RhG-CSF Ameliorates Neuronal Apoptosis Via The MTOR/p70S6K Signaling Pathway In Neonatal Hypoxia-ischemia In Rats

ObjectivesTo investigate the effects of recombinant human Granulocyte-colony stimulating factor(rhG-CSF)on neuronal apoptosis after neonatal hypoxia-ischemia and the possible mechanism involved by investigating the mTOR/p70S6K signaling pathway.MethodsSeven-day-old Sprague-Dawley rats(n=90)were randomly assigned into 5 groups,sham group,hypoxia-ischemia(HI)group,rhG-CSF group,rapamycin(RAPA)group and Ethanol group.Pups were subjected to unilateral carotid artery ligation followed by 2hrs of hypoxia or sham surgery.HI animals received normal saline,rhG-CSF(50?g/kg),rhG-CSF combined with rapamycin(250?g/kg)or Ethanol(vehicle for rapamycin).Pups were euthanized 48hrs post-HI to quantify brain infarct volume.The pathomorphologic changes of the hippocampal CA1 area and cortex were observed by Nissl staining.Neuronal cell death was determined using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL).mTOR,activated mTOR(p-mTOR),P70S6K,activated p70S6K(p-p70S6K),Cleaved Caspase-3(CC3),Bax,and Bcl-2were quantified using Western blot analysis.ResultsTTC scans showed that the model group had significant brain infraction area compared with sham group(P<0.05).And the brain infraction area reduced significantly in the rhG-CSF and Ethanol group(P<0.05).Compared with the rhG-CSF group,the brain infraction area significantly increased in the rapamycin group(P<0.05).But no statistical significance in brain infraction volume was found in rapamycin group compared with the model group(P>0.05).Compared with the sham group,the other groups showed an obvious loose structure,cellular swelling,even dissolution and disappearance of Nissl bodies in some neurons in hippocampal CA1 region and cortical(P<0.05).Of which,the HI and rapamycin group hurt the most,the Nissl stained neurons decreased significantly,followed by the rhG-CSF and Ethanol group.Compared with the HI group,the Nissl stained neurons significantly increased in the rhG-CSF group(P<0.05).rhG-CSF treatment resulted in significant reduction of neuronal apoptosis of hippocampal CA1 area and cortex(P<0.05)after neonatal hypoxia-ischemia damage in rats.However,rapamycin administration reversed the neuroprotective effect of rhG-CSF.Compared with the rhG-CSF group,rapamycin administration increased the apoptosis index in hippocampal CA1 area and cortex(P<0.05).Furthermore,rhG-CSF increased p-mTOR,p-p70S6K and Bcl-2 expression and also decreased CC3 and Bax expression levels in the ipsilateral hemisphere,which were all reversed by rapamycin.ConclusionsThis study demonstrates that rhG-CSF attenuated caspase activation and reduced brain injury by up-regulating the activity of mTOR/P70S6K signaling pathway after experimental HI in rat pups,which is a potential target for treating HI-induced brain injury.