缺血后处理在大鼠心肌线粒体比较蛋白质组学的研究
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摘要
目的：
建立大鼠心肌缺血再灌注损伤模型,观察缺血后处理对缺血再灌注损伤大鼠心脏功能的影响；分离纯化心肌线粒体并提取线粒体蛋白行2D-DIGE比较蛋白质组学分析与鉴定,探讨缺血后处理方式对心肌线粒体蛋白表达的影响。
方法：
1.将SD大鼠随机分为正常组(Nor)、缺血再灌注损伤组(Con)、缺血后处理组(IPO)、5-羟葵酸拮抗缺血后处理组(5-HD+IPO),每组6只。采用Langendorff灌注装置建立各组大鼠离体心脏模型,各组均先灌注K-H液平衡20min。Nor组：续灌K-H液100min; Con组：灌注4℃ST.Thomas停跳液后缺血40min,复灌K-H液60min；IPO组：灌注4℃ST.Thomas停跳液后缺血40min,于复灌开始前予以复灌K-H液10s、停灌10s的循环6次,续灌K-H液58min; 5-HD+IPO组：灌注4℃ST.Thomas停跳液后缺血40min,行5-羟葵酸灌注5min,继以K-H液复灌10s、停灌10s循环6次,续灌K-H液53min。观察并记录各组平衡末和续灌末心率(HR)、左室发展压(LVDP)、左室舒张末压(LVDEP)、以及最大dp/dt等心功能的变化。
2.以差速离心法及Nycodenz密度梯度离心法提取、纯化大鼠心肌线粒体,并以Western blot法验证其纯度。
3.按照2中方法获取纯化的心肌线粒体；提取各组线粒体蛋白并行纯化后定量；采用Cydye染料对各组荧光标记后行2D-DIGE分离。采用DeCyder V6.0软件分析对比组差异表达蛋白点并将差异蛋白质斑点进行全自动斑点处理工作站处理；最后进行MALDI-TOF-MS鉴定和数据检索。
结果：
1.平衡末各组间心功能无明显差异；续灌末Nor, IPO组心功能显著优于Con组,5-HD+IPO组与Con组比较心功能无明显差异。
2.采用Nycodenz不连续密度梯度离心法提纯了大鼠心肌线粒体,并以Western blot法证实了所获线粒体纯度较高。
3.对各组进行2D-DIGE,得到聚焦良好、相似性高的图谱,在各组胶上平均检测出752±49个蛋白斑点；各组分析鉴定后得到28个较好的差异蛋白点图谱,本研究重点关注其中差异1.5倍以上的5个蛋白。
4.(1)Nor和Con组比较鉴定出：spot554 (NDUFA10, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10, mitochondrial; NADH脱氢酶亚基)表达水平降低3.84倍；spot555 (rCG55630,isoform CRA,推测为NADH脱氢酶亚基)表达水平降低3.76倍。
(2)IPO和Con组比较鉴定出：spot554(NDUFA10)、spot254(SDHA, Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial;琥珀酸脱氢酶—黄素蛋白)两种蛋白表达水平显著分别降低3.33倍、1.68倍；spot645 (CoQ9,合成CoQ的必须酶)表达水平降低3.43倍、spot648 (CoQ9)表达水平升高2.42倍。
(3) 5-HD+IPO和IPO组比较鉴定出：spot254 (SDHA)表达水平升高1.65倍。
结论：
1.缺血后处理能改善大鼠心脏功能；而5-羟葵酸拮抗缺血后处理能抑制缺血后处理改善心功能的作用。
2. Nycodenz不连续密度梯度离心法可获得纯度较高心肌线粒体。
3.(1)心肌缺血再灌注损伤影响NDUFA10的活性。
(2)缺血后处理可维持线粒体NADH呼吸链稳定。
(3) SDHA和线粒体敏感性K+通道(mitoKATP)存在着密切的关系；两者相互作用共同参与了缺血后处理内源性保护机制。
(4)缺血后处理用于缺血再灌注心肌时可能造成CoQ9发生剪切或修饰。
Objective:
Constructing rat ischemia/reperfusion injury(IRI) model to investigate the protective effect of ischemic postconditioning on ischemic rat myocardium which suffered IRI. Myocardial mitochondria was purified and the mitochondrial protein line 2D-DIGE comparative was performed,to explore the effect of protein expression in mitochondria following ischemic postconditioning.
Methods:
1 Sprague-Dawley rats were divided randomly into 4 groups(n=6, each group), i.e. the normal (Nor), the control (Con), the ischemic postconditioning (IPO),5-hydroxydecanoate plus postconditioning group (5-HD+IPO). Langendorff apparatus were used to establish the model of myocardial ischemia reperfusion injury. Each group of rats were perfused with K-H for 20 minutes for equilibration. Group Nor:went on perfusion for another 100 minutes after equilibration. Group Con:perfusing 4℃ST.Thomas solution to make the heart stop beating after equilibration, and then underwent 40 minutes global ischemia, followed by 60 minutes reperfusion. Group IPO:after 40 minutes of global ischemia, then reperfusion for 10 secnds, and arrested for 10 secnds.The above procedures were repeated 6 times, reperfusion 58 minutes. Group 5-HD+IPO:perfused with 5-HD(100μmol/Lof K-Hsol) for 5min and then postconditioning was administered as that in IPO group, then perfuse 53 minutes. Investigating the cardiac function of each group at the point of equilibration and reperfusion end such as HR、LVDP、LVDEPand the max dp/dt.
2 The myocardial mitochondrial were purified by using Nycodenz density gradient centrifugation and verified purity by western blot.
3 The technique of 2D-DIGE and MALO-TOF-MS were utilised to investigate the myocardium mitochandrial protein.
Results:
1 The cardiac function:no obvious difference between each group after equilibrium,and group Nor,IPO were better than group Con.Intersetly,5-HD+IPO and IPO have no obvious differ.
2 Using Nycodenz to purify the myocardial mitochondrial and enrich the degree of mitochondria.
3 Well focused and distinct 2-DE maps with good reproducibility were obtained, means of 752±49 protein spots were detected.28 good protein maps were found. We pay close attention to five protein spots of them.
4 (1) Group Nor and the Con identified:The expression level of spot554 (NDUFA10, NADH dehydrogenase Asia base)reduces 3.84 times. spot555 (rCG55630, isoform CRA, is extrapolated as the NADH dehydrogenase Asia base) expresses the level to reduce 3.76 times.
(2) Group IPO and the Con compares appraises:spot554 (NDUFA10) and spot254 (SDHA, succinic acid dehydrogenase-flavine protein) expression level remarkable reduces 3.33 time,1.68 times separately. spot645 (CoQ9) express the level to reduce 3.43 times. spot648 (CoQ9) expresses the level to elevate 2.42 times.
(3) 5-HD+IPO and the IPO group compares appraises:The spot254 (SDHA) expression level elevates 1.65 times.
Conclusion:
1 Ischemic postconditioning can improve rat myocardium;whereas this protective effects can be antagonistic by 5-hydroxy decanoate.
2 Nycodenz density gradient centrifugation is a useful technique to isolate and purify mitochondria.
3(1) Ischemia reperfusion injury can affect the NDUFA10.
(2) Ischemic postconditioning can maintain the stability of mitochondrial NADH respiratory chain.
(3) There is familiar relationship between SDHA and mitoKATP;Both of them may together participate in the protection mechanism of endogenous IPO.
(4) CoQ9 may be sheared or modified of Ischemic postconditioning.
建立大鼠心肌缺血再灌注损伤模型,观察缺血后处理对缺血再灌注损伤大鼠心脏功能的影响；分离纯化心肌线粒体并提取线粒体蛋白行2D-DIGE比较蛋白质组学分析与鉴定,探讨缺血后处理方式对心肌线粒体蛋白表达的影响。
方法：
1.将SD大鼠随机分为正常组(Nor)、缺血再灌注损伤组(Con)、缺血后处理组(IPO)、5-羟葵酸拮抗缺血后处理组(5-HD+IPO),每组6只。采用Langendorff灌注装置建立各组大鼠离体心脏模型,各组均先灌注K-H液平衡20min。Nor组：续灌K-H液100min; Con组：灌注4℃ST.Thomas停跳液后缺血40min,复灌K-H液60min；IPO组：灌注4℃ST.Thomas停跳液后缺血40min,于复灌开始前予以复灌K-H液10s、停灌10s的循环6次,续灌K-H液58min; 5-HD+IPO组：灌注4℃ST.Thomas停跳液后缺血40min,行5-羟葵酸灌注5min,继以K-H液复灌10s、停灌10s循环6次,续灌K-H液53min。观察并记录各组平衡末和续灌末心率(HR)、左室发展压(LVDP)、左室舒张末压(LVDEP)、以及最大dp/dt等心功能的变化。
2.以差速离心法及Nycodenz密度梯度离心法提取、纯化大鼠心肌线粒体,并以Western blot法验证其纯度。
3.按照2中方法获取纯化的心肌线粒体；提取各组线粒体蛋白并行纯化后定量；采用Cydye染料对各组荧光标记后行2D-DIGE分离。采用DeCyder V6.0软件分析对比组差异表达蛋白点并将差异蛋白质斑点进行全自动斑点处理工作站处理；最后进行MALDI-TOF-MS鉴定和数据检索。
结果：
1.平衡末各组间心功能无明显差异；续灌末Nor, IPO组心功能显著优于Con组,5-HD+IPO组与Con组比较心功能无明显差异。
2.采用Nycodenz不连续密度梯度离心法提纯了大鼠心肌线粒体,并以Western blot法证实了所获线粒体纯度较高。
3.对各组进行2D-DIGE,得到聚焦良好、相似性高的图谱,在各组胶上平均检测出752±49个蛋白斑点；各组分析鉴定后得到28个较好的差异蛋白点图谱,本研究重点关注其中差异1.5倍以上的5个蛋白。
4.(1)Nor和Con组比较鉴定出：spot554 (NDUFA10, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10, mitochondrial; NADH脱氢酶亚基)表达水平降低3.84倍；spot555 (rCG55630,isoform CRA,推测为NADH脱氢酶亚基)表达水平降低3.76倍。
(2)IPO和Con组比较鉴定出：spot554(NDUFA10)、spot254(SDHA, Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial;琥珀酸脱氢酶—黄素蛋白)两种蛋白表达水平显著分别降低3.33倍、1.68倍；spot645 (CoQ9,合成CoQ的必须酶)表达水平降低3.43倍、spot648 (CoQ9)表达水平升高2.42倍。
(3) 5-HD+IPO和IPO组比较鉴定出：spot254 (SDHA)表达水平升高1.65倍。
结论：
1.缺血后处理能改善大鼠心脏功能；而5-羟葵酸拮抗缺血后处理能抑制缺血后处理改善心功能的作用。
2. Nycodenz不连续密度梯度离心法可获得纯度较高心肌线粒体。
3.(1)心肌缺血再灌注损伤影响NDUFA10的活性。
(2)缺血后处理可维持线粒体NADH呼吸链稳定。
(3) SDHA和线粒体敏感性K+通道(mitoKATP)存在着密切的关系；两者相互作用共同参与了缺血后处理内源性保护机制。
(4)缺血后处理用于缺血再灌注心肌时可能造成CoQ9发生剪切或修饰。
Objective:
Constructing rat ischemia/reperfusion injury(IRI) model to investigate the protective effect of ischemic postconditioning on ischemic rat myocardium which suffered IRI. Myocardial mitochondria was purified and the mitochondrial protein line 2D-DIGE comparative was performed,to explore the effect of protein expression in mitochondria following ischemic postconditioning.
Methods:
1 Sprague-Dawley rats were divided randomly into 4 groups(n=6, each group), i.e. the normal (Nor), the control (Con), the ischemic postconditioning (IPO),5-hydroxydecanoate plus postconditioning group (5-HD+IPO). Langendorff apparatus were used to establish the model of myocardial ischemia reperfusion injury. Each group of rats were perfused with K-H for 20 minutes for equilibration. Group Nor:went on perfusion for another 100 minutes after equilibration. Group Con:perfusing 4℃ST.Thomas solution to make the heart stop beating after equilibration, and then underwent 40 minutes global ischemia, followed by 60 minutes reperfusion. Group IPO:after 40 minutes of global ischemia, then reperfusion for 10 secnds, and arrested for 10 secnds.The above procedures were repeated 6 times, reperfusion 58 minutes. Group 5-HD+IPO:perfused with 5-HD(100μmol/Lof K-Hsol) for 5min and then postconditioning was administered as that in IPO group, then perfuse 53 minutes. Investigating the cardiac function of each group at the point of equilibration and reperfusion end such as HR、LVDP、LVDEPand the max dp/dt.
2 The myocardial mitochondrial were purified by using Nycodenz density gradient centrifugation and verified purity by western blot.
3 The technique of 2D-DIGE and MALO-TOF-MS were utilised to investigate the myocardium mitochandrial protein.
Results:
1 The cardiac function:no obvious difference between each group after equilibrium,and group Nor,IPO were better than group Con.Intersetly,5-HD+IPO and IPO have no obvious differ.
2 Using Nycodenz to purify the myocardial mitochondrial and enrich the degree of mitochondria.
3 Well focused and distinct 2-DE maps with good reproducibility were obtained, means of 752±49 protein spots were detected.28 good protein maps were found. We pay close attention to five protein spots of them.
4 (1) Group Nor and the Con identified:The expression level of spot554 (NDUFA10, NADH dehydrogenase Asia base)reduces 3.84 times. spot555 (rCG55630, isoform CRA, is extrapolated as the NADH dehydrogenase Asia base) expresses the level to reduce 3.76 times.
(2) Group IPO and the Con compares appraises:spot554 (NDUFA10) and spot254 (SDHA, succinic acid dehydrogenase-flavine protein) expression level remarkable reduces 3.33 time,1.68 times separately. spot645 (CoQ9) express the level to reduce 3.43 times. spot648 (CoQ9) expresses the level to elevate 2.42 times.
(3) 5-HD+IPO and the IPO group compares appraises:The spot254 (SDHA) expression level elevates 1.65 times.
Conclusion:
1 Ischemic postconditioning can improve rat myocardium;whereas this protective effects can be antagonistic by 5-hydroxy decanoate.
2 Nycodenz density gradient centrifugation is a useful technique to isolate and purify mitochondria.
3(1) Ischemia reperfusion injury can affect the NDUFA10.
(2) Ischemic postconditioning can maintain the stability of mitochondrial NADH respiratory chain.
(3) There is familiar relationship between SDHA and mitoKATP;Both of them may together participate in the protection mechanism of endogenous IPO.
(4) CoQ9 may be sheared or modified of Ischemic postconditioning.
引文
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1. Zhao ZQ, Corvera JS, Halkos ME, et al. Inhabition of myocardial injury by ischemic postconditioning during reperfusion:comparision with ischemic preconditioning. Physiol Heart Circ Physiol,2003,285(2):H579-H588.
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3. Juhaszova M, Zorov DB, Yaniv Y, et al. Role of glycogen synthase kinase-3beta in cardioprotection. Circ Res,2009,104(11):1240-1252.
4. Nishida H, Sato T, Nomura M, et al. Glimepiride Treatment Upon Reperfusion Limits Infarct Size via the Phosphatidylinositol 3-Kinase/Akt Pathway in Rabbit Hearts. Pharmacol Sci,2009,109(2):251-256.
5. Zatta AJ, Kin H, Yoshishige D, et al. Evidence that cardioprotection by postconditioning involves preservation of myocardial opioid content and selective opioid receptor activation. Physiol Hear Circ Physiol,2008,294(3):H1444-H1451.
6. Couvreur N, Tissier R, Pons, et al. The ceiling effect of pharmacological postconditioning with the phytoestrogen genistein is reversed by the GSK3 beta inhibitor SB-216763 through mitochondrial ATP-dependent potassium channel opening. Pharmacol Exp Ther,2009,329(3):1134-1141.
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1. Zhao ZQ, Corvera JS, Halkos ME, et al. Inhabition of myocardial injury by ischemic postconditioning during reperfusion:comparision with ischemic preconditioning. Physiol Heart Circ Physiol,2003,285(2):H579-H588.
2. Tisser R, Waintranb X, Couvreur N, et al. Pharmacological postconditioning with the phytoestrogen genistein. Me cell cardiol,2007,42(1):79-87.
3. Juhaszova M, Zorov DB, Yaniv Y, et al. Role of glycogen synthase kinase-3beta in cardioprotection. Circ Res,2009,104(11):1240-1252.
4. Nishida H, Sato T, Nomura M, et al. Glimepiride Treatment Upon Reperfusion Limits Infarct Size via the Phosphatidylinositol 3-Kinase/Akt Pathway in Rabbit Hearts. Pharmacol Sci,2009,109(2):251-256.
5. Zatta AJ, Kin H, Yoshishige D, et al. Evidence that cardioprotection by postconditioning involves preservation of myocardial opioid content and selective opioid receptor activation. Physiol Hear Circ Physiol,2008,294(3):H1444-H1451.
6. Couvreur N, Tissier R, Pons, et al. The ceiling effect of pharmacological postconditioning with the phytoestrogen genistein is reversed by the GSK3 beta inhibitor SB-216763 through mitochondrial ATP-dependent potassium channel opening. Pharmacol Exp Ther,2009,329(3):1134-1141.
7. Penna C, Mancardi D, Rainondo S, et al. The paradigm of postcondintioning to protect the heart. Cell Mol Med,2008,12(2):435-458.
8. Kocsis GF, Pipis J, Fekete V, et al. Lavastatin interferes with the infact size limiting effect of ischemic preconditioning and postconditioning in rat hearts. Am J Physiol Heart Circ physiol,2008,294(5):H2406-H2409.
9. Efthymiou CA, Mocanu MM, Yellon DM. A trovastatin and myocardial reperfusion injury new pleiotropic effect inplicating multiple prosurvival signling. Cardiovasc Pharmac,2005,45(3):247-252.
10.Yao Y, Li L, Li L, et al. Sevoflurane postconditioning protects chronically-infarcted rat hearts against ischemia-reperfusion injury by activation of pro-survival kinases and inhibition of mitochondrial permeability transition pore opening upon reperfusion. Biol Phaim Bull,2009,32(11):1854-1861.
11.Paillard M, Gomez L, Augeul L, et al. Postconditioning inhibits mPTP opening independent of oxidative phosphorylation and membrane potential. Mol Cell Cardiol, 2009,46(6):902-909.
12.Purdom-Dickinson SE, Lin Y, Dedek M, et al. Induction of Antioxidant and Detoxification Response by Oxidantsin Cardiomyocytes:Evidence from Gene Expression Profiling and Activation of Nrf2 Transcription Factor. Mol Cell Cardiol, 2007,42(1):159-176.
13.Abe M, Takiguchi Y, Ichimaru S, et al. Comparison of the protective effect of Nacety lcysteine by different treatments on rat myocardial ischemia-reperfusion injury. Pharmacolgy,2008,106(4):571-577.
14.Tsutsumi YM, Yokoyama T, Horikawa Y, et al. Reactive oxygen species trigger ischaemic and pharmacological postconditioning in vitro characterization. Life Sci, 2007,81(15):1223-1227.
15. Yang L, Yu T. Prolonged donor heart preservation with pinacidil:The role of mitochondria and the mitochondrial adenosine triphosphate-sensitive potassium channe. Thora Cardiovasc Surg,2010,1399(4):1057-1063.
16.Zang WJ, Sun L, Yu XJ, et al. Cardioprotection of ischemic postconditioning and pharmacological post-treamtment with adenosine or acetylcholine. Sheng LI XUE Bao, 2007,59(50):593-600.
17.Fujita M, Asanuma H, Hirata A, et al. Prolonged transient acidosis during early reperfusion contributes to the cardioprotective effects of postconditioning. Am J Physiol Heart Circ Physiol,2007,292(4):H2004-H2008.
18.陈其彬,喻田,傅小云,等.二氮嗪预处理对大鼠离体心脏缺血/再灌注时心肌线粒体通透性转换孔的影响.中华麻醉学杂志,2008(1)：25-28.
19.Penna C, Mancardi D, Raimondo S, et al. The paraddigm of postconditioning to protect the heart. Cell Mol Med,2008,12(2):435-458.
20.Ge ZD, Pravdic D, Bienengraeber M, et al. Isoflurane postconditioning protects against reperfusion injury by preventing mitochondrial permeability transition by an endothelial nitric oxide synthase-dependent mechanism. Anesthesiology,2010,112(1): 73-85.
21.He W, Zhang FJ, Wang SP, et al. Postconditioning of sevoflurane and propofol is associated with mitochondrial permeability transition pore. Zhejiang Univci B,2008, 9(2):100-108.
22.H6nisch A, Theuring N, Ebner B, et al. Postconditioning with levosimendan reduces the infarct size involving the PI3K pathway and KATP-channel activation but is independent of PDE-Ⅲ inhibition. Basic Res Cardiol,2010,105(2):155-167.
23.Mudalagiri NR, disalvo C, Kolvekar S, et al. Erythropoietin pretects human myocardium aginst hypoxia-reoxygenation injury via PI3-kinase and ERK1/2 activation. Br J Parmacol,2008,153(1):50-56.
24. Penna C, Mancardi D, Rastaldo R, et al. Intermittent activation of bradykin in B2 Receptors and mitochondrial KATP channel trigger cardiac postconditioning through redox signaling. Cardiovase Res,2007,75(1):168-177.
25 Kim N, Lee Y, Kim H, et al. Potential biomarkers for ischemic heart damage identified in mitochondrial proteins by comparative proteomics. Proteomics,2006,6(4): 1237-1249.