芪参益气方抗急性心肌缺血作用的蛋白质组学研究
摘要
蛋白质组学的诞生与发展,为研究蛋白质表达和功能变化提供了强有力的技术支持,使人们可以从系统的角度了解药物在蛋白质水平上对机体的作用。中药具有多组分、多靶点、整合调节的作用特点,运用蛋白质组学技术发现中药活性物质可能的作用靶点,了解中药作用途径,将有助于在分子层次上诠释传统中医药理论的现代生物学意义。本文以急性心肌缺血大鼠的心肌蛋白质组为研究对象,首先通过实验研究建立和优化心肌蛋白质组的双向电泳分析方法;然后利用该方法对芪参益气方的抗急性心肌缺血作用机制进行研究,主要研究内容如下:
1.大鼠心肌蛋白质组分析方法研究:从大鼠急性心肌缺血模型中获取缺血区心肌组织,研究建立蛋白提取、纯化等方法。通过对心肌蛋白质组分析过程中各关键参数的优化(包括上样量,IPG胶条规格,等电聚焦条件和SDS-PAGE电压等条件),建立了心肌蛋白质组学双向电泳分析方法。运用该方法对维拉帕米作用下急性心梗大鼠的心肌蛋白组进行分析,结果表明该法能较理想地分离出差异蛋白,并获取了15个差异蛋白进行质谱鉴定。
2.芪参益气方抗急性心肌缺血作用的蛋白质组学研究:对中药方剂芪参益气方抗心肌缺血作用开展药效学实验,并利用已建立的方法对芪参益气方给药3天与7天的心肌缺血大鼠心肌蛋白质组进行分析,研究发现35个蛋白差异点。对这些差异表达的蛋白进行质谱鉴定后发现其与能量代谢、抗氧化应激等作用有关。研究结果表明,芪参益气方的心肌保护作用与恢复心肌损伤过程中的能量供应、对抗氧化应激等作用有关。
通过以上研究,本论文成功建立了心肌蛋白质组学的实验研究方法,利用该法对芪参益气方的抗急性心肌缺血作用进行了蛋白质组学研究,研究结果为揭示该方剂多途径作用机制提供了一定的线索。
Proteomics is a new emerging technique, which offers a systematical view on the variation of expression and function of protein under certain stimuli. It helps people get new insight of drug action on the proteo-level. The traditional Chinese medicine has the characteristic of the multi-component, multi-targets and synergistic effects. Using the proteomic technique to identify the potential mechanism of traditional Chinese medicine will contribute to understand the way that the traditional Chinese medicine works by. It will also benefit to reveal modern biological meaning of the theory of traditional Chinese medicine on the molecular level. In this thesis, cardiac proteome of the acute myocardial ischemic rats were studied. A two dimensional electrophoresis (2-DE) based proteomic analytical method was developed by a series of comparative experiments. Subsequently, the cardioprotective effects of Qi-Shen-Yi-Qi formula in- acute myocardial ischemic rats were studied by the routine pharmacological assays and cardiac proteome analysis. The main research contents were as followed:
1、Firstly, a 2-DE based method for the analysis of rat myocardial proteome was established. The tissue of cardiac muscle were obtained from the rats with acute myocardial ischemia. The method of myocardial proteomic sample preparation (include protein extraction and purification) was developed. Several key parameters in 2-DE analysis were optimized, including sample amount, IPG strips, voltage for focusing and SDS-PAGE. The established method was applied in analyze proteome of ischemic hearts being treated with verapamil. 15 differential proteins were well separated. The results indicated the present method was reliable and stable, which can be used to characterize myocardial proteome.
2、The proteomic study on cardioprotective effects of Qi-Shen-Yi-Qi formula in acute myocardial ischemic rats. The pharmacological assays were performed to determine serum enzyme levels in each experimental group. The proteomic analysis was also progressed by the built 2-DE method to rats cardiac proteome which was treated with Qi-Shen-Yi-Qi formula for 3 and 7 days after heart ischemia. The result showed that the expression of 35 proteins were changed significantly. Using MALDI-TOF-MS, these 35 proteins were identified by database searching. These proteins can be related to energy metabolism and mitochondrial function, anti-oxidative stress property, and so on. The findings showed that the myocardial protective effects of the qishenyiqi formula in myocardial damage process are related to the recovery of energy supply as well as anti-oxidative stress property.
In this thesis, the method of myocardial proteome was built successfully, and the proteomic research on the anti-ischemic effect of the Qi-Shen-Yi-Qi formula was progressed by this built method, the result afforded some clues in multiple targets mechanism of the traditional Chinese medicine.
1.大鼠心肌蛋白质组分析方法研究:从大鼠急性心肌缺血模型中获取缺血区心肌组织,研究建立蛋白提取、纯化等方法。通过对心肌蛋白质组分析过程中各关键参数的优化(包括上样量,IPG胶条规格,等电聚焦条件和SDS-PAGE电压等条件),建立了心肌蛋白质组学双向电泳分析方法。运用该方法对维拉帕米作用下急性心梗大鼠的心肌蛋白组进行分析,结果表明该法能较理想地分离出差异蛋白,并获取了15个差异蛋白进行质谱鉴定。
2.芪参益气方抗急性心肌缺血作用的蛋白质组学研究:对中药方剂芪参益气方抗心肌缺血作用开展药效学实验,并利用已建立的方法对芪参益气方给药3天与7天的心肌缺血大鼠心肌蛋白质组进行分析,研究发现35个蛋白差异点。对这些差异表达的蛋白进行质谱鉴定后发现其与能量代谢、抗氧化应激等作用有关。研究结果表明,芪参益气方的心肌保护作用与恢复心肌损伤过程中的能量供应、对抗氧化应激等作用有关。
通过以上研究,本论文成功建立了心肌蛋白质组学的实验研究方法,利用该法对芪参益气方的抗急性心肌缺血作用进行了蛋白质组学研究,研究结果为揭示该方剂多途径作用机制提供了一定的线索。
Proteomics is a new emerging technique, which offers a systematical view on the variation of expression and function of protein under certain stimuli. It helps people get new insight of drug action on the proteo-level. The traditional Chinese medicine has the characteristic of the multi-component, multi-targets and synergistic effects. Using the proteomic technique to identify the potential mechanism of traditional Chinese medicine will contribute to understand the way that the traditional Chinese medicine works by. It will also benefit to reveal modern biological meaning of the theory of traditional Chinese medicine on the molecular level. In this thesis, cardiac proteome of the acute myocardial ischemic rats were studied. A two dimensional electrophoresis (2-DE) based proteomic analytical method was developed by a series of comparative experiments. Subsequently, the cardioprotective effects of Qi-Shen-Yi-Qi formula in- acute myocardial ischemic rats were studied by the routine pharmacological assays and cardiac proteome analysis. The main research contents were as followed:
1、Firstly, a 2-DE based method for the analysis of rat myocardial proteome was established. The tissue of cardiac muscle were obtained from the rats with acute myocardial ischemia. The method of myocardial proteomic sample preparation (include protein extraction and purification) was developed. Several key parameters in 2-DE analysis were optimized, including sample amount, IPG strips, voltage for focusing and SDS-PAGE. The established method was applied in analyze proteome of ischemic hearts being treated with verapamil. 15 differential proteins were well separated. The results indicated the present method was reliable and stable, which can be used to characterize myocardial proteome.
2、The proteomic study on cardioprotective effects of Qi-Shen-Yi-Qi formula in acute myocardial ischemic rats. The pharmacological assays were performed to determine serum enzyme levels in each experimental group. The proteomic analysis was also progressed by the built 2-DE method to rats cardiac proteome which was treated with Qi-Shen-Yi-Qi formula for 3 and 7 days after heart ischemia. The result showed that the expression of 35 proteins were changed significantly. Using MALDI-TOF-MS, these 35 proteins were identified by database searching. These proteins can be related to energy metabolism and mitochondrial function, anti-oxidative stress property, and so on. The findings showed that the myocardial protective effects of the qishenyiqi formula in myocardial damage process are related to the recovery of energy supply as well as anti-oxidative stress property.
In this thesis, the method of myocardial proteome was built successfully, and the proteomic research on the anti-ischemic effect of the Qi-Shen-Yi-Qi formula was progressed by this built method, the result afforded some clues in multiple targets mechanism of the traditional Chinese medicine.
引文
[1]李春阳,李林.蛋白质组学技术对中医药研究的影响[J].中国中药杂志,2005,30(14):1062-1064.
[2]申定珠,李家邦,蒋荣鑫.证候蛋白质组学与中医证候学相关性探讨[J].中国中西医结合杂志,2006,24(4):366-368.
[3]钟小兰,吕志平,钱令嘉.肝郁证模型大鼠血清蛋白质组的差异表达研究[J].中华中医药杂志,2006,21(7):399-404.
[4]李炜,陈敏,周凌燕.甲亢肝阳上亢证大鼠肾上腺组织蛋白质组的双向电泳分析[J].中国医师杂志,2007,9(1):20-24.
[5]吴红金,马增春,高月.蛋白质组学技术对冠心病血瘀证相关蛋白的研究[J].中西医结合心脑血管病杂志,2005,3(3):189-191.
[6]牟君,杨泽松,杨德兰.抑郁大鼠海马的双向凝胶电泳图谱分析[J].中国神经精神疾病杂志,2006,32(3):219-221.
[7]郭平,王升启.芍药苷对人骨髓基质细胞HFCL蛋白质表达的作用[J].中草药,2006,37(8):1206-1210.
[8]董燕,冯冲,马伟忠.苍耳子对小鼠脾淋巴细胞蛋白质组影响的双向电泳分析[J].中药新药与临床药理,2005,16(5):324-327.
[9]谢文光,马晓昌,邵宁生.赤芍治疗热毒血瘀证的血清蛋白质组变化的初步研究[J].中国中西医结合杂志,2005,25(6):520-523.
[10]张伟,曾园山,汪洋.灵芝孢子促进大鼠受损伤脊髓运动神经元存活及其轴突再生相关蛋白质组学的初步研究[J].中西医结合学报,2006,4(3):298-300.
[11]Wang.Y.,Y.H.Cheung,Z.Yang,Proteomic approach to study the cytotoxicity of dioscin(saponin)[J],Proteomics,2006.
[12]Ong,E.S.,S.M.Len,and A.C.H.Lee.Proteomic analysis of mouse liver for the evaluation of effects of Scutellariae radix by liquid chromatography with tandem mass spectrometry[J].Rapid Commun.Mass Spectrom,2004.18:2522.-2528.
[13]吴伟康,李劲平,罗汉川.四逆汤抗心肌缺血作用的相关蛋白谱研究[J].中国病理生理杂志,2005,21(3):506-508.
[14]刘丽丽,马增春,王宇光.四物汤对环磷酰胺致血虚证小鼠骨髓蛋白质组的影响[J].中国中药杂志,2006,31(14):1172-1175.
[15]马增春,高月,谭洪玲.复方丹参片对冠心病患者血浆蛋白影响的蛋白质组学研究[J].中国中药 杂志,2006,31(9):766-768.
[16]李连达,张荣利,刘成源.双龙方对大鼠心肌梗塞的治疗作用[J].中药新药与临床药理,2004,15(3):149-153.
[17]叶能胜,赵艳峰,张荣利.双龙方干预骨髓间充质干细胞分化过程的蛋白表达[J].中成药,2007,29(1):24-27.
[18]孙丽红,李鸿珠,韩丽萍.青蒿素对离体大鼠心肌缺血/再灌注损伤的保护作用[J].中国中药杂志,2007,32(15):1547-1551.
[19]Wang,Y.,L.Liu,C.Hu.Effects of Salviae Mitiorrhizae and Cortex Moutan extract on the rat heart after myocardial infarction[J].A proteomic study,biochemical pharmacology,2007,74:415-420.
[20]Tom Berkelman,Stenstedt T.2-D Electrophoresis[M]:Principles and Methods:Amersham Biosciences,2002.
[21]陈主初,梁宋平.肿瘤蛋白质组学[M],2002,长沙:湖南科学技术出版社.
[22]双向电泳实验手册[M],GE Healthcare公司.
[23]沈朋,范骁辉,曾真,程翼宇.基于图像特征和数学形态学的蛋白质组双向电泳分析法[J].中国科学B辑:化学,2005,35(1):44-50.
[24]Redfeam D.P.,Skanes A.C.,Lane J.,et al Signal-Averaged P Wave Reflects Change in Atrial Electrophysiological Substrate Afforded by Verapamil Following Cardioversion from Atrial Fibrillation.Pacing Clin Elecrophvsiol[J],2006,10:1089-1095.
[25]Cooper-DeHoff R.M.,Aranda J.M.,Gaxiola E.,et al.Blood pressure control and cardiovascular outcomes in high-risk Hispanic patients-Findings From the International Verapamil SR/Trandolapril Study.Am Heart[J],2006,151(5):1072-1079.
[26]Shaheen N.,Mahboob T.Antihypertensive and metabolic effects of verapamil:role of Na-K-ATPase and electrolytes homeostasis in male and female rats..Pak J Pharm Sci.[J],2004,17(2):1-11.
[27]Hansen J.F.Treatment with verapamil after an acute myocardial infarction.Review of the Danish studies on verapamil in myocardial infarction(DAVIT I and II)..Drugs.[J],1991,42:43-53.
[28]Yamaguchi F.,Sanbe A.,Takeo S.Cardiac Sarcoplasmic Reticular Function in Rats with Chronic Heart Failure Following Myocardial Infarction Cardiac Sarcoplasmic Reticular Function in Rats with Chronic Heart Failure Following Myocardial Infarction.J Mol Cell Cardiol.[J],1997,29:753-763.
[29]Jensen O.N.,Wilm M.,Shevchenko A.,et al.Sample Preparation Methods for Mass Spectrometric Peptide Mapping Directly from 2-DE Gels.Methods Mol Biol.[J],1999,112:513-530.
[30]Frolov V.A.,Drozdova G.Ao,Rieger P.,et al Initial Mechanisms of the Development of Hypertonic Heart..Bull Exp Biol Med.[J],2005,139(3):277-278.
[31]Lesnefsky E.J.,Slabe T.J.,Stoll M.S.,et al.Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria.Am J Physiol Heart Circ Physiol.[J],2001,280:H2770-H2778.
[32]Lepore D.A.,Knight K.R.,Anderson R.L.,et al.Role of priming stresses and Hsp70 in protection from ischemia-reperfusion injury in cardiac and skeletal muscle.Cell Stress Chap.[J],2001,6:93-96.
[33]祝维峰.通络宁治疗冠心病心绞痛的研究[J].山东中医学院学报,1994,18(5):311-317.
[34]王金荣,王知佳等.辨证辨病治疗冠心病108例临床观察[J].辽宁中医杂志,2001,28(8):475-476.
[35]方祝元,顾学兰.复方三七口服液治疗气虚血瘀型冠心病临床研究[J],中国中医急症,2000,9(6):247-248.
[36]王秀娥,梁华龙.舒心通脉片治疗冠心病心绞痛临床研究[J].山东中医杂志,1997,16(8):346-347.
[37]天津天士力制药股份有限公司.治疗冠心病心绞痛的中药制剂及其制备方法,专利号200310107278.0
[38]Yamaguchi F,Sanbe A,Takeo S.Cardiac sarcoplasmic reticular function in rats with chronic heart failure following myocardial infarction[J].J Mol Cell Cardiol,1997,29:753-763.
[39]Yamakuchi M,Greer JJ,Cameron S J,Matsushita K,Morrell CN,Talbot-Fox K,et al.HMG-CoA reductase inhibitors inhibit endothelial exocytosis and decrease myocardial infarct size[J].Circ Res,2005,96:1185-1192.
[40]Kelly RA,Balligand JL,Smith TW.Nitric oxide and cardiac function[J].Circ Res,1996,79:363-380.
[41]Kelm M,Schrader J.Control of coronary vascular tone by nitric oxide[J].Circ Res,1990,66:1561-1575.
[42]Gorg A,Postel W,Weser J,Günther S,Strahler JR,Hanash SM,et al.Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer[J].Electrophoresis,1987,8:122-124.
[43]Jensen ON,Wilm M,Shevchenko A,Mann M.Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels[J].Methods Mol Biol,1999,112:513-530.
[44]Maytin M,Leopold J,Loscalzo J.Oxidant stress in the vasculature[J].Curr Atheroscler Rep,1999,1: 156-164.
[45] Lesnefsky EJ, Slabe TJ, Stoll MS, Minkler PE, Hoppel CL. Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria[J]. Am J Physiol Heart Circ Physiol, 2001,280: H2770-2778.
[46] Lesnefsky EJ, Chen Q, Moghaddas S, Hassan MO, Tandler B, Hoppel CL. Blockade of electron transport during ischemia protects cardiac mitochondria[J]. J Biol Chem, 2004,279:47961-47967.
[47] Lepore DA, Knight KR, Anderson RL, Morrison WA. Role of priming stresses and Hsp70 in protection from ischemia-reperfusion injury in cardiac and skeletal muscle[J]. Cell Stress Chaperones, 2001, 6:93-96.
[48] Sun J, Huang SH, Tan BK, Whiteman M, Zhu YC, Wu YJ, et al. Effects of purified herbal extract of Salvia miltiorrhiza on ischemic rat myocardium after acute myocardial infarction[J].Life Sci, 2005, 76:2849-2860.
[2]申定珠,李家邦,蒋荣鑫.证候蛋白质组学与中医证候学相关性探讨[J].中国中西医结合杂志,2006,24(4):366-368.
[3]钟小兰,吕志平,钱令嘉.肝郁证模型大鼠血清蛋白质组的差异表达研究[J].中华中医药杂志,2006,21(7):399-404.
[4]李炜,陈敏,周凌燕.甲亢肝阳上亢证大鼠肾上腺组织蛋白质组的双向电泳分析[J].中国医师杂志,2007,9(1):20-24.
[5]吴红金,马增春,高月.蛋白质组学技术对冠心病血瘀证相关蛋白的研究[J].中西医结合心脑血管病杂志,2005,3(3):189-191.
[6]牟君,杨泽松,杨德兰.抑郁大鼠海马的双向凝胶电泳图谱分析[J].中国神经精神疾病杂志,2006,32(3):219-221.
[7]郭平,王升启.芍药苷对人骨髓基质细胞HFCL蛋白质表达的作用[J].中草药,2006,37(8):1206-1210.
[8]董燕,冯冲,马伟忠.苍耳子对小鼠脾淋巴细胞蛋白质组影响的双向电泳分析[J].中药新药与临床药理,2005,16(5):324-327.
[9]谢文光,马晓昌,邵宁生.赤芍治疗热毒血瘀证的血清蛋白质组变化的初步研究[J].中国中西医结合杂志,2005,25(6):520-523.
[10]张伟,曾园山,汪洋.灵芝孢子促进大鼠受损伤脊髓运动神经元存活及其轴突再生相关蛋白质组学的初步研究[J].中西医结合学报,2006,4(3):298-300.
[11]Wang.Y.,Y.H.Cheung,Z.Yang,Proteomic approach to study the cytotoxicity of dioscin(saponin)[J],Proteomics,2006.
[12]Ong,E.S.,S.M.Len,and A.C.H.Lee.Proteomic analysis of mouse liver for the evaluation of effects of Scutellariae radix by liquid chromatography with tandem mass spectrometry[J].Rapid Commun.Mass Spectrom,2004.18:2522.-2528.
[13]吴伟康,李劲平,罗汉川.四逆汤抗心肌缺血作用的相关蛋白谱研究[J].中国病理生理杂志,2005,21(3):506-508.
[14]刘丽丽,马增春,王宇光.四物汤对环磷酰胺致血虚证小鼠骨髓蛋白质组的影响[J].中国中药杂志,2006,31(14):1172-1175.
[15]马增春,高月,谭洪玲.复方丹参片对冠心病患者血浆蛋白影响的蛋白质组学研究[J].中国中药 杂志,2006,31(9):766-768.
[16]李连达,张荣利,刘成源.双龙方对大鼠心肌梗塞的治疗作用[J].中药新药与临床药理,2004,15(3):149-153.
[17]叶能胜,赵艳峰,张荣利.双龙方干预骨髓间充质干细胞分化过程的蛋白表达[J].中成药,2007,29(1):24-27.
[18]孙丽红,李鸿珠,韩丽萍.青蒿素对离体大鼠心肌缺血/再灌注损伤的保护作用[J].中国中药杂志,2007,32(15):1547-1551.
[19]Wang,Y.,L.Liu,C.Hu.Effects of Salviae Mitiorrhizae and Cortex Moutan extract on the rat heart after myocardial infarction[J].A proteomic study,biochemical pharmacology,2007,74:415-420.
[20]Tom Berkelman,Stenstedt T.2-D Electrophoresis[M]:Principles and Methods:Amersham Biosciences,2002.
[21]陈主初,梁宋平.肿瘤蛋白质组学[M],2002,长沙:湖南科学技术出版社.
[22]双向电泳实验手册[M],GE Healthcare公司.
[23]沈朋,范骁辉,曾真,程翼宇.基于图像特征和数学形态学的蛋白质组双向电泳分析法[J].中国科学B辑:化学,2005,35(1):44-50.
[24]Redfeam D.P.,Skanes A.C.,Lane J.,et al Signal-Averaged P Wave Reflects Change in Atrial Electrophysiological Substrate Afforded by Verapamil Following Cardioversion from Atrial Fibrillation.Pacing Clin Elecrophvsiol[J],2006,10:1089-1095.
[25]Cooper-DeHoff R.M.,Aranda J.M.,Gaxiola E.,et al.Blood pressure control and cardiovascular outcomes in high-risk Hispanic patients-Findings From the International Verapamil SR/Trandolapril Study.Am Heart[J],2006,151(5):1072-1079.
[26]Shaheen N.,Mahboob T.Antihypertensive and metabolic effects of verapamil:role of Na-K-ATPase and electrolytes homeostasis in male and female rats..Pak J Pharm Sci.[J],2004,17(2):1-11.
[27]Hansen J.F.Treatment with verapamil after an acute myocardial infarction.Review of the Danish studies on verapamil in myocardial infarction(DAVIT I and II)..Drugs.[J],1991,42:43-53.
[28]Yamaguchi F.,Sanbe A.,Takeo S.Cardiac Sarcoplasmic Reticular Function in Rats with Chronic Heart Failure Following Myocardial Infarction Cardiac Sarcoplasmic Reticular Function in Rats with Chronic Heart Failure Following Myocardial Infarction.J Mol Cell Cardiol.[J],1997,29:753-763.
[29]Jensen O.N.,Wilm M.,Shevchenko A.,et al.Sample Preparation Methods for Mass Spectrometric Peptide Mapping Directly from 2-DE Gels.Methods Mol Biol.[J],1999,112:513-530.
[30]Frolov V.A.,Drozdova G.Ao,Rieger P.,et al Initial Mechanisms of the Development of Hypertonic Heart..Bull Exp Biol Med.[J],2005,139(3):277-278.
[31]Lesnefsky E.J.,Slabe T.J.,Stoll M.S.,et al.Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria.Am J Physiol Heart Circ Physiol.[J],2001,280:H2770-H2778.
[32]Lepore D.A.,Knight K.R.,Anderson R.L.,et al.Role of priming stresses and Hsp70 in protection from ischemia-reperfusion injury in cardiac and skeletal muscle.Cell Stress Chap.[J],2001,6:93-96.
[33]祝维峰.通络宁治疗冠心病心绞痛的研究[J].山东中医学院学报,1994,18(5):311-317.
[34]王金荣,王知佳等.辨证辨病治疗冠心病108例临床观察[J].辽宁中医杂志,2001,28(8):475-476.
[35]方祝元,顾学兰.复方三七口服液治疗气虚血瘀型冠心病临床研究[J],中国中医急症,2000,9(6):247-248.
[36]王秀娥,梁华龙.舒心通脉片治疗冠心病心绞痛临床研究[J].山东中医杂志,1997,16(8):346-347.
[37]天津天士力制药股份有限公司.治疗冠心病心绞痛的中药制剂及其制备方法,专利号200310107278.0
[38]Yamaguchi F,Sanbe A,Takeo S.Cardiac sarcoplasmic reticular function in rats with chronic heart failure following myocardial infarction[J].J Mol Cell Cardiol,1997,29:753-763.
[39]Yamakuchi M,Greer JJ,Cameron S J,Matsushita K,Morrell CN,Talbot-Fox K,et al.HMG-CoA reductase inhibitors inhibit endothelial exocytosis and decrease myocardial infarct size[J].Circ Res,2005,96:1185-1192.
[40]Kelly RA,Balligand JL,Smith TW.Nitric oxide and cardiac function[J].Circ Res,1996,79:363-380.
[41]Kelm M,Schrader J.Control of coronary vascular tone by nitric oxide[J].Circ Res,1990,66:1561-1575.
[42]Gorg A,Postel W,Weser J,Günther S,Strahler JR,Hanash SM,et al.Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer[J].Electrophoresis,1987,8:122-124.
[43]Jensen ON,Wilm M,Shevchenko A,Mann M.Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels[J].Methods Mol Biol,1999,112:513-530.
[44]Maytin M,Leopold J,Loscalzo J.Oxidant stress in the vasculature[J].Curr Atheroscler Rep,1999,1: 156-164.
[45] Lesnefsky EJ, Slabe TJ, Stoll MS, Minkler PE, Hoppel CL. Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria[J]. Am J Physiol Heart Circ Physiol, 2001,280: H2770-2778.
[46] Lesnefsky EJ, Chen Q, Moghaddas S, Hassan MO, Tandler B, Hoppel CL. Blockade of electron transport during ischemia protects cardiac mitochondria[J]. J Biol Chem, 2004,279:47961-47967.
[47] Lepore DA, Knight KR, Anderson RL, Morrison WA. Role of priming stresses and Hsp70 in protection from ischemia-reperfusion injury in cardiac and skeletal muscle[J]. Cell Stress Chaperones, 2001, 6:93-96.
[48] Sun J, Huang SH, Tan BK, Whiteman M, Zhu YC, Wu YJ, et al. Effects of purified herbal extract of Salvia miltiorrhiza on ischemic rat myocardium after acute myocardial infarction[J].Life Sci, 2005, 76:2849-2860.