RhG-CSF Ameliorates Neuronal Apoptosis Through Anti-apoptotic AKT/caspase9 Signal Pathway Following Cerebral Ischemia/ Reperfusion Injury In Rats

Objective:To discuss the nerve protective mechanism of recombinant human granulocyte colony-stimulating factor(rhG-CSF) and dynamic observe the proteins expression changes of p-AKT、caspase9、caspase3 after cerebral ischemia-reperfusion injury in rats.Methods:74 adult male SD rats were randomly divided into four groups:the sham-operated(SO)group, the middle cerebral artery occlusion/reperfusion(MCAO /R)group,rhG-CSF group and observation(OG)group. The temporary middle cerebral artery occlusion reperfusion model was made by the suture method. In rhG-CSF group, rats were injected a single dose of 50 ug/kg rhG-CSF subcutaneously at the time of reperfusion and per 6 hours after reperfusion. The SO group and MCAO /R group received the same volume of saline.At the 24-hour time point, some rats were executed after scored with Longa 5-point scale.Then the morphological changes of cortical neurons were observed by HE, the expressions of p-AKT 、caspase9 were detected by immune-histochemistry and western blot analysis, the apoptosis cells were shown by TUNEL method. The others were executed at the different time of reperfusion(2h、6h、24h、48h、72h), and the p-AKT 、caspase9 and caspase3 proteins were detected by immune-histochemistry.Results:1. At the 24-hour time point, the rats were normal in the SO group, and neurological outcome scores of the rhG-CSF group(1.58±0.67)were lower than the MCAO/R group(2.25±0.62,p=0.022).The results of p-AKT 、caspase9 proteins’ expression were consistent between immune-histochemistry with western blot. Compared with the SO group, the mean gray values of the expressions of p-AKT 、 caspase9 increased significantly in the rhG-CSF group and in the MCAO/R group(p<0.01).The mean gray values of the expressions of p-AKT in the rhG-CSF group were significantly increased compared with MCAO/R group( p<0.01), caspase9 instead. Compared with the MCAO/R group[( 31.80±2.91) %], the percentage of apoptotic cells decreased significantly in the rhG-CSF group[(22.13±2.74)%] and were hardly to be found in the SO group.2. The p-AKT 、caspase9 and caspase3 proteins expressed differently at the different time of reperfusion.Conclusion:rhG-CSF could protect brain from ischemia/reperfusion injury in rats and its neuroprotective mechanism may be through the AKT/caspase9 signaling pathway to reduce nerve cell apoptosis.