脑红蛋白在脊髓损伤后星型胶质细胞内的表达及意义
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摘要
目的观察脊髓损伤(SCI)后星形胶质细胞(AS)内脑红蛋白(Ngb)的表达,并探讨其意义。方法将30只SD大鼠随机分为SCI组(25只)和假手术组(5只),SCI组应用改良Allen's法制备大鼠SCI模型,HE染色观察SCI后病理改变,免疫荧光法观察SCI后AS内Ngb的动态表达变化。采用过氧化氢(H2O2)构建大鼠脊髓AS氧化应激损伤模型,MTT法检测不同浓度(0、50、100、150、200、400μmol/L)H2O2处理AS 6、12 h后的活力变化。流式细胞术观察不同浓度H2O2处理6、12 h后AS产生氧自由基(ROS)的能力。实时荧光定量PCR法测定AS氧化应激损伤后Ngb mRNA表达。结果 HE染色显示大鼠SCI后呈现出由急性损伤逐步转变为胶质瘢痕形成的自然修复过程。免疫荧光结果显示SCI后AS内的Ngb表达呈先升后降趋势,并在SCI后14 d达到峰值。不同浓度(50、100、150、200、400μmol/L)H2O2处理6、12 h后,模型组AS活力均低于对照组(P均<0. 05)。不同浓度H2O2处理6、12 h后AS产生ROS增加。随着H2O2浓度(50、100、150、200、400μmol/L)增加及暴露时间(6、12 h)延长,AS的Ngb mRNA表达呈现先升后降趋势,呈现时间-剂量依赖效应(P均<0. 05)。结论 SCI后脊髓AS内Ngb的表达增加。Ngb可能参与了SCI后AS的抗氧化应激损伤,从而发挥内源性细胞保护作用。
Objective To investigate the expression and significance of neuroglobin( Ngb) in astrocytes( AS) after spinal cord injury( SCI). Methods Thirty SD rats were randomly divided into two groups: the SCI group and the non-operative group. Modified Allen's method was used to prepare the SCI model. HE staining was carried out to observe the pathological changes,and immunofluorescence assay was performed to detect the expression changes of Ngb in AS after SCI. AS incubated with hydrogen peroxide was utilized to mimic the cellular oxidative stress injury. The activity of AS was detected by MTT after 6-hour and 12-hour treatment with different concentrations of H2 O2(0,50,100,150,200,and 400 μmol/L). Oxygen free radical( ROS) was measured by flow cytometry after treatment with different concentrations of H2 O2. The Ngb mRNA level after oxidative stress was determined by real-time fluorescent quantitative PCR. Results HE staining showed that the wound repair represented a natural healing process that gradually changed from acute injury to glical scar formation. The immunofluorescence assay demonstrated that Ngb expression in AS first increased,then decreased,and reached the peak at 14 days after SCI. The activity of AS was significantly lower than that of controls after 6-hour and 12-hour treatment with different concentrations of H2 O2(0,50,100,150,200,and 400 μmol/L)(P < 0. 05). Different concentrations of H2 O2 promoted ROS production at 6 and 12 h. With the increase of H2 O2 concentration and exposure time,the Ngb mRNA level showed a trend of increasing first and decreasing lately after oxidative stress injury in AS with a time-dose dependent manner( P < 0. 05). Conclusion The expression of Ngb increases in AS after SCI,and Ngb may be involved in the anti-oxidative stress injury in AS after SCI,thereby exerting endogenous cytoprotective effects.
Objective To investigate the expression and significance of neuroglobin( Ngb) in astrocytes( AS) after spinal cord injury( SCI). Methods Thirty SD rats were randomly divided into two groups: the SCI group and the non-operative group. Modified Allen's method was used to prepare the SCI model. HE staining was carried out to observe the pathological changes,and immunofluorescence assay was performed to detect the expression changes of Ngb in AS after SCI. AS incubated with hydrogen peroxide was utilized to mimic the cellular oxidative stress injury. The activity of AS was detected by MTT after 6-hour and 12-hour treatment with different concentrations of H2 O2(0,50,100,150,200,and 400 μmol/L). Oxygen free radical( ROS) was measured by flow cytometry after treatment with different concentrations of H2 O2. The Ngb mRNA level after oxidative stress was determined by real-time fluorescent quantitative PCR. Results HE staining showed that the wound repair represented a natural healing process that gradually changed from acute injury to glical scar formation. The immunofluorescence assay demonstrated that Ngb expression in AS first increased,then decreased,and reached the peak at 14 days after SCI. The activity of AS was significantly lower than that of controls after 6-hour and 12-hour treatment with different concentrations of H2 O2(0,50,100,150,200,and 400 μmol/L)(P < 0. 05). Different concentrations of H2 O2 promoted ROS production at 6 and 12 h. With the increase of H2 O2 concentration and exposure time,the Ngb mRNA level showed a trend of increasing first and decreasing lately after oxidative stress injury in AS with a time-dose dependent manner( P < 0. 05). Conclusion The expression of Ngb increases in AS after SCI,and Ngb may be involved in the anti-oxidative stress injury in AS after SCI,thereby exerting endogenous cytoprotective effects.
引文
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[14]Li J,Guo W,Xiong M,et al. Erythropoietin facilitates the recruit-ment of bone marrow mesenchymal stem cells to sites of spinal cordinjury[J]. Exp Ther Med,2017,13(5):1806-1812.
[15]Bermudez S,Khayrullina G,Zhao Y. NADPH oxidase isoform ex-pression is temporally regulated and may contribute to microglial/macrophage polarization after spinal cord injury[J]. Mol Cell Neu-rosci,2016,77:53-64.
[16]Amri F,Ghouili I,Amri M,et al. Neuroglobin protects astroglialcells from hydrogen peroxide induced oxidative stress and apoptoticcell death[J]. J Neurochem,2017,140(1):151-169.
[17]Arbo BD,Benetti F,Ribeiro MF. Astrocytes as a target for neuro-protection:Modulation by progesterone and dehydroepiandrosterone[J]. Progress Neurobiol,2016,144:27-47.
[18]Nathan FM. Environmental cues determine the fate of astrocytes af-ter spinal cord injury[J]. Neural Regen Res,2017,12(12):1964-1970.
[19] Fabrizius A,Andre D,Laufs T,et al. Critical re-evaluation ofneuroglobin expression reveals conserved patterns among mammals[J]. Neurosciense,2016,3337:339-354.
[20]Douiri S,Bahdoudi S,Hamdi Y,et al. Involvement of endogenousantioxidant systems in the protective activity of pituitary adenylatecyclase-activating polypeptide against hydrogen peroxide-inducedoxidative damages in cultured rat astrocytes[J]. J Neurochem,2016,137(6):913-930.
[2]Siddiqui AM,Khazaei M,Fehlings MG. Translating mechanisms ofneuroprotection,regeneration,and repair to treatment of spinalcord injury[J]. Prog Brain Res,2015,218:15-54.
[3]Fatima G,Sharma VP,Das SK. Oxidative stress and antioxidativeparameters in patients with spinal cord injury:implications in thepathogenesis of disease[J]. Spinal Cord,2015,53(1):3-6.
[4]Yu Z,Poppe JL,Wang X. mitochondrial mechanisms of neuroglo-bin's neuroprotection[J]. Oxid Med Cell Longev,2013,2013:1-11.
[5]Stepien KM,Heaton R,Rankin S,et al. Evidence of oxidativestress and secondary mitochondrial dysfunction in metabolic andnon-metabolic disorders[J]. J Clin Med,2017,6(7):pii:E71.
[6]Guo G. Hypoxia/reoxygenation differentially modulates NF-kappaBactivation and i NOSexpression in astrocytes and microglia[J]. An-tioxid Redox Signal,2006,8(5-6):911-918.
[7]Okada S,Nakamura M,Katoh H,et al. Conditional ablation ofStat3 or Socs3 discloses a dual role for reactive astrocytes after spi-nal cord injury[J]. Nat Med,2006,12(7):829-834.
[8]Anderson MA,Burda JE,Ren Y,et al. Astrocyte scar formationaids central nervous system axon regeneration[J]. Nature,2016,532(7598):195-200.
[9]Watson JL,Hala TJ,Putatunda R,et al. Persistent at-level ther-mal hyperalgesia and tactileallodynia accompany chronic neuronaland astrocyte activation in superficial dorsal horn following mousecervical contusion spinal cord inury[J]. PLo S One,2014,9(9):e109099.
[10] Brittain T. The anti-apoptotic role of neuroglobin[J]. Cells,2012,1(4):1133-1155.
[11]Ascenzi P,di Masi A,Leboffe L,et al. Neuroglobin:From struc-ture to function in health and disease[J]. Mol Aspects Med,2016,52:1-48.
[12]Baez E,Echeverria V,Cabezas R,et al. Protection by Neuroglo-bin Expression in Brain Pathologies[J]. Front Neurol,2016,7:146.
[13]Della Valle B,Hempel C,Kurtzhals JA. In vivo expression of neu-roglobin in reactive astrocytes during neuropathology in murinemodels of traumatic brain injury,cerebral malaria,and autoimmuencephalitis[J]. Glia,2010,58(10):1220-1227.
[14]Li J,Guo W,Xiong M,et al. Erythropoietin facilitates the recruit-ment of bone marrow mesenchymal stem cells to sites of spinal cordinjury[J]. Exp Ther Med,2017,13(5):1806-1812.
[15]Bermudez S,Khayrullina G,Zhao Y. NADPH oxidase isoform ex-pression is temporally regulated and may contribute to microglial/macrophage polarization after spinal cord injury[J]. Mol Cell Neu-rosci,2016,77:53-64.
[16]Amri F,Ghouili I,Amri M,et al. Neuroglobin protects astroglialcells from hydrogen peroxide induced oxidative stress and apoptoticcell death[J]. J Neurochem,2017,140(1):151-169.
[17]Arbo BD,Benetti F,Ribeiro MF. Astrocytes as a target for neuro-protection:Modulation by progesterone and dehydroepiandrosterone[J]. Progress Neurobiol,2016,144:27-47.
[18]Nathan FM. Environmental cues determine the fate of astrocytes af-ter spinal cord injury[J]. Neural Regen Res,2017,12(12):1964-1970.
[19] Fabrizius A,Andre D,Laufs T,et al. Critical re-evaluation ofneuroglobin expression reveals conserved patterns among mammals[J]. Neurosciense,2016,3337:339-354.
[20]Douiri S,Bahdoudi S,Hamdi Y,et al. Involvement of endogenousantioxidant systems in the protective activity of pituitary adenylatecyclase-activating polypeptide against hydrogen peroxide-inducedoxidative damages in cultured rat astrocytes[J]. J Neurochem,2016,137(6):913-930.
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