescent staining was applied toobserve the damage of neurons, and the detection of LDH and Tunel were applied to showroughly death and apoptosis at OGD6,9,12,24,30h. Selected the appropriate time pointwith the highest apoptotic efficiency as the best observation time point.3.In order to make sure the inhibitory effect of apelin-13on apoptosis induced byoxygen-glucose deprivation, primary cultured cortical neurons were pretreated withapelin-13(10,1,0.1nmol/L) for30min, then exposed to OGD and apelin-13of the sameconcentration for12h. Morphologic observation of injuried neurons was performed with inverted microscope and fluorescence staining of MAP-2respectively. The detection ofLDH was applied to show roughly death. Tunel, NeuN and Hoechst33342staining wasused to calculate the percentage of apoptotic cells and observe the surviving cells.Detection the expression level of caspase-3, bcl-2and bax was performed by Real TimeRT-PCR.4.The role of apelin-13in the modulation of cAMP expression level was determinedby enzyme immunoassay, and apelin-13’s effect was compared to PDE4inhibitorRolipram.5.Neurons were treated with OGD, apelin-13and PKA inhibitor H89(10μmol/L), thenthe phosphorylation level of CREB was detected by Western Blot in order to determine theeffect of PKA inhibitor H89on phospho-CREB level and make sure the influence ofapelin-13on cAMP/PKA/CREB signaling pathway, and whether this effect wasPKA-dependent.Results1.Cortical neurons of neonatal rats cultured for7days, then identified withNeuronspecific enolase. The percentage of neurons was (90±6.164414)%. It was relativelypure neurons culture, and it could be used for next experiments.2.In the OGD model, neurons showed obvious damage via observation under invertedmicroscope and by MAP-2fluorescence staining. Along with the prolongation ofoxygen-glucose deprivation, LDH leakage increased in a time-dependent manner, and theneuron apoptosis occurred. Tunel staining showed the apoptosis rate of OGD12h groupwas52.83±3.53%, which was significantly higher than other groups. So we choseOGD12h as the best observation time point in this model.3.Apelin-13could inhibit ischemic hypoxic injury of cortical neurons induced byOGD. Surviving neurons increased by observation under inverted microscope and viaMAP-2fluorescence staining. LDH leakage reduced significantly(P<0.01). Apelin-13could suppress apoptosis induced by OGD: Tunel staining illustrated that apelin-13couldreduce the percent of apoptotic cells(P<0.05); and apelin-13could depress caspase-3expression (P<0.05), raise BCL-2expression (P<0.05), and could reduce bax expression(P<0.01).4.The negative control group exposed to apelin-13for3hours, and its intracellularcAMP concentration didn’t show alteration(P＞0.05); and the apelin-13significantlyinduced intracellular cAMP concentration increase in OGD+apelin-13group(P<0.01),similar to Rolipram. Interestingly, induction of intracellular cAMP level decreased insteadly in neurons treated with apelin-13and Rolipram at the same time. The cAMPlevel in OGD+apelin-13+Rolipram group was significantly lower than the OGD+apelin-13and OGD+Rolipram group (P<0.05).5.After treatment with apelin-13for3hours, intracellular phospho-CREB level didn’tshow obviously change in negative control group(P>0.05); phospho-CREB level in OGDgroup was significantly lower than that in control group(P<0.01); OGD exposed toapelin-13for3hours, and p-CREB level increased significantly (P<0.05); but treatmentwith apelin-13and PKA inhibitor H89at the same time, p-CREB level significantlydecreased (P<0.01).Conclusion1.It is feasible to imitate the hypoxia ischemia model of neurons in vitro which caninduce neurons apoptosis effectively via culturing with glucose-free Earle’s Solution andsealing with steriled paraffin liquid.2.Apelin-13could inhibit neurons apoptosis induced by oxygen-glucose deprivation.3.The inhibitory effect of apelin-13on apoptosis induced by OGD may associatedwith cAMP/PKA/CREB signaling pathway.