Opposing Roles For NF-κB Factors P65 And C-rel In Neuroprotective Effects Of G-CSF On Hypoxic-ischemic Brain Injury In Neonatal Rats

Objectives1.To investigate the neuroprotective effects of granulocyte colony stimulating factor(G-CSF)on neonatal rat hypoxic-ischemi.c brain damage/injury(HIBD)model;2.To explore the activity changes of two of NF-κB family members(p65 and c-Rel)in neonatal rat HIBD model with or without G-CSF intervention,so as to analyze the role of NF-κB signaling pathway in the neuroprotective effects of G-CSF on HIBD.MethodsFifty-four SD rats in postnatal day 7 were randomly divided into three groups:Sham operation group(Sham group),hypoxic-ischemic brain damage group(HIBD group)and G-CSF treated group(G-CSF group),with 18 rats in each group.HIBD models were established in the latter two groups by unilateral carotid arterial ligation and hypoxia treatment according to Rice-Vannucci method,while the Sham group underwent similar operation without arterial ligation and hypoxia.The G-CSF group was given G-CSF 50 μg/kg intraperitoneally at 1 and 24 hours after modeling,while the Sham group and the HIBD group were given saline only.At 48th hour after modeling,all pubs were subjected to the following items.Body weight of each rat was measured.All rats received 3 neurobehavioral tests:Longa,Grip and suspension test.TTC(2,3,5-triphenyltetrazole chloride)staining was used to evaluate the size of cerebral infarction.Nissl staining was used to observe the pathomorphology in the hippocampus and cortex.Nuclear translocation of p65 and c-Rel was assessed by immunohistochemistry.The level of MDA and SOD activity in ipsilateral brain tissue were detected.The protein levels of CC-3 and Bcl-2 in ipsilateral brain tissue were detected by western blot(WB).Results1.Ponderal growth:The weight growth in the Sham group was significantly higher than that in the HIBD group and the G-CSF group(both P<0.05),while there was no statistical significance in the weight gain between the HIBD group and the G-CSF group(P>0.05).2.Neurobehavioral tests:In terms of Longa and Grip test,the results of Sham group and G-CSF group were significantly better than those of HIBD group(both P<0.05),while the difference between Sham group and G-CSF group was not statistically significant(P>0.05).In terms of suspension test,the suspension time of Sham group was significantly longer than that of HIBD group and G-CSF group(both P<0.05),and the suspension time of G-CSF group was longer than that of HIBD group(P<0.05).3.Cerebral infarction:The percentage of cerebral infarct area in HIBD group and G-CSF group was significantly higher than that in Sham group(both P<0.05),and the percentage of cerebral infarct area in G-CSF group was decreased compared with that in HIBD group(P<0.05).4.Pathomorphology comparison by Nissl staining:Compared with the Sham group,the number of neurons in the HIBD group and G-CSF group significantly decreased,and the cell body was shrunk and deformed.The number of Nissl body decreased,with the staining getting shallow and blurred.Pyknosis,fragmentation or dissolution of nuclei can be found in some cell.Compared with HIBD group,G-CSF group had more neurons in hippocampal CA1 region and cortical region,wi.th the nucleus morphology and cell body more regular,meanwhile Nissl bodies appeared larger and clearer.5.Activity of p65 and c-Rel:P65:Both in the hippocampus CA1 region and cortex region,the nuclear translocation rate of P65 in Sham group was significantly lower than that in HIBD group and G-CSF group(both P<0.05).Compared with HIBD group,the nuclear translocation rate of P65 in G-CSF group decreased(P<0.05).C-Rel:The nuclear translocation rate of c-Rel in both hippocampus CA1 area and cortex area in Sham group was significantly lower-than that in HIBD group and G-CSF group(both P<0.05).Compared with HIBD group,The nuclear translocation rate of c-Rel in.G-CSF group increased(P<0.05).6.Oxidative stress:Compared with Sham group,MDA level in the ipsilateral br-ain tissue of HIBD group and G-CSF group significantly increased,while SOD activity significantly decreased(all P<0.05).Compared with HIBD group,MDA level in G-CSF group partially decreased,while SOD activity partially increased(all P<0.05).7.Apoptosis-associated proteins:Compared with the Sham group,the protein level of CC3 in HIBD group and G-CSF group significantly increased,while that of Bcl-2 signifi cantly decreased(all P<0.05).Compared with the HIBD group,the protein level of CC3 in G-CSF group decreased partially,while that of Bcl-2 increased(P<0.05).Conclusions1.G-CSF plays a neuroprotective role in neonatal HIBD rat models;2.Both p65 and c-Rel were significantly activated in brain tissues of HIBD model,and G-CSF may play a neuroprotective role partially by decreasing the transcriptional activity of p65 while increasing that of c-Rel.3.p65 and c-Rel may play opposing roles in HIBD.