二氮嗪预处理对大鼠海马神经元缺氧复氧后细胞凋亡和c-Jun氨基末端激酶表达的影响
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摘要
【目的】探讨线粒体内膜ATP敏感性钾通道(mitoKATP通道)选择性开放剂二氮嗪预处理对原代大鼠海马神经元遭受缺氧复氧损伤后细胞凋亡和c-Jun氨基末端激酶(JNK)信号转导通路的影响以及可能的联系。
【方法】出生时间<24 h SD大鼠100只,体重5～6 g,断头取海马组织。海马神经元培养至9～10 d,随机分为5组:对照组(A组)、二氮嗪0 umol/ L组(B组)、二氮嗪30 umol/ L组(C组)、二氮嗪100 umol/ L组(D组)、mitoKATP通道特异性阻断剂5-羟葵酸100 umol/ L +二氮嗪100 umol/ L组(E组)。各组细胞每天给二氮嗪预处理l h,连续3 d后,接受缺氧4 h和复氧24 h。用四唑盐(MTT)比色法分析海马神经元存活率,蛋白质印迹法检测Bc1-2、Bax和JNK蛋白表达水平的变化。
【结果】①与A组比较,其余4组大鼠海马神经元缺氧复氧损伤后存活率明显减少(P<0.01);Bc1-2、Bax、JNK蛋白表达水平升高(P<0.01,P<0.05);②与B组比较,C和D组的存活率大大增加(分别P<0.05,P<0.01),其中C组存活率升高5%,D组存活率升高12%;C和D组的Bc1-2蛋白表达明显增高(分别P<0.05,P<0.01),C组增加25%,D组增加100%;Bax和JNK蛋白表达程度下降(P<0.05),其中Bax蛋白在C组减少22%,在D组减少42%,JNK蛋白在C组减少19%,在D组减少35%;但B组与E组比较均无显著性差异(P>0.05);③C组与D组各指标比较有显著性差异(P<0.05,P<0.01)。与C组比较,D组的细胞存活率增加7%,Bc1-2蛋白表达增高25%,Bax蛋白表达减少27%,JNK蛋白表达减少19%。
【结论】MitoKATP通道特异性开放剂二氮嗪预处理可减少大鼠海马神经元缺氧复氧后细胞凋亡,增加大鼠海马神经元对缺氧复氧损伤的耐受性。JNK信号转导途径参与神经元凋亡。二氮嗪可能通过JNK信号通路,下调JNK蛋白的表达,上调Bcl-2的表达,下调Bax的表达,从而减轻缺氧复氧后海马神经元凋亡。
Objective To investigate the effects and possible relationship of diazoxide (a selective opener of mitochondrial ATP-sensitive potassium channel,mitoKATP) precon-ditioning on the expressions of apoptosis and c-Jun N-terminal kinase (JNK) signali-ng transduction pathway in primary cultured rat hippocampal neurons in vitro cause-d by anoxia-reoxygenation injury.
Methods 100 Sprage-Dawley rats (<24 h after birth) weighing 5～6 g were decapitated,hippocampal tissue was isolated.After being cultured for 9～10 d,the hippocampal neurons were randomly assigned to one of 5 groups:Control group (Gr-oup A),diazoxide 0,30,100 umol/ L (Group B,C,D) and 5-hydroxydecanoate (a specific inhibitor of mitoKATP) 100 umol/ L + diazoxide l00 umol/ L (Group E). The neurons were treated with diazoxide l h per day for 3 d before being subjected to 4 h oxygen deprivation followed by 24 h reoxygenation.The neuronal viability was measured by MTT assay.Expressions and variability of Bcl-2、Bax and JNK proteins were detected by Western blot analysis.
Results The results obtained are as follows:①The survival rates of the rat hippocampal neurons subjected to anoxia-reoxygenation were diminished than that inGroup A significantly (P<0.01) .Bc1-2、Bax and JNK proteins were up-regulated than those in Group A (P<0.01,P<0.05);②Compared with that in Group B,the neuronal survival rates rise in both Group C and Group D (P<0.01,P<0.05) .The survival rate in Group C was increased by about 5% and by nearly 12% in Group D.The expression of Bcl-2 protein in both Group C and Group D was upgraded (P<0.05,P<0.01)by approximately 25% and 100% each.Bax and JNK proteins were decreased (P<0.05) .The expression of Bax protein was descended by almost 22% and 42% each,while JNK protein was lower by 19% and 35% respectively. No difference was detected between Group B and Group E (P>0.05);③There was significant difference between Group C and Group D (P<0.05,P<0.01) .By contra-st to Group C,the neuronal survival rate in Group D was advanced by almost 7%,Bcl-2 expression was increaseed by 25%,whereas Bax and JNK expressions were decreased by 27% and 19% respectively.
Conclusion Diazoxide (a selective opener of mitoKATP) preconditioning may at-tenuate neuronal apoptosis and enhance the tolerance against anoxia-reoxygenation injury in primary cultured hippocampal neurons.JNK signaling transduction pathway plays a vivtal role in the neuronal apoptosis.Diazoxide may lower the expression of JNK protein,and increase cell survival ratio through up-regulation of Bcl-2 and down-regulation of Bax expression by JNK signaling transduction pathway.
【方法】出生时间<24 h SD大鼠100只,体重5～6 g,断头取海马组织。海马神经元培养至9～10 d,随机分为5组:对照组(A组)、二氮嗪0 umol/ L组(B组)、二氮嗪30 umol/ L组(C组)、二氮嗪100 umol/ L组(D组)、mitoKATP通道特异性阻断剂5-羟葵酸100 umol/ L +二氮嗪100 umol/ L组(E组)。各组细胞每天给二氮嗪预处理l h,连续3 d后,接受缺氧4 h和复氧24 h。用四唑盐(MTT)比色法分析海马神经元存活率,蛋白质印迹法检测Bc1-2、Bax和JNK蛋白表达水平的变化。
【结果】①与A组比较,其余4组大鼠海马神经元缺氧复氧损伤后存活率明显减少(P<0.01);Bc1-2、Bax、JNK蛋白表达水平升高(P<0.01,P<0.05);②与B组比较,C和D组的存活率大大增加(分别P<0.05,P<0.01),其中C组存活率升高5%,D组存活率升高12%;C和D组的Bc1-2蛋白表达明显增高(分别P<0.05,P<0.01),C组增加25%,D组增加100%;Bax和JNK蛋白表达程度下降(P<0.05),其中Bax蛋白在C组减少22%,在D组减少42%,JNK蛋白在C组减少19%,在D组减少35%;但B组与E组比较均无显著性差异(P>0.05);③C组与D组各指标比较有显著性差异(P<0.05,P<0.01)。与C组比较,D组的细胞存活率增加7%,Bc1-2蛋白表达增高25%,Bax蛋白表达减少27%,JNK蛋白表达减少19%。
【结论】MitoKATP通道特异性开放剂二氮嗪预处理可减少大鼠海马神经元缺氧复氧后细胞凋亡,增加大鼠海马神经元对缺氧复氧损伤的耐受性。JNK信号转导途径参与神经元凋亡。二氮嗪可能通过JNK信号通路,下调JNK蛋白的表达,上调Bcl-2的表达,下调Bax的表达,从而减轻缺氧复氧后海马神经元凋亡。
Objective To investigate the effects and possible relationship of diazoxide (a selective opener of mitochondrial ATP-sensitive potassium channel,mitoKATP) precon-ditioning on the expressions of apoptosis and c-Jun N-terminal kinase (JNK) signali-ng transduction pathway in primary cultured rat hippocampal neurons in vitro cause-d by anoxia-reoxygenation injury.
Methods 100 Sprage-Dawley rats (<24 h after birth) weighing 5～6 g were decapitated,hippocampal tissue was isolated.After being cultured for 9～10 d,the hippocampal neurons were randomly assigned to one of 5 groups:Control group (Gr-oup A),diazoxide 0,30,100 umol/ L (Group B,C,D) and 5-hydroxydecanoate (a specific inhibitor of mitoKATP) 100 umol/ L + diazoxide l00 umol/ L (Group E). The neurons were treated with diazoxide l h per day for 3 d before being subjected to 4 h oxygen deprivation followed by 24 h reoxygenation.The neuronal viability was measured by MTT assay.Expressions and variability of Bcl-2、Bax and JNK proteins were detected by Western blot analysis.
Results The results obtained are as follows:①The survival rates of the rat hippocampal neurons subjected to anoxia-reoxygenation were diminished than that inGroup A significantly (P<0.01) .Bc1-2、Bax and JNK proteins were up-regulated than those in Group A (P<0.01,P<0.05);②Compared with that in Group B,the neuronal survival rates rise in both Group C and Group D (P<0.01,P<0.05) .The survival rate in Group C was increased by about 5% and by nearly 12% in Group D.The expression of Bcl-2 protein in both Group C and Group D was upgraded (P<0.05,P<0.01)by approximately 25% and 100% each.Bax and JNK proteins were decreased (P<0.05) .The expression of Bax protein was descended by almost 22% and 42% each,while JNK protein was lower by 19% and 35% respectively. No difference was detected between Group B and Group E (P>0.05);③There was significant difference between Group C and Group D (P<0.05,P<0.01) .By contra-st to Group C,the neuronal survival rate in Group D was advanced by almost 7%,Bcl-2 expression was increaseed by 25%,whereas Bax and JNK expressions were decreased by 27% and 19% respectively.
Conclusion Diazoxide (a selective opener of mitoKATP) preconditioning may at-tenuate neuronal apoptosis and enhance the tolerance against anoxia-reoxygenation injury in primary cultured hippocampal neurons.JNK signaling transduction pathway plays a vivtal role in the neuronal apoptosis.Diazoxide may lower the expression of JNK protein,and increase cell survival ratio through up-regulation of Bcl-2 and down-regulation of Bax expression by JNK signaling transduction pathway.
引文
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[8] Rajapakse N,Shimizu K,Kis B,et a1.Activation of mitochondrial ATP-sens-itive potassium channels prevents neuronal cell death after ischemia in neonatal rats [J] .Neurosci Lett,2002,327(3):208-212.
[9] Shimizu K,Lacza Z,Rajapakse N,et a1.MitoK(ATP)opener,diazoxide, reduces neuronal damage after middle cerebral artery occlusion in the rat [J] .Am J Physiol Heart Circ Physiol,2002,283(3):Hl005-H1011.
[10] Harris MH,Thompson CB.The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability [J] .Cell Death Differ,2000,7(12):l182-l191.
[11] Ouyang YB,Giffard RG. .Cellular neuroprotective mechanisms in cerebral isc-hemia:Bcl-2 family proteins and protection of mitochondrial function [J] .CellCalcium,2004,36(3-4):303-311.
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