单磷酸腺苷激活的蛋白激酶在新生大鼠缺氧缺血性脑损伤中的动态变化被引量：3

Dynamic changes in adenosine monophosphate-activated protein kinase in neonatal rats with hypoxic ischemic encephalopathy

原文传递

导出

摘要目的探讨缺氧缺血损伤后神经细胞内单磷酸腺苷激活的蛋白激酶（adenosine monophosphate activated protein kinase，AMPK）的表达变化。方法以生后24h内和7日龄新生大鼠为研究对象，采用原代培养的新生大鼠大脑皮层神经元糖氧剥夺（oxygen glucose deprivation，OGD）模型及新生大鼠缺氧缺血性脑病（hypoxic ischemic encephalopathy，HIE）模型，乳酸脱氢酶（1acticdehydrogenase，LDH）法检测神经元损伤程度，噻唑蓝[3-（4，5-dimethylthiazol-2-y1）-2，5-diphenyltetrazolium bromide，MTTl检测细胞活力；于OGD及HIE损伤后不同时间点提取组织或细胞蛋白，蛋白质印迹法检测细胞中磷酸化单磷酸腺苷激活的蛋白激酶（phosphorylated adenosine monophosphate activated protein kinase，P—AMPK）／AMPK、磷酸化蛋白激酶（phosphorylated activated protein kinase，P—Akt）／Akt、Cleaved Caspase-3／Caspase-3的表达水平变化。采用Student—t检验进行统计学分析。结果（1）与对照组相比，OGD组LDH水平在2、4、8和24h均明显高于对照组（P值均〈0．05），MTT吸光度低于对照组（P值均〈0．05）。（2）与对照组比较，OGD组P—AMPK水平明显增高，OGD组P—AMPK浓度在30min及2、4、8和24h呈时间依赖性增加，2组上述各时间点的差异均有统计学意义（P值均〈0．05）。HIE组脑组织P—AMPK表达水平明显高于对照组（HIE组1、3和7d分别为0．345±0．038、0．387±0．112和0．618±0．075，对照组为0．132±0．032），差异有统计学意义（P值均〈0．05）。（3）OGD组P—Akt水平在损伤初期（30min）高于对照组（分别为0．991±0．134与0．304±0．050），于2h达峰值（1．183±0．107）．差异有统计学意义（P值均〈0．05），随后迅速下降至正常水平；OGD组Cleaved Caspase-3表达水平明显高于对照组，且呈时间依赖性增加（P值均〈0．05）。结论缺氧缺血损伤后神经细胞内AMPK活性增高，这-趋势随暴露时间延长而更加明显。 Objective To determine the dynamic changes in adenosine monophosphate-activated protein kinase （AMPK） in neurons of neonatal rats suffering from hypoxic ischemic brain injury. Methods Twenty-four-hour old and seven-day old neonatal rats were used in this study. A classic primary cortical neuron oxygen glucose deprivation （OGD） model and neonatal rat hypoxic ischemic encephalopathy （HIE） model were employed. Lactic dehydrogenase （LDH） and 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） were used to evaluate neuron viability and damage. The expression of phosphorylated adenosine monophosphate-activated protein kinase （P-AMPK）, phosphorylated activated protein kinase （P-Akt） and Cleaved Caspase-3 in neurons and brain tissue was measured by Western blot at different time points after OGD or HIE. The Student-t test was used for statistical analysis. Results （1） Compared with the control group, LDH levels at 2, 4, 8 and 24 h after OGD were higher （all P〈0.05） and optical absorption levels of MTT were lower （all P〈0.05）. （2） Levels of P-AMPK in the OGD group were higher than those in the control group, and showed a time-dependent increase at 30 min and 2, 4, 8 and 24 h （all P〈0.05）. The expression levels of P-AMPK in the HIE group were higher than those in the control group （0.345±0.038, 0.387±0.112 and 0.618±0.075 at 1, 3 and 7 days after HIE, and 0.132±0.032 in the control group, all P〈0.05）. （3） The levels of P-Akt increased above the control levels at 30 min （0.991 ±0.134 vs 0.304±0.050）, reached a maximum level at 2 h （1.183 ± 0.107）, and then gradually declined, whereas the levels of Cleaved Caspase-3 started to increase at 30 rain, and remained elevated at 24 h （all P〈0.05）. Conclusion Following hypoxic ischemic brain damage, the expression of P-AMPK is significantly increased in both in vivo and in vitro studies in a time-dependent manner.

作者容志惠刘伟李文斌蔡保欢常立文

机构地区华中科技大学同济医学院附属同济医院儿科

出处《中华围产医学杂志》 CAS CSCD 2016年第8期603-607,共5页 Chinese Journal of Perinatal Medicine

基金第48批留学回国人员科研启动基金湖北省自然科学基金（2013CFB085）

关键词缺氧缺血脑 AMP活化蛋白激酶类动物新生大鼠 Hypoxia-ischemia, brain AMP-activated protein kinases Animals, newborn Rats

分类号 R742 [医药卫生—神经病学与精神病学]

引文网络
相关文献

参考文献15

1Johnston MV. Plasticity in the developing brain: implications for rehabilitation[J]. Dev Disabil Res Rev, 2009,15(2):94-101.
2Skoff RP, Bessert D, Barks JD, et al. Plasticity of neurons and glia following neonatal hypoxic-ischemic brain injury in rats[J]. Neurochem Res, 2007,32(2):331-342.
3Spasic, Callaerts P, Norga KK. AMP-activated protein kinase (AMPK) molecular crossroad for metabolic control and survival of neurons[J]. Neuroscientist, 2009,15(4):309-316.
4Thornton C, Rousset CI, Kichev A, et al. Molecular mechanisms of neonatal brain injury[J]. Neurol Res Int, 2012,2012:506320.
5Pan R, Rong Z, She Y, et al. Sodium pyruvate reduces hypoxic?ischemic injury to neonatal rat brain[J]. Pediatr Res, 2012, 72(5): 479-489.
6Rong Z, Pan R, Xu Y, et al. Hesperidin pretreatment protects hypoxia-ischemic brain injury in neonatal rat[J]. Neuroscience, 2013,255:292-299.
7Rong Z, Pan R, Chang L, et al. Combination treatment with ethyl pyruvate and IGF-I exerts neuroprotective effects against brain injury in a rat model of neonatal hypoxic-ischemic encephalopathy[J]. Int J Mol Med, 2015,36(1):195-203.
8刘伟,李文斌,陈治军,容志惠,常立文.胰岛素样生长因子-1对新生大鼠神经细胞氧化损伤保护作用的研究[J].中国当代儿科杂志,2014,16(2):203-207. 被引量：8
9Graham EM, Ruis KA, Hartman AL, et al. A systematic review of the role of intrapartum hypoxia-ischemia in the causation of neonatal encephalopathy[J]. Am J Obstet Gynecol, 2008,199(6):587-595.
10Verklan MT. The chilling details: hypoxic-ischemic encephalopathy[J]. J Perinat Neonatal Nurs, 2009,23(1):59-68; quiz 69-70.

二级参考文献13

1Maruta T,Noshi M,Tanouchi A. H2O2-triggered retrograde signaling from chloroplasts to nucleus plays specific role in response to stress[J].{H}Journal of Biochemistry,2012,(15):11717-11729.
2Dringen R,Pawlowski PG,Hirrlinger J. Peroxide detoxification by brain cells[J].{H}Journal of Neuroscience Research,2005,(1-2):157-165.
3Alonso A,Gonzalez C. Neuroprotective role of estrogens:relationship with insulin/IGF-1 signaling[J].Front Biosci(Elite Ed),2012.607-619.
4Netchine I,Azzi S,Le Bouc Y. IGF-1 molecular anomalies demonstrate its critical role in fetal,postnatal growth and brain development[J].{H}Best Practice and Research Clinical Endocrinology and Metablism,2011,(1):181-190.
5Hodge RD,D'Ercole AJ,O'Kusky JR. Insulin-like growth factor-Ⅰ (IGF-Ⅰ) inhibits neuronal apoptosis in the developing cerebral cortex in vivo[J].Int J Dcv Neurosci,2007,(4):233-241.
6Zhong J,Lee WH. Hydrogen peroxide attenuates insulinlike growth factor-1 neuroprotective effect,prevented by minocycline[J].{H}Neurochemistry International,2007,(6-7):398-404.
7Pereira MD,Ksiazek K,Menezes R. Oxidative stress in neurodegenerative diseases and ageing[J].Oxid Med Cell Longev,2012.796360.
8Shi DY,Xie FZ,Zhai C. The role of cellular oxidative stress in regulating glycolysis energy metabolism in hepatoma cells[J].{H}Molecular Cancer Therapautics,2009.32.
9Jin H,Kanthasamy A,Anantharam V. Transcriptional regulation of pro-apoptotic protein kinase Cdelta:implications for oxidative stress-induced neuronal cell death[J].{H}Journal of Biological Chemistry,2011,(22):19840-19859.
10Beresewicz M,Majewska M,Makarewicz D. Changes in the expression of insulin-like growth factor 1 variants in the postnatal brain development and in neonatal hypoxia-ischaemia[J].{H}International Journal of Neuroscience,2010,(1):91-97.