缺氧缺糖/复氧复糖HT22细胞损伤的动态观察
|
摘要
目的探讨缺氧缺糖/复氧复糖不同时间点小鼠海马神经元细胞系HT22细胞损伤情况。方法取对数生长期的HT22细胞,随机分为6组:正常对照组、缺氧缺糖/复氧复糖(OGD/R) 0 h组(OGD/R 0 h)、缺氧缺糖/复氧复糖12 h组(OGD/R 12 h)、缺氧缺糖/复氧复糖24 h组(OGD/R 24 h)、缺氧缺糖/复氧复糖48 h组(OGD/R 48 h)、缺氧缺糖/复氧复糖72 h组(OGD/R 72 h)。除正常对照组外,其余各组细胞均在缺氧缺糖6 h后进行复氧复糖。倒置显微镜下观察细胞形态,细胞计数试剂盒8(CCK-8)法检测细胞存活率,乳酸脱氢酶(LDH)法检测细胞损伤情况,Bax、Bcl-2免疫荧光染色检测细胞凋亡情况。结果正常组HT22细胞呈两极或多极,突起明显,突起之间相互交织成网状,细胞膜光滑且完整,胞体折光性强,OGD/R各组细胞胞体轴突减少,细胞皱缩,胞质凝聚。与正常组比较,OGD/R各组细胞存活率均显著降低(P<0.05),OGD/R 12 h至OGD/R 24 h达到最低(P<0.05);OGD/R 12 h及OGD/R 24 h组LDH显著升高(P<0.05),OGD/R 0 h、48 h、72 h组均有升高趋势。除OGD/R 0 h组和OGD/R 72 h组外,OGD/R各组Bax/Bcl-2比值均较正常组显著升高(P<0.05),峰值出现于OGD/R 24 h。结论缺氧缺糖/复氧复糖细胞损伤是一个复杂的动态过程,随着复氧复糖时间的延长,细胞损伤不断加重,24 h达到顶峰,随后细胞损伤情况逐渐缓解。
Objective To investigate the damage of hippocampal neuron cell line HT22 at different time points after oxygen and glucose deprivation/reoxygenation.Methods Logarithmic growth phase of HT22 cells was randomly divided into six groups,normal control group,OGD/R 0 hour,OGD/R 12 hours,OGD/R 24 hours,OGD/R 48 hours,OGD/R 72 hours groups.All groups were treated with reoxygenation after deprived of oxygen and glucose for 6 hours except normal group.The cell morphology was observed by invertded microscope.Cell counting kit-8(CCK-8) method was used to test the cell survival rate.Lactate dehydrogenase(LDH) method was used to detect cell damage.Bax and Bcl-2 immunofluorescence were used to detect apoptosis.Results HT22 cells in normal group were bipolar or multipolar.Obvious synapses and multiple synapses were woven into a network.Cell membrane was smooth and complete and the refractivity of cell was strong.But the axons decreased,cell was shrinkage and cytoplasmic aggregation in HT22 in OGD/R groups,and the cell viability was significantly reduced(P<0.05),the lowest point appeared in OGD/R 12 hours to OGD/R 24 hours;OGD/R 12 hours and OGD/R 24 hours LDH increased significantly(P<0.05),OGD/R 0 hour,48 hours,and 72 hours increased.The ratios of Bax/Bcl-2 in OGD/R groups were significantly increased(P<0.05) except for OGD/R 0 hour and OGD/R 72 hours,and peaked at OGD/R 24 hours.Conclusion Injury of cells afer OGD/R is a complex and dynamic process.Cell injury is aggravating as the reoxygenation time increased and reached a peak in OGD/R 24 hours,from then on,cell damage gradually decreased.
Objective To investigate the damage of hippocampal neuron cell line HT22 at different time points after oxygen and glucose deprivation/reoxygenation.Methods Logarithmic growth phase of HT22 cells was randomly divided into six groups,normal control group,OGD/R 0 hour,OGD/R 12 hours,OGD/R 24 hours,OGD/R 48 hours,OGD/R 72 hours groups.All groups were treated with reoxygenation after deprived of oxygen and glucose for 6 hours except normal group.The cell morphology was observed by invertded microscope.Cell counting kit-8(CCK-8) method was used to test the cell survival rate.Lactate dehydrogenase(LDH) method was used to detect cell damage.Bax and Bcl-2 immunofluorescence were used to detect apoptosis.Results HT22 cells in normal group were bipolar or multipolar.Obvious synapses and multiple synapses were woven into a network.Cell membrane was smooth and complete and the refractivity of cell was strong.But the axons decreased,cell was shrinkage and cytoplasmic aggregation in HT22 in OGD/R groups,and the cell viability was significantly reduced(P<0.05),the lowest point appeared in OGD/R 12 hours to OGD/R 24 hours;OGD/R 12 hours and OGD/R 24 hours LDH increased significantly(P<0.05),OGD/R 0 hour,48 hours,and 72 hours increased.The ratios of Bax/Bcl-2 in OGD/R groups were significantly increased(P<0.05) except for OGD/R 0 hour and OGD/R 72 hours,and peaked at OGD/R 24 hours.Conclusion Injury of cells afer OGD/R is a complex and dynamic process.Cell injury is aggravating as the reoxygenation time increased and reached a peak in OGD/R 24 hours,from then on,cell damage gradually decreased.
引文
[1]National Cardiovascular Center.Report on Cardiovascular Disease in China[M].Beijing:Encyclopedia of China Publishing House,2014:96-97.(in Chinese)国家心血管病中心.中国心血管病报告[M].北京:中国大百科全书出版社,2014:96-97.
[2]Piironen K,Tiainen M,Mustanoja S,et al.Mild hypothermia after intravenous thrombolysis in patients with acute stroke:a randomized controlled trial[J].Stroke,2014,45(2):486-491.
[3]Bennett DA,Krishnamurthi RV,Barkercollo S,et al.The global burden of ischemic stroke:findings of the GBD 2010 study[J].Glob Heart,2014,9(1):107-112.
[4]Khandelwal P,Yavagal DR,Sacco RL.Acute Ischemic Stroke Intervention[J].J Am Coll Cardiol,2016,67(22):2631-2644.
[5]Kotlga D,Golab-Janowska M,Masztalewicz M,et al.The emotional stress and risk of ischemic stroke[J].Neurol Neurochir Pol,2016,50(4):265-270.
[6]Hou YC,liou KT,Chen CM,et al.Preventive effect of silymarin in cerebral ischemia-reperfusion-induced brain injury in rats possibly through impairing NF-κB and STAT-1 activation[J].Phytomedicine,2010,17(12):963-973.
[7]Wang J,Han D,Sun M,et al.Cerebral ischemic postconditioning induces autophagy inhibition and a HMGB1 secretion attenuation feedback loop to protect against ischemia reperfusion injury in an oxygen glucose deprivation cellular model[J].Mol Med Rep,2016,14(5):4162-4172.
[8]Jin XF,Zhang Y,Zhou XH,et al.Effects of astragalosideⅣon apoptosis of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation[J].Chinese Pharmacological Bulletin,2016,32(10):1411-1415.(in Chinese)靳晓飞,张颖,周晓红,等.黄芪甲苷对缺氧缺糖/复氧复糖PC12细胞凋亡的影响[J].中国药理学通报,2016,32(10):1411-1415.
[9]Taylor RC,Cullen SP,Martin SJ.Apoptosis:controlled demolition at the cellular level[J].Nat Rev Mol Cell Biol,2008,9(3):231-241.
[10]Kalinichenko SG,Niu M.Morphological characteristic of apoptosis and its significance in neurogenesis[J].Morfologiia,2007,131(2):16-28.
[11]Song XY,Hu JF,Chen NH.Neurons apoptosis and cerebral ischemia[J].Chinese Pharmacological Bulletin,2012,28(3):307-310.(in Chinese)宋修云,胡金凤,陈乃宏.神经细胞凋亡与脑缺血疾病[J].中国药理学通报,2012,28(3):307-310.
[12]Pohl SO,Agostino M,Dharmarajan A,et al.Crosstalk between cellular redox state and the anti-apoptotic protein Bcl-2[J].Antioxid Redox Signal,2018,29(13):1215-1236.
[13]Loor G,Kondapalli J,Iwase H,et al.Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion[J].Biochim Biophys Acta,2011,1813(7):1382-1394.
[14]Kristen AV,Ackermann K,Buss S,et al.Inhibition of apoptosis by the intrinsic but not the extrinsic apoptotic pathway in myocardial ischemia-reperfusion[J].Cardiovasc Pathol,2013,22(4):280-286.
[15]Liu QS,Deng R,Li S,et al.Ellagic acid protects against neuron damage in ischemic stroke through regulating the ratio of Bcl-2/Bax expression[J].Appl Physiol Nutr Metab,2017,42(8):855-860.
[16]Yan FX,Qian T,Gao WJ,et al.Effect of astragalus injection on the expression of Caspase-3 after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of rats[J].Acta Anatomica Sinica,2011,42(1):14-21.(in Chinese)闫凤霞,钱涛,高维娟,等.黄芪注射液抑制缺氧缺糖/复氧复糖大鼠海马神经元Caspase-3表达[J].解剖学报,2011,42(1):14-21.
[2]Piironen K,Tiainen M,Mustanoja S,et al.Mild hypothermia after intravenous thrombolysis in patients with acute stroke:a randomized controlled trial[J].Stroke,2014,45(2):486-491.
[3]Bennett DA,Krishnamurthi RV,Barkercollo S,et al.The global burden of ischemic stroke:findings of the GBD 2010 study[J].Glob Heart,2014,9(1):107-112.
[4]Khandelwal P,Yavagal DR,Sacco RL.Acute Ischemic Stroke Intervention[J].J Am Coll Cardiol,2016,67(22):2631-2644.
[5]Kotlga D,Golab-Janowska M,Masztalewicz M,et al.The emotional stress and risk of ischemic stroke[J].Neurol Neurochir Pol,2016,50(4):265-270.
[6]Hou YC,liou KT,Chen CM,et al.Preventive effect of silymarin in cerebral ischemia-reperfusion-induced brain injury in rats possibly through impairing NF-κB and STAT-1 activation[J].Phytomedicine,2010,17(12):963-973.
[7]Wang J,Han D,Sun M,et al.Cerebral ischemic postconditioning induces autophagy inhibition and a HMGB1 secretion attenuation feedback loop to protect against ischemia reperfusion injury in an oxygen glucose deprivation cellular model[J].Mol Med Rep,2016,14(5):4162-4172.
[8]Jin XF,Zhang Y,Zhou XH,et al.Effects of astragalosideⅣon apoptosis of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation[J].Chinese Pharmacological Bulletin,2016,32(10):1411-1415.(in Chinese)靳晓飞,张颖,周晓红,等.黄芪甲苷对缺氧缺糖/复氧复糖PC12细胞凋亡的影响[J].中国药理学通报,2016,32(10):1411-1415.
[9]Taylor RC,Cullen SP,Martin SJ.Apoptosis:controlled demolition at the cellular level[J].Nat Rev Mol Cell Biol,2008,9(3):231-241.
[10]Kalinichenko SG,Niu M.Morphological characteristic of apoptosis and its significance in neurogenesis[J].Morfologiia,2007,131(2):16-28.
[11]Song XY,Hu JF,Chen NH.Neurons apoptosis and cerebral ischemia[J].Chinese Pharmacological Bulletin,2012,28(3):307-310.(in Chinese)宋修云,胡金凤,陈乃宏.神经细胞凋亡与脑缺血疾病[J].中国药理学通报,2012,28(3):307-310.
[12]Pohl SO,Agostino M,Dharmarajan A,et al.Crosstalk between cellular redox state and the anti-apoptotic protein Bcl-2[J].Antioxid Redox Signal,2018,29(13):1215-1236.
[13]Loor G,Kondapalli J,Iwase H,et al.Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion[J].Biochim Biophys Acta,2011,1813(7):1382-1394.
[14]Kristen AV,Ackermann K,Buss S,et al.Inhibition of apoptosis by the intrinsic but not the extrinsic apoptotic pathway in myocardial ischemia-reperfusion[J].Cardiovasc Pathol,2013,22(4):280-286.
[15]Liu QS,Deng R,Li S,et al.Ellagic acid protects against neuron damage in ischemic stroke through regulating the ratio of Bcl-2/Bax expression[J].Appl Physiol Nutr Metab,2017,42(8):855-860.
[16]Yan FX,Qian T,Gao WJ,et al.Effect of astragalus injection on the expression of Caspase-3 after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of rats[J].Acta Anatomica Sinica,2011,42(1):14-21.(in Chinese)闫凤霞,钱涛,高维娟,等.黄芪注射液抑制缺氧缺糖/复氧复糖大鼠海马神经元Caspase-3表达[J].解剖学报,2011,42(1):14-21.