全脑缺血再灌注大鼠海马CA1区Ngb与Caspase-3的相关性研究
|
摘要
目的：通过观察全脑缺血再灌注后Sprague-Dawley(SD)大鼠海马CA1区脑红蛋白(Ngb)与Caspase-3的时间变化关系,分析脑红蛋白与Caspase-3表达时间的规律性及其两者的相关性机制,为临床抗缺血损伤提供参考依据。
方法：随机将60只健康SD大鼠雌雄各半随机分为全脑缺血／再灌注(I/R)组,假手术(SO)组。每组按其再灌注的时间又分为3、6、12、24、48小时(h)共5个观察时间点,每个时间点6只大鼠,采用改良的Pulsinelli等四血管阻塞法(椎动脉留置针法)建立大鼠脑I/R损伤的模型。两组于再灌注后腹腔注射生理盐水(0.5ml/kg),并每6h注射一次,在相应的时间点断头取脑。用免疫组化法和原位杂交法检测海马CA1区锥体细胞中Ngb及Caspase-3的表达,测量其平均光密度值(OD),用苏木精-伊红(HE)染色检测存活锥体细胞数,用TUNEL原位末端标记法检测凋亡锥体细胞,并计算凋亡率。
结果：在SO组,Caspase-3蛋白及Caspase-3 mRNA、Ngb蛋白及Ngb mRNA各时间点表达的平均光密度值对比差异无统计学意义(P>0.05),存活神经元细胞数及细胞凋亡率的各时间点比较差异均无统计学意义(P>0.05)。在I/R组,Caspase-3蛋白及Caspase-3 mRNA在缺血再灌注后3小时开始表达,24小时达高峰,48小时后逐渐下降,24小时与3、6、12、48小时比较差异有统计学意义(P<0.05)；Ngb蛋白及Ngb mRNA在缺血再灌注后3、6小时弥散表达,3、12、24、48小时与SO组比较差异有统计学意义(P<0.05)；存活神经元细胞数及细胞凋亡率的各时间点比较差异均有统计学意义(P<0.05)。SO组各时间点的Caspase-3和Ngb比较差异无统计学意义(P>0.05)。I/R组各时间点的Caspase-3和Ngb比较差异有统计学意义(P<0.05)。Ngb与Caspase-3呈负相关(r=-0.882,P<0.05)；Ngb与细胞凋亡率呈负相关(r=-0.984,p<0.01); Caspase-3与细胞凋亡率呈正相关(r=0.994,P<0.01)。
结论：(1)脑缺血再灌注损伤后Caspase-3呈时间递增性表达,Ngb呈递减性表达;
(2) Caspase-3与Ngb呈负相关性,Ngb可能抑制细胞的凋亡。
Objective:To investigate the expression of neuroglobin (Ngb) and Caspase-3 in hippocampus CA1 region of the Sprague-Dawley (SD) rats with global cerebral ischemia/reperfusion(I/R) injury and analyze the expression regularity of Ngb and Caspase-3 in differernt time,meanwhile analyze the relevance of Ngb and Caspase-3 distribution;discuss the effect mechanism of neuroglobin and Caspase-3.provide a reference for clinical.
Methods:60 healthy SD rats were randomly divided into 2 groups:sham operation (SO) group and I/R group.each group was assigned into 5 observing time-points: 3,6,12,24,48 hours (h) according to reperfusion time. The SD rats model of global cerebral ischemica/reperfusion was produced by means of simple Pulsinelli-Brierley's four arteries occlusion method.SO and I/R groups were both intraperitoneally injected with 0.9% Sodium chloride (dose:0.5ml/kg) per six hours after reperfusion.Then SD rats'cerebrums were taken out of their skull at each of observing time-point.The expression activation of Caspase-3 and Ngb protein in pyramidal cells in hippocampus comuammonis (CA)1 region were examined by immunohistochemical method (SABC) and Caspase-3 mRNA and Ngb mRNA by in situ hybridization, measured their average optical density(OD),and hematoxylin and eosin (HE) staining was also performed to detect the number of surviving pyramidal cells,and terminal-deoxynucleotidy transferase medatd dUTP nick end labeling (TUNEL) was used to detect apoptotic pyramidal cells (positive cells), And calculated the apoptotic rate of cells.
Results:SO group,the expression of average optical density (OD) of Caspase-3 protein and Caspase-3 mRNA,Ngb and Ngb mRNA were not statistically significant(P>0.05);the number of surviving neurons and apoptotic rate of cells was no significant changes,there was no statistically significant (P>0.05);In the I/R group, Caspase-3 protein and Caspase-3 mRNA began to express at 3h after reperfusion,at the top at 24h,decreased gradually after 48h,24h has variability conspicuous statistically significant compared with3、6、12、48h (P<0.05);Ngb protein and Ngb mRNA's dispersively expressed at 3h,6h after reperfusion,3、12、24、48h compared with the SO group was statistically significant(p<0.05);the number of surviving neurons and apoptotic rate of cells was significant changes,there was statistically significant (P<0.05).In SO group,Caspase-3 compared with the Ngb was not statistically significant(P>0.05). In the I/R group was statistically significant(P<0.05). Correlated analysis showed that there was negative correlation between the expression levels of Ngb and Caspase-3(r=-0.882,p<0.05),Ngb and apoptotic rate of cells(r =-0.984, P<0.01);positive correlation between Caspase-3 and apoptotic rate of cells (r =0.994, P<0.01).
Conclusion:(1) After cerebral ischemia-reperfusion injury Caspase-3'expression was increased with time,meanwhile Ngb was reduced; (2) Caspase-3 has a negative correlation with the Ngb after global cerebal ischemia/reperfusion injury,neuroglobin may inhibit neuronal apoptosis.
方法：随机将60只健康SD大鼠雌雄各半随机分为全脑缺血／再灌注(I/R)组,假手术(SO)组。每组按其再灌注的时间又分为3、6、12、24、48小时(h)共5个观察时间点,每个时间点6只大鼠,采用改良的Pulsinelli等四血管阻塞法(椎动脉留置针法)建立大鼠脑I/R损伤的模型。两组于再灌注后腹腔注射生理盐水(0.5ml/kg),并每6h注射一次,在相应的时间点断头取脑。用免疫组化法和原位杂交法检测海马CA1区锥体细胞中Ngb及Caspase-3的表达,测量其平均光密度值(OD),用苏木精-伊红(HE)染色检测存活锥体细胞数,用TUNEL原位末端标记法检测凋亡锥体细胞,并计算凋亡率。
结果：在SO组,Caspase-3蛋白及Caspase-3 mRNA、Ngb蛋白及Ngb mRNA各时间点表达的平均光密度值对比差异无统计学意义(P>0.05),存活神经元细胞数及细胞凋亡率的各时间点比较差异均无统计学意义(P>0.05)。在I/R组,Caspase-3蛋白及Caspase-3 mRNA在缺血再灌注后3小时开始表达,24小时达高峰,48小时后逐渐下降,24小时与3、6、12、48小时比较差异有统计学意义(P<0.05)；Ngb蛋白及Ngb mRNA在缺血再灌注后3、6小时弥散表达,3、12、24、48小时与SO组比较差异有统计学意义(P<0.05)；存活神经元细胞数及细胞凋亡率的各时间点比较差异均有统计学意义(P<0.05)。SO组各时间点的Caspase-3和Ngb比较差异无统计学意义(P>0.05)。I/R组各时间点的Caspase-3和Ngb比较差异有统计学意义(P<0.05)。Ngb与Caspase-3呈负相关(r=-0.882,P<0.05)；Ngb与细胞凋亡率呈负相关(r=-0.984,p<0.01); Caspase-3与细胞凋亡率呈正相关(r=0.994,P<0.01)。
结论：(1)脑缺血再灌注损伤后Caspase-3呈时间递增性表达,Ngb呈递减性表达;
(2) Caspase-3与Ngb呈负相关性,Ngb可能抑制细胞的凋亡。
Objective:To investigate the expression of neuroglobin (Ngb) and Caspase-3 in hippocampus CA1 region of the Sprague-Dawley (SD) rats with global cerebral ischemia/reperfusion(I/R) injury and analyze the expression regularity of Ngb and Caspase-3 in differernt time,meanwhile analyze the relevance of Ngb and Caspase-3 distribution;discuss the effect mechanism of neuroglobin and Caspase-3.provide a reference for clinical.
Methods:60 healthy SD rats were randomly divided into 2 groups:sham operation (SO) group and I/R group.each group was assigned into 5 observing time-points: 3,6,12,24,48 hours (h) according to reperfusion time. The SD rats model of global cerebral ischemica/reperfusion was produced by means of simple Pulsinelli-Brierley's four arteries occlusion method.SO and I/R groups were both intraperitoneally injected with 0.9% Sodium chloride (dose:0.5ml/kg) per six hours after reperfusion.Then SD rats'cerebrums were taken out of their skull at each of observing time-point.The expression activation of Caspase-3 and Ngb protein in pyramidal cells in hippocampus comuammonis (CA)1 region were examined by immunohistochemical method (SABC) and Caspase-3 mRNA and Ngb mRNA by in situ hybridization, measured their average optical density(OD),and hematoxylin and eosin (HE) staining was also performed to detect the number of surviving pyramidal cells,and terminal-deoxynucleotidy transferase medatd dUTP nick end labeling (TUNEL) was used to detect apoptotic pyramidal cells (positive cells), And calculated the apoptotic rate of cells.
Results:SO group,the expression of average optical density (OD) of Caspase-3 protein and Caspase-3 mRNA,Ngb and Ngb mRNA were not statistically significant(P>0.05);the number of surviving neurons and apoptotic rate of cells was no significant changes,there was no statistically significant (P>0.05);In the I/R group, Caspase-3 protein and Caspase-3 mRNA began to express at 3h after reperfusion,at the top at 24h,decreased gradually after 48h,24h has variability conspicuous statistically significant compared with3、6、12、48h (P<0.05);Ngb protein and Ngb mRNA's dispersively expressed at 3h,6h after reperfusion,3、12、24、48h compared with the SO group was statistically significant(p<0.05);the number of surviving neurons and apoptotic rate of cells was significant changes,there was statistically significant (P<0.05).In SO group,Caspase-3 compared with the Ngb was not statistically significant(P>0.05). In the I/R group was statistically significant(P<0.05). Correlated analysis showed that there was negative correlation between the expression levels of Ngb and Caspase-3(r=-0.882,p<0.05),Ngb and apoptotic rate of cells(r =-0.984, P<0.01);positive correlation between Caspase-3 and apoptotic rate of cells (r =0.994, P<0.01).
Conclusion:(1) After cerebral ischemia-reperfusion injury Caspase-3'expression was increased with time,meanwhile Ngb was reduced; (2) Caspase-3 has a negative correlation with the Ngb after global cerebal ischemia/reperfusion injury,neuroglobin may inhibit neuronal apoptosis.
引文
[1]颜建云,吴伟康.脑缺血损伤的分子机制研究进展[J].中国病理生理杂志2003,19(3)：423-426.
[2]Guo Y,Xu X,Li Q,et al, Anti-inflammation effects of picroside 2 in cerebral ischemic injury rats[J].Behav Brain Funct,2010,6:43.
[3]Pandya JD,Sullivan PG, Pettigrew LC.Focal cerebral ischemia and mitochondrial dysfunctio in the TNFa-transgenic rat[J].Brain Res.2011,1384:151～160.
[4]Li JS,Liu JX,Tian YS,et al.Effects of Naomaitong combined with mobilization of bone marrow mesenchymal stem cells on neuron apoptosis and expressions of Fas,FasL and caspase-3 proteins in rats with cerebral ischemia [J].Zhong Xi Yi Jie He Xue Bao,2009,7(9):860～867
[5]Thomas Brittain,Joanna Skommer,Raychaudhuri S,et al.An Antiapoptotic Neuroprotective Role for Neuroglobin[J].International Journal of Molecular Sciences,2010,11(6):2306-2321.
[6]张兴毅,韩江全,宋国林等.建建立大鼠全脑缺血模型制作的方法改进[J].西部医学.2010,22(1)：18-23.
[7]Ahmed BY,Chakravarthy S,Eggers R,et al.Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors[J].BMC Neurosci,2004,5(1):4-14.
[8]Vincenzo M,Michelangelo I,Carolina M,et al.The protective effect of M40401,a superoxide dismutase mimetic,on post-ischemic brain damage in Monglolian gerbils[J].BMC Pharmacology,2003,6(16):1-9.
[9]Zhang M,Ma YF,Gan JX,et al.Basic fibroblast growth factor alleviates brain injury following global ischemia reperfusion in rabbits[J].J Zhejiang Univ Sci B,2005, 613(7):637～643.
[10]Schmidt-Kastner R,Paschen W,Ophoff BG,et al.A modified four-vessel occlusi-on model for inducing incomplete forebrain ischemia in rats[J]. Stroke,1989,20(7) :938～946.
[11]阳生光,苏科,张兴毅等.改良式大鼠全脑缺血再灌注模型的制作及海马回核因子-K B的表达研究[J].中国全科医学杂志.2010,13(18)：2007-2009.
[12]Shang A,Zhou D,Wang L,et al.Increased neuroglobin levels in the cerebral cortex and serum after ischemia-reperfusion insults[J].Brain Research,2006,1078(1): 219～226.
[13]刘昊.脑红蛋白的研究进展[J].华北煤炭医学院学报,2006,8(6)：777-778.
[14]Fordele E,Thus L,Martinet W,et.al.Neuroglobinand cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death[J].Neurosci Lett,2006,410(2):146～151.
[15]Li RC,Guo SZ,Lee SK,et al.Neuroglobin protects neurons against oxidative stress in global ischemia[J].J Cereb Blood Flow Metab,2010,30(11):1874～1882.
[16]Burmester T,W eich B,Reinhardt S et al.A vertebrate globin expressed in the brain[J].Nature,2000,407(6803):520.
[17]张成岗,尚爱加,周定标等.脑缺血再灌注损伤后大脑皮质和海马区脑红蛋白的表达变化[J].解放军医学杂志,2006,3(11)：1060-1062.
[18]Hundahl CA,Allen GC,Nyengaard JR,et al.Neuroglobin in the rat brain:localizati-on[J].Neuroendocrinology,2008,88(3):173～182.
[19]Powers JM.P53-mediated apoptosis,neuroglobin overexpression,and globin deposits in a patient with hereditary ferritinopathy[J].Neuropathol Exp Neurol, 2006,65(7):716～721
[20]Laufs TL,Wystubs,ReussS,et al.Neuron-specific expression of neuroglobin in mammals[J].Neurosic Lett,2004,362(2):83～86.
[21]Yunjuan Sun,Kunlin Jin,Xiao Ou Mao,et al.Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury[J].PNAS,2001,98(26):15306-15308.
[22]Li RC,Morris MW,Lee SK,et al.Neuroglobin protects PC 12 cells against oxidative stress [J]. Brain Res,2008,23,1190:159～66.
[23]Brunori M,Giuffre A,Nienaush K,et al.Neurogbloin,nitric-oxide and oxygen functional pathways and conformational changes[J].Proc Natl Acad Sci U S A,2005,102(24):8483～8488.
[24]Wakasugi K,Kitatsuji C,Morishimalna l.possible neuroproprotective mechanism Of human neuroglobin[J].Ann N Y Acad Sci,2005,1053:220～230.
[25]徐文琳,王春丽,张永亮等.脑红蛋白与Na', K'-ATP酶阶亚基的相互作用及作 用位点的鉴定.生物化学与生物物理学报[J].2003,35：823-827.
[26]Chen H,Kim GS,Okami N,et.al,NADPH oxidase is involved in post-ischemic brain inflammation[J].Neurobiol Dis,2011,42(3):341～348.
[27]Dewild S,BurmesterT, Pesce A,et al.The hunman brain hexacoordinated neurglob-in three-dimensional structure[J].Micron,2004,35(1):63～65.
[28]Bergeron M,Gidday J M,Yu A Y,et.al,Role of hypoxia- inducible-facter-1 in hypoxia-induced ischemic tolerance in noneatal rat brain[J].Ann Neurol,2000, 48(3):285～296.
[29]王航雁,王静,张荣媛,等缺血再灌注损伤大鼠脑组织脑红蛋白表达的变化[J].实用儿科临床杂志,2008,23(21)：1680-1682.
[30]Fordel E,Geuens E,Dewilde S,et al.Hypoxia/ischemia and the regulation of neuroglobin and cytoglobin expression[J].IUBMB Life,2004,56(11-12):681～687.
[31]Fan TJ,Han LH,Cong RS,et al.Caspase family proteases and apoptosis[J].Acta Biochim Biophys Sin,2005,37(11):719～727.
[32]Green DR.Overview:apoptotic signaling pathways in the immune syste- m.[J] Immunol Rev.2003,193:5-9.
[33]Gibson ME,Han BH,Choi J,et al.BAX contributes to apoptotic-like death following neonatal hypoxia-ischemia:evidence for distinct apoptosis pathwa-ys[J].Mol Med,2001,7(9):644～655.
[34]Weisleder N,Taffet GE,Capetanaki Y,et al.BCL-2 overexpression corrects mito-chondrial defects and ameliorates inherited desmin null cardiomyopathy[J].Proc Natl Acad Sci USA,2004,101(3):769～774.
[35]Pandey P,Salch A,Nakazawa A,et.al,Negative regulation of cytochromec mediated oligomeriza -tion of Apaf-1 and activation of procaspase-9 by heart shock protein90[J]. EMBOJ,2000,19(2):4996-5006.
[36]王华梅,李军荣,吴家幂,等.大鼠脑缺血再灌注后Caspase-3、Bcl-2和Bax的表达[J].神经疾病与精神卫生,2008,8(3)：190-192.
[37]Raychaudhuri S,Skommer J,Henty K,et al.Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death[J].Apoptosis,2010,15 (4):401～411.
[38]陈浩,臧贺川,伊红丽,等.一氧化碳中毒大鼠脑红蛋白表达变化及对半胱氨 酸天冬氨酸蛋白酶3表达的影响[J].中国临床康复,2006,10(2)：73-76.
[39]Duong TT,Witting PK,Antao ST,et al.Multiple protective activities of neuroglobin in cultured neuronal cells exposed to hypoxia re-oxygenation injury [J].J Neurochem,2009,108(5):1143～1154.
[40]李佩尧,张成岗.细胞内低氧感受器：缺氧诱导因子脯氨酰羟化酶研究进展[J].生理科学进展,2007,38(1)：62-64.
[41]Li L,Qu Y,Mao M,Zhang L,et al.Relationship between hypoxia inducible factor 1 alpha:expression and neuron apoptosis during hypoxia ischemia brain damage in neonatal rats[J].Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi.2007,21(12):1326-1329.
[42]Hong LZ,Zhao XY,Zhang HL.p53-mediated neuronal cell death in ischemic brain injury[J].Neuroscience bulletin,2010,26(3):232～240.
[43]陈婷芳.脑红蛋白神经保护功能及其机制的初步研究[D].北京：中国人民解放军军事医学科学院,2008.
[1]Burmester T,Weich B,Reinhardt S,et al.A vertebrate globin expressed in the brain[J].Nature,2000,407(6803):520.
[2]颜建云,吴伟康.脑缺血损伤的分子机制研究进展[J].中国病理生理杂志,2003,19(3)：423-426.
[3]Yao J,Xu Y,Ji F,et al. Protective effects of MLIF analogs on cerebral ischemia-reperfusio-n injury in rats[J].Peptides,2011,32(5):1047～1054.
[4]Jantzie LL,Cheung PY,Johnson ST,et al.Cerebral amino acid profiles after hypoxia-reoxygenation and N-acetylcysteine treatment in the newborn piglet[J].Neonatology.2010,97(3):195～203.
[5]Thomas Brittain,Joanna Skommer,Raychaudhuri S,et al.An Antiapoptotic Neurop-rotective Role for Neuroglobin[J],International Journal of Molecular Sciences, 2010,11(6):2306～2321.
[6]KunlinJin,XiaoOuMao,Khan AA,et al.Neuroglobin Protects Against Nitric Oxide Toxicity[J].Neurosci Lett,2008,430(2):135～137.
[7]Bates B,Hirt L,Thomas SS,et al.Neurotrophin-3 promotes cell death in duced in Cerebral ischemia,oxygen-glucosed eprivation,and oxidative stress:possible in volveent of oxygen free radi-Gals[J].NeurobiolD is,2002,9(1):24～37.
[8]Sun M,Gu Y, Zhao Y,et al.Protective functions of taurine against experimental stroke through depressing mitochondria-mediated cell death in rats[J].Amino Acids,2011,40(5):1419～1429.
[9]Aoki T,Sumii T,Mori T,et al.Blood-brain barrier disruptionand matrixm etalloproteinas-9 expression during reperfusion injury:mechanicalv ersus embolic focal ischemia in spontaneously hypertensive rats[J].S troke,2002,33(11):2711～ 2777.
[10]Tu YF,Tsai YS, Wang LW,et al.Overweight Worsens Apoptosis,Neuroinflammati-on and Blood-Brain Barrier Damage after Hypoxic Ischemia in Neonatal Brain through JNK Hyperactivation[J].JNeuroinflammation,2011,28(1):40.
[11]赵泽燕.内质网应激与未成熟脑组织缺氧缺血性脑损伤[J].中华医学写作杂志,2005,12：1236-1239.
[12]Wang HK,Park UJ, Kim SY,et al.Free radical production in CA1 neurons induces MIP-1 alpha expression,microglia recruitment, and delayed neuronal death after transient forebrain ischemia[J].J Neurosci,2008,28(7):1721～1727.
[13]Khan AA,Wang Y,Sun Y,et al.Neuroglobin-overexpressing transgenic mice are resistant to cerebral and myocardial ischemia[J].Proc Natl Acad Sci USA,2006, 103(47):17944～17948.
[14]Susanna Herold,Angela Fago, Weber RE,et al.Reactivity Studies of the Fe(Ⅲ) and Fe(II)NO Forms of Human Neuroglobin Reveal a Potential Role against Oxidative Stress[J].2004,279:22841～22847.
[15]Brunori M,Giuffre A,Nienaush K,et al.Neurogbloin,nitric-oxide,and oxygen:fun-ctional pathways and conformational changes[J].Proc Natl Acad Sci USA,2005, 102(24):8483～8488.
[16]Wakasugi K,Kitatsuji C,Morishimalna Ⅰ.possible neuroproprotective mechanism Of human neuroglobin[J]Ann N Y Acad Sci,2005,1053:220-230.
[17]Hamdane D,Kiger I,DewiMe S,et al.Coupling of the heine and an internal disulfide bond in human neuroglobin[J].Micron,2004,35(1):59～62.
[18]邓美玉,张成岗,王航雁等.脑红蛋白mRNA在大鼠脑内的定位[J].解剖学报,2003,34：85-89.
[19]Hundahl CA,Allen GC,Nyengaard JR,Dewilde S,et al.Neuroglobin in the rat brain:localization[J].Neuroendocrinology,2008,88(3):173～182.
[20]Schmidt M,Giessl A,Laufs T,et al.How does the eye breathe?Evdence for neuroglobin-mediated oxygen supply in the matnmalianretina JI[J]. BiolChem, 2003,278(3):1932～1935.
[21]Powers JM.P53-mediated apoptosis,neuroglobin overexpression,and globin deposits in a patient with hereditary ferritinopathy[J].Neuropathol Exp Neurol, 2006,65:716～721.
[22]LaufsTL,Wystubs,ReussS,et al.Neuron-specific expression of neuroglobin in mammals[J].Neurosic Lett,2004,362(2):83～86.
[23]Maurizio Brunori,Alessandro Giuffre,Nienhaus K,et al.Neuroglobin,nitric oxide and oxygen:Functional pathways and conformational changes[J].Biochemistry,2005 ,102(24):8483～8488.
[24]张荣媛,尚爱加.脑红蛋白在大鼠脑皮层缺血半影区表达的变化[J].军医进修学院学报,2008,29(6)：526-527.
[25]Li W,Wu Y,Ren C,Lu Y,et al.The activity of recombinant human neuroglobin as an antioxidant and free radical scavenger[J].Proteins,2011,79(1):115～125.
[26]Antao ST,Duong TT,Aran R,et al.Neuroglobin overexpression in cultured human neuronal cells protects against hydrogen peroxide insult via activating phosphoinositide-3 kinase and opening the mitochondrial K(ATP) channel [J]. antioxid Redox Signal,2010,13(6):769～781.
[27]Li RC,Guo SZ,Lee SK,et al.Neuroglobin protects neurons against oxidative stress in global ischemia[J].J Cereb Blood Flow Metab.,2010,30(11):1874～1882
[28]Hua S,Antao ST,Corbett A,et al.The significance of neuroglobin in the brain[J].Curr Med Chem,2010,17(2):160～172.
[29]Nicolis S,Monzani E,Ciaccio C,et al.Does human neuroglobin act only as a scavenger reactivity and endogenous modification by nitrite and hydrogen peroxide[J].J Biochem,2007,407(1):89～99.
[30]Wakasugi K,Morishima Ⅰ.Ideti of residues in human neuroglobin crucial for guanine nucl-eotide dissociation inhibitor activity[J].Biochemistry,2005,44(8): 2943～2948.
[31]Duong TT,Witting PK,Antao ST,et al.Multiple protective activities of neuroglobin in cultured neuronal cells exposed to hypoxia re-oxygenation injury[J].J Neurochem,2009,108(5):1143～1154.
[32]Li W,Wu Y,Ren C,et al.The activity of recombinant human neuroglobin as an antioxidant and free radical scavenger[J].Proteins,2011,79(1):115～125.
[33]Xiao ying Wang MD,PhD,Jianxiang Liu PhD,et al.Effects of Neuroglobin Over-expression on Acute Brain Injury and Long-Term Outcomes After Focal Cerebral Ischemia[J].Stroke,2008,39(6):1869～1874.
[34]Jianxiang Liu,Zhanyang Yu,Lee SR,et al.Effects of neuroglobin over-expression on mitochondrial function and oxidative stress following hypoxia/reoxygenation in cultured neurons[J].J Neurosci Res.2009,87(1):164～170.
[35]Zhanyang Yu,Jianxiang Liu,Xing C,et al.Neuroglobin-overexpression Alters Hypoxic response Gene Expression in Primary Neuron Culture Following Oxygen Glucose deprivation[J].Neuroscience,2009,162(2):396～403.
[36]Raychaudhuri S,Skommer J,Henty K,et al.Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death[J].Apoptosis,2010,15(4) :401～411.
[37]Watanabe S,Wakasugi K.Neuroprotective function of human neuroglobin is correlated with its guanine nucleotide dissociation inhibitor activity[J].Biochem biophys Res Commun,2008,369(2):695～700.
[38]Wakasugi K,Kitatsuji C,Morishimalna l.possible neuroproprotective mechanism of human neuroglobin[J].Ann N Y Acad Sci,2005,1053:220-230.
[39]Wakasugi K,Nakano T,et al.Human neuroglobin interaets with flotillin-1, a lipid raft micro domain associated protein[J].Bioch Biophy Reseom,2004,318(2):453～ 460.
[40]Bergeron M,Gidday J M,Yu A Y,et al.Role of hypoxia-inducible-facter-1 in hypoxia-in-duced ischemic tolerance in noneatal rat brain[J].Ann Neurol,2000,48 (3):285～296.
[41]徐文琳,王春丽,张永亮,等.脑红蛋白与Na+, K+-ATP酶阶亚基的相互作用及作用位点的鉴定[J].生物化学与生物物理学报,2003,35：823-827.
[2]Guo Y,Xu X,Li Q,et al, Anti-inflammation effects of picroside 2 in cerebral ischemic injury rats[J].Behav Brain Funct,2010,6:43.
[3]Pandya JD,Sullivan PG, Pettigrew LC.Focal cerebral ischemia and mitochondrial dysfunctio in the TNFa-transgenic rat[J].Brain Res.2011,1384:151～160.
[4]Li JS,Liu JX,Tian YS,et al.Effects of Naomaitong combined with mobilization of bone marrow mesenchymal stem cells on neuron apoptosis and expressions of Fas,FasL and caspase-3 proteins in rats with cerebral ischemia [J].Zhong Xi Yi Jie He Xue Bao,2009,7(9):860～867
[5]Thomas Brittain,Joanna Skommer,Raychaudhuri S,et al.An Antiapoptotic Neuroprotective Role for Neuroglobin[J].International Journal of Molecular Sciences,2010,11(6):2306-2321.
[6]张兴毅,韩江全,宋国林等.建建立大鼠全脑缺血模型制作的方法改进[J].西部医学.2010,22(1)：18-23.
[7]Ahmed BY,Chakravarthy S,Eggers R,et al.Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors[J].BMC Neurosci,2004,5(1):4-14.
[8]Vincenzo M,Michelangelo I,Carolina M,et al.The protective effect of M40401,a superoxide dismutase mimetic,on post-ischemic brain damage in Monglolian gerbils[J].BMC Pharmacology,2003,6(16):1-9.
[9]Zhang M,Ma YF,Gan JX,et al.Basic fibroblast growth factor alleviates brain injury following global ischemia reperfusion in rabbits[J].J Zhejiang Univ Sci B,2005, 613(7):637～643.
[10]Schmidt-Kastner R,Paschen W,Ophoff BG,et al.A modified four-vessel occlusi-on model for inducing incomplete forebrain ischemia in rats[J]. Stroke,1989,20(7) :938～946.
[11]阳生光,苏科,张兴毅等.改良式大鼠全脑缺血再灌注模型的制作及海马回核因子-K B的表达研究[J].中国全科医学杂志.2010,13(18)：2007-2009.
[12]Shang A,Zhou D,Wang L,et al.Increased neuroglobin levels in the cerebral cortex and serum after ischemia-reperfusion insults[J].Brain Research,2006,1078(1): 219～226.
[13]刘昊.脑红蛋白的研究进展[J].华北煤炭医学院学报,2006,8(6)：777-778.
[14]Fordele E,Thus L,Martinet W,et.al.Neuroglobinand cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death[J].Neurosci Lett,2006,410(2):146～151.
[15]Li RC,Guo SZ,Lee SK,et al.Neuroglobin protects neurons against oxidative stress in global ischemia[J].J Cereb Blood Flow Metab,2010,30(11):1874～1882.
[16]Burmester T,W eich B,Reinhardt S et al.A vertebrate globin expressed in the brain[J].Nature,2000,407(6803):520.
[17]张成岗,尚爱加,周定标等.脑缺血再灌注损伤后大脑皮质和海马区脑红蛋白的表达变化[J].解放军医学杂志,2006,3(11)：1060-1062.
[18]Hundahl CA,Allen GC,Nyengaard JR,et al.Neuroglobin in the rat brain:localizati-on[J].Neuroendocrinology,2008,88(3):173～182.
[19]Powers JM.P53-mediated apoptosis,neuroglobin overexpression,and globin deposits in a patient with hereditary ferritinopathy[J].Neuropathol Exp Neurol, 2006,65(7):716～721
[20]Laufs TL,Wystubs,ReussS,et al.Neuron-specific expression of neuroglobin in mammals[J].Neurosic Lett,2004,362(2):83～86.
[21]Yunjuan Sun,Kunlin Jin,Xiao Ou Mao,et al.Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury[J].PNAS,2001,98(26):15306-15308.
[22]Li RC,Morris MW,Lee SK,et al.Neuroglobin protects PC 12 cells against oxidative stress [J]. Brain Res,2008,23,1190:159～66.
[23]Brunori M,Giuffre A,Nienaush K,et al.Neurogbloin,nitric-oxide and oxygen functional pathways and conformational changes[J].Proc Natl Acad Sci U S A,2005,102(24):8483～8488.
[24]Wakasugi K,Kitatsuji C,Morishimalna l.possible neuroproprotective mechanism Of human neuroglobin[J].Ann N Y Acad Sci,2005,1053:220～230.
[25]徐文琳,王春丽,张永亮等.脑红蛋白与Na', K'-ATP酶阶亚基的相互作用及作 用位点的鉴定.生物化学与生物物理学报[J].2003,35：823-827.
[26]Chen H,Kim GS,Okami N,et.al,NADPH oxidase is involved in post-ischemic brain inflammation[J].Neurobiol Dis,2011,42(3):341～348.
[27]Dewild S,BurmesterT, Pesce A,et al.The hunman brain hexacoordinated neurglob-in three-dimensional structure[J].Micron,2004,35(1):63～65.
[28]Bergeron M,Gidday J M,Yu A Y,et.al,Role of hypoxia- inducible-facter-1 in hypoxia-induced ischemic tolerance in noneatal rat brain[J].Ann Neurol,2000, 48(3):285～296.
[29]王航雁,王静,张荣媛,等缺血再灌注损伤大鼠脑组织脑红蛋白表达的变化[J].实用儿科临床杂志,2008,23(21)：1680-1682.
[30]Fordel E,Geuens E,Dewilde S,et al.Hypoxia/ischemia and the regulation of neuroglobin and cytoglobin expression[J].IUBMB Life,2004,56(11-12):681～687.
[31]Fan TJ,Han LH,Cong RS,et al.Caspase family proteases and apoptosis[J].Acta Biochim Biophys Sin,2005,37(11):719～727.
[32]Green DR.Overview:apoptotic signaling pathways in the immune syste- m.[J] Immunol Rev.2003,193:5-9.
[33]Gibson ME,Han BH,Choi J,et al.BAX contributes to apoptotic-like death following neonatal hypoxia-ischemia:evidence for distinct apoptosis pathwa-ys[J].Mol Med,2001,7(9):644～655.
[34]Weisleder N,Taffet GE,Capetanaki Y,et al.BCL-2 overexpression corrects mito-chondrial defects and ameliorates inherited desmin null cardiomyopathy[J].Proc Natl Acad Sci USA,2004,101(3):769～774.
[35]Pandey P,Salch A,Nakazawa A,et.al,Negative regulation of cytochromec mediated oligomeriza -tion of Apaf-1 and activation of procaspase-9 by heart shock protein90[J]. EMBOJ,2000,19(2):4996-5006.
[36]王华梅,李军荣,吴家幂,等.大鼠脑缺血再灌注后Caspase-3、Bcl-2和Bax的表达[J].神经疾病与精神卫生,2008,8(3)：190-192.
[37]Raychaudhuri S,Skommer J,Henty K,et al.Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death[J].Apoptosis,2010,15 (4):401～411.
[38]陈浩,臧贺川,伊红丽,等.一氧化碳中毒大鼠脑红蛋白表达变化及对半胱氨 酸天冬氨酸蛋白酶3表达的影响[J].中国临床康复,2006,10(2)：73-76.
[39]Duong TT,Witting PK,Antao ST,et al.Multiple protective activities of neuroglobin in cultured neuronal cells exposed to hypoxia re-oxygenation injury [J].J Neurochem,2009,108(5):1143～1154.
[40]李佩尧,张成岗.细胞内低氧感受器：缺氧诱导因子脯氨酰羟化酶研究进展[J].生理科学进展,2007,38(1)：62-64.
[41]Li L,Qu Y,Mao M,Zhang L,et al.Relationship between hypoxia inducible factor 1 alpha:expression and neuron apoptosis during hypoxia ischemia brain damage in neonatal rats[J].Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi.2007,21(12):1326-1329.
[42]Hong LZ,Zhao XY,Zhang HL.p53-mediated neuronal cell death in ischemic brain injury[J].Neuroscience bulletin,2010,26(3):232～240.
[43]陈婷芳.脑红蛋白神经保护功能及其机制的初步研究[D].北京：中国人民解放军军事医学科学院,2008.
[1]Burmester T,Weich B,Reinhardt S,et al.A vertebrate globin expressed in the brain[J].Nature,2000,407(6803):520.
[2]颜建云,吴伟康.脑缺血损伤的分子机制研究进展[J].中国病理生理杂志,2003,19(3)：423-426.
[3]Yao J,Xu Y,Ji F,et al. Protective effects of MLIF analogs on cerebral ischemia-reperfusio-n injury in rats[J].Peptides,2011,32(5):1047～1054.
[4]Jantzie LL,Cheung PY,Johnson ST,et al.Cerebral amino acid profiles after hypoxia-reoxygenation and N-acetylcysteine treatment in the newborn piglet[J].Neonatology.2010,97(3):195～203.
[5]Thomas Brittain,Joanna Skommer,Raychaudhuri S,et al.An Antiapoptotic Neurop-rotective Role for Neuroglobin[J],International Journal of Molecular Sciences, 2010,11(6):2306～2321.
[6]KunlinJin,XiaoOuMao,Khan AA,et al.Neuroglobin Protects Against Nitric Oxide Toxicity[J].Neurosci Lett,2008,430(2):135～137.
[7]Bates B,Hirt L,Thomas SS,et al.Neurotrophin-3 promotes cell death in duced in Cerebral ischemia,oxygen-glucosed eprivation,and oxidative stress:possible in volveent of oxygen free radi-Gals[J].NeurobiolD is,2002,9(1):24～37.
[8]Sun M,Gu Y, Zhao Y,et al.Protective functions of taurine against experimental stroke through depressing mitochondria-mediated cell death in rats[J].Amino Acids,2011,40(5):1419～1429.
[9]Aoki T,Sumii T,Mori T,et al.Blood-brain barrier disruptionand matrixm etalloproteinas-9 expression during reperfusion injury:mechanicalv ersus embolic focal ischemia in spontaneously hypertensive rats[J].S troke,2002,33(11):2711～ 2777.
[10]Tu YF,Tsai YS, Wang LW,et al.Overweight Worsens Apoptosis,Neuroinflammati-on and Blood-Brain Barrier Damage after Hypoxic Ischemia in Neonatal Brain through JNK Hyperactivation[J].JNeuroinflammation,2011,28(1):40.
[11]赵泽燕.内质网应激与未成熟脑组织缺氧缺血性脑损伤[J].中华医学写作杂志,2005,12：1236-1239.
[12]Wang HK,Park UJ, Kim SY,et al.Free radical production in CA1 neurons induces MIP-1 alpha expression,microglia recruitment, and delayed neuronal death after transient forebrain ischemia[J].J Neurosci,2008,28(7):1721～1727.
[13]Khan AA,Wang Y,Sun Y,et al.Neuroglobin-overexpressing transgenic mice are resistant to cerebral and myocardial ischemia[J].Proc Natl Acad Sci USA,2006, 103(47):17944～17948.
[14]Susanna Herold,Angela Fago, Weber RE,et al.Reactivity Studies of the Fe(Ⅲ) and Fe(II)NO Forms of Human Neuroglobin Reveal a Potential Role against Oxidative Stress[J].2004,279:22841～22847.
[15]Brunori M,Giuffre A,Nienaush K,et al.Neurogbloin,nitric-oxide,and oxygen:fun-ctional pathways and conformational changes[J].Proc Natl Acad Sci USA,2005, 102(24):8483～8488.
[16]Wakasugi K,Kitatsuji C,Morishimalna Ⅰ.possible neuroproprotective mechanism Of human neuroglobin[J]Ann N Y Acad Sci,2005,1053:220-230.
[17]Hamdane D,Kiger I,DewiMe S,et al.Coupling of the heine and an internal disulfide bond in human neuroglobin[J].Micron,2004,35(1):59～62.
[18]邓美玉,张成岗,王航雁等.脑红蛋白mRNA在大鼠脑内的定位[J].解剖学报,2003,34：85-89.
[19]Hundahl CA,Allen GC,Nyengaard JR,Dewilde S,et al.Neuroglobin in the rat brain:localization[J].Neuroendocrinology,2008,88(3):173～182.
[20]Schmidt M,Giessl A,Laufs T,et al.How does the eye breathe?Evdence for neuroglobin-mediated oxygen supply in the matnmalianretina JI[J]. BiolChem, 2003,278(3):1932～1935.
[21]Powers JM.P53-mediated apoptosis,neuroglobin overexpression,and globin deposits in a patient with hereditary ferritinopathy[J].Neuropathol Exp Neurol, 2006,65:716～721.
[22]LaufsTL,Wystubs,ReussS,et al.Neuron-specific expression of neuroglobin in mammals[J].Neurosic Lett,2004,362(2):83～86.
[23]Maurizio Brunori,Alessandro Giuffre,Nienhaus K,et al.Neuroglobin,nitric oxide and oxygen:Functional pathways and conformational changes[J].Biochemistry,2005 ,102(24):8483～8488.
[24]张荣媛,尚爱加.脑红蛋白在大鼠脑皮层缺血半影区表达的变化[J].军医进修学院学报,2008,29(6)：526-527.
[25]Li W,Wu Y,Ren C,Lu Y,et al.The activity of recombinant human neuroglobin as an antioxidant and free radical scavenger[J].Proteins,2011,79(1):115～125.
[26]Antao ST,Duong TT,Aran R,et al.Neuroglobin overexpression in cultured human neuronal cells protects against hydrogen peroxide insult via activating phosphoinositide-3 kinase and opening the mitochondrial K(ATP) channel [J]. antioxid Redox Signal,2010,13(6):769～781.
[27]Li RC,Guo SZ,Lee SK,et al.Neuroglobin protects neurons against oxidative stress in global ischemia[J].J Cereb Blood Flow Metab.,2010,30(11):1874～1882
[28]Hua S,Antao ST,Corbett A,et al.The significance of neuroglobin in the brain[J].Curr Med Chem,2010,17(2):160～172.
[29]Nicolis S,Monzani E,Ciaccio C,et al.Does human neuroglobin act only as a scavenger reactivity and endogenous modification by nitrite and hydrogen peroxide[J].J Biochem,2007,407(1):89～99.
[30]Wakasugi K,Morishima Ⅰ.Ideti of residues in human neuroglobin crucial for guanine nucl-eotide dissociation inhibitor activity[J].Biochemistry,2005,44(8): 2943～2948.
[31]Duong TT,Witting PK,Antao ST,et al.Multiple protective activities of neuroglobin in cultured neuronal cells exposed to hypoxia re-oxygenation injury[J].J Neurochem,2009,108(5):1143～1154.
[32]Li W,Wu Y,Ren C,et al.The activity of recombinant human neuroglobin as an antioxidant and free radical scavenger[J].Proteins,2011,79(1):115～125.
[33]Xiao ying Wang MD,PhD,Jianxiang Liu PhD,et al.Effects of Neuroglobin Over-expression on Acute Brain Injury and Long-Term Outcomes After Focal Cerebral Ischemia[J].Stroke,2008,39(6):1869～1874.
[34]Jianxiang Liu,Zhanyang Yu,Lee SR,et al.Effects of neuroglobin over-expression on mitochondrial function and oxidative stress following hypoxia/reoxygenation in cultured neurons[J].J Neurosci Res.2009,87(1):164～170.
[35]Zhanyang Yu,Jianxiang Liu,Xing C,et al.Neuroglobin-overexpression Alters Hypoxic response Gene Expression in Primary Neuron Culture Following Oxygen Glucose deprivation[J].Neuroscience,2009,162(2):396～403.
[36]Raychaudhuri S,Skommer J,Henty K,et al.Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death[J].Apoptosis,2010,15(4) :401～411.
[37]Watanabe S,Wakasugi K.Neuroprotective function of human neuroglobin is correlated with its guanine nucleotide dissociation inhibitor activity[J].Biochem biophys Res Commun,2008,369(2):695～700.
[38]Wakasugi K,Kitatsuji C,Morishimalna l.possible neuroproprotective mechanism of human neuroglobin[J].Ann N Y Acad Sci,2005,1053:220-230.
[39]Wakasugi K,Nakano T,et al.Human neuroglobin interaets with flotillin-1, a lipid raft micro domain associated protein[J].Bioch Biophy Reseom,2004,318(2):453～ 460.
[40]Bergeron M,Gidday J M,Yu A Y,et al.Role of hypoxia-inducible-facter-1 in hypoxia-in-duced ischemic tolerance in noneatal rat brain[J].Ann Neurol,2000,48 (3):285～296.
[41]徐文琳,王春丽,张永亮,等.脑红蛋白与Na+, K+-ATP酶阶亚基的相互作用及作用位点的鉴定[J].生物化学与生物物理学报,2003,35：823-827.