Endogenous Expression Of Neuroglobin In Astrocytes Contributes To Cell Protection Against Oxidative Stress In Spinal Cord Injury

Background:Spinal cord injury（SCI）is a serious trauma disease of the central nervous system,which is largely refractory to therapy in the medical area all over the world.It brings heavy burden to the family and society.As a traditional treatment of SCI,surgical decompression with drug therapy has limited effect,while the endogenous protective effect after SCI has paid more and more attention.Neuroglobin（Ngb）,as an endogenous oxygen-supply protein,has the effect of anti-oxidative stress and anti-apoptosis.The role of Astrocyte（AS）in SCI has been controversial.However,the expression of Ngb in AS and its function have not been reported lately after SCI.Objective:To investigate the dynamic expression of neuroglobin in astrocytes after spinal cord injury and the relative Ngb,GFAP,CAT and SOD₂ mRNA expression of astrocytes after H₂O₂ induced oxidative stress injury.Methods:Modified Allen’s method was used to prepare the rat SCI model.HE staining was carried out to observe the pathological changes and immunofluorescence assay was performed to detect the dynamic expression of Ngb in astrocytes after SCI.Astrocytes incubated with hydrogen peroxide was utilized to mimic the cellular oxidative stress injury.The activity of astrocytes was detected by MTT assay after 6 h and 12 h treatment with different concentrations of H₂O₂.LDH release was used to test the toxicity of H₂O₂ on astrocytes.Flow cytometry was applied to observe the apoptosis of astrocytes exposure by H₂O₂.Oxygen free radical（ROS）produced by astrocytes was measured by flow cytometry after H₂O₂ treatment.The gene expression of Ngb,GFAP,CAT and SOD₂ after oxidative stress was determined by real-time fluorescence quantitative PCR.Results:HE staining showed that the wound repair presented a natural healing process including spinal cord destruction,neurodegeneration and necrosis,glial cell hyperplasia and scar formation in rats after SCI.The immunofluorescence assay demonstrated that the expression of Ngb in AS after SCI appeared to increase first and then decreased,while Ngb reached the peak at the fourteenth day after SCI.MTT assay indicated that the activity of AS was significantly lower than that of controls after H₂O₂ treatment with different concentrations in the 6 h and 12 h（6 h F=137.363,P<0.01;12 h F=143.617,P<0.001）.With the increase of H₂O₂ concentration,the LDH release of AS gradually increased compared with the control group（6 h F=120.899,P<0.001;12 h F=207.375,P<0.001）.This shows that hydrogen peroxide can destroy the permeability of the cellular membrane,thereby promoting the release of LDH.AnnexinV/PI apoptosis staining showed that H₂O₂ can induce apoptosis of AS.Compared with the control group（H₂O₂ 0μmol/L）,different H₂O₂ concentrations（50,100,200,400μmol/L）exposed for 6 h or 12 h,the percentage of apoptotic cells gradually increased（from 2.72%to 17.62%,23.29%,28.36%,34.67%over 6 h and from6.58%to 26.19%,36.01%,50.33%,59.24%over 12 h）（P<0.05）,presenting a time-dose dependent effect.Flow cytometry confirmed that H₂O₂ can promote ROS production of AS,which leads to oxidative stress injury.The qRT-PCR results showed that with the increase of H₂O₂ concentration and exposure time,the expression levels of Ngb,CAT and SOD₂ mRNA showed a trend of increasing first and decreasing lately after oxidative stress injury in AS.The peak value of the aboved gene expression reached at 50μmol/L or 100μmol/L,while the GFAP mRNA expression level continued to decrease totally,which may be related to the apoptosis effect of high concentration of H₂O₂.Conclusion:The increased expression of Ngb in AS after SCI was involved in the natural repair process of SCI.Oxidative stress of AS induced by H₂O₂ can promote the expression of Ngb,CAT and SOD₂ genes.Like CAT and SOD₂,Ngb can fight against oxidative stress injury in the AS,thereby exerting endogenous cytoprotective effects.