Role And Mechanism Of Neuroglobin In Axonal Outgrowth Of Neurons After Ischemia

Ischemic stroke is a common cerebrovascular disease,with high incidence,high recurrence rate and high mortality rate,seriously affecting the patients' health and daily life,even imposing a huge economic burden to the family and society.Axonal retraction,or fracture caused by ischemia-reperfusion is the main cause of neurological dysfunction.Axonal outgrowth assisted the central nervous system(CNS)to form new synapses and establish a new neural network and played an important role in the pathophysiology of ischemia.Thus,treatment of neuroprotection and neuronal recovery has been the focus of current preclinical studies,but yet has not found an effective treatment in promoting axonal regeneration to improve neurological function.Neuroglobin(Ngb),a monomeric globin of 17 kDa,primarily expressed in neurons of human and mouse.Ngb shows a very high affinity for O2 and barely easy to dissolve.Ngb not only combined with O2,CO,NO or other small molecules,but also with G-protein or other proteins and regulate its activity.Most evidences have demonstrated that overexpression of Ngb has protective effects on CNS injury such as ischemia/hypoxia/oxidative stress,suggesting that Ngb as a neuroprotective factor has potential prospects for the treatment of CNS injury such as ischemic stroke.In this study,the model of MCAo/reperfusion and OGD/reoxygenation of primary cultured neurons were used as experimental materials and subjects to simulate ischemic stroke.Then,animal biology,cell biology and molecular biology were used to investigate the regulation of Ngb on axonal outgrowth and its mechanism in ischemia.We found that the expression of Ngb was gradually increased at different time points during ischemia/reperfusion in MCAo model and accumulated at the growth cone of neurites in ischemic lesions,suggesting a positive correlation between Ngb and axonal outgrowth in ischemia.Knocking down the endogenous Ngb significantly decreased the length of neurites in primary cortical neurons suffered OGD/reoxygenation,while overexpressing of Ngb promoted neurite outgrowth.In order to further explore the molecular mechanisms of Ngb in promoting the length of axons,Ngb was overexpressed in primary cultured cortical neurons suffered OGD/reoxygenation and inhibitors of different signaling pathways were added.It was found that p38 MAPK signal pathway inhibitor SB203580 could significantly reverse the function of Ngb.In addition,IF,Western blotting,BiFC and GST pull down assay showed that Ngb activated p38 phosphorylation by binding to p38 in OGD/reoxygenation model.Interestingly,the binding effect of H64L with p38 was significantly enhanced compared with wild-type Ngb and other mutants and significantly activated the phosphorylation of p38 during OGD/reoxygenation.We also found that the 7-122 fragment was required for Ngb/p38 interaction by GST pull down assay.Furthermore,T77A and S145A significantly promoted the interaction with p38 and obviously activated the phosphorylation of p38 by GST pull down and Western blotting assay.Finally,TAT-Ngb,a penetrating peptide expressed in prokaryotic expressing vector,could significantly activated the expression of GAP43 and obviously reversed the axonal retraction of primary cultured neurons after OGD/reoxygenation.In the study of CNS injury,such as ischemic stroke and spinal cord injury,promoting neuronal axonal regeneration after ischemia has been shown to significantly improve neurological function.The role promoting axonal regeneration and the mechanism under ischemic stress will accelerate the recovery of brain function of ischemic stroke.