Mechanism Of Macroautophagy Regulated By RhG-CSF Involving In Repairing With Neuron After Ischemia-hypoxia

Background and objective:Cerebrovascular disease has become one of the higher mortality and morbidity disease in our county, and ischemic disease accounts for70-80%of cerebrovascular disease, so positive and effective clinical intervention has a significant social value. RhG-CSF (Recombinant human granulocyte colony-stimulating factor) is a hematopoietic growth factor, and has the ability to promote proliferation and differentiation of neutrophil progenitor cell. Recently, however more and more foreign experimental evidence shows that it plays an important role in the neural tissue repairing and functional recovery, such as promoting the regeneration of nerve cells, anti-apoptosis of neuron, promoting angiogenesis, anti-inflammatory, immunomodulatory. However, specific mechanisms of rhG-CSF protect neuron with cerebral ischemic are not yet clear,and the effect of macroautophagy are also rarely reported.we observed ratio changes of LC3Ⅱ/LC3Ⅰ with neurons after Cerebral ischemia in vivo and in vitro, and explored the mechanism of macroautophagy regulated by RhG-CSF and the possible neuroprotective mechanism,and provide a theoretical basis for clinical application.Methods:Establishing model of cell after hypoxia by taking oxygen-glucose deprivation(OGD).After intervening in cells by different concentration of rhG-CSF, ratio change of LC3Ⅱ/LC3Ⅰ was measured by Western blot, macroautophagy vesicle change was analyzed by monodansylcadaverine(MDC), cell activity was investigated by3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium(MTT). Focal cerebral ischemia model was established by modified suture-occluded method, neurological deficit was scored(NDS) before and after the intervention of rhG-CSF, ratio change of LC3II/LC3I of cortical neurons in rat was observed by Western blot, change of LC3expression was detected by immunohistochemistry.Results:With the treatment of rhG-CSF after hypoxia of cell model, LC3II/LC3I ratios in low and high dose group raised compared with no dosing group; Compared with low dose group,LC3II/LC3I ratio in high dose group raised, and the difference was statistically significant (p<0.05).MDC staining showed a dose-dependent manner that with the increasing of concentration of rhG-CSF administration, the number of macroautophagy vesicles gradually raised. It showed that with the increasing of the concentration of rhG-CSF administration, autophagy activity enhanced corresponding,the difference was statistically significant (p<0.05); Compared with model group, neurological function score in rhG-CSF group decreased, the difference was statistically significant (p<0.05);After treatment than before, neurological function score in rat was lower,the difference was statistically significant (p<0.05);It showed that the role of rhG-CSF in repairing of the neuron was positive; Compared with sham group, LC3II/LC3I ratio raised in the rat model group.Compared with sham and model group,LC3II/LC3I ratio raised in rhG-CSF group in rat,the difference was statistically significant (p<0.05). It showed that the intervention with rhG-CSF raised macroautophagy activity after focal cerebral ischemia in rat.Conclusion:rhG-CSF may enhance the role of macroautophagy in neuron after focal ischemic, and have a role to promote activity of neuronal cell. It show that there is a trend that with the increasing of concentration of rhG-CSF, the role enhanced. After focal cerebral ischemia,rhG-CSF intervention may involve in repairing of the neuron by raising level of macroautophagy.