EGCG预处理在心肌细胞缺氧/复氧损伤中的保护作用及其细胞内PI3-K/Akt信号通路的研究
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摘要
目的:以原代培养的SD新生乳鼠心肌细胞为研究对象,建立心肌细胞缺氧/复氧损伤模型,在细胞水平模拟心肌缺血再灌注损伤,探讨表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)预处理对心肌细胞缺氧/复氧(Anoxia/Reoxygenation,A/R)损伤的保护作用及其与细胞内PI3K-Akt信号转导通路的关系。
方法:实验用SD新生大鼠(1～3 d),雌雄不拘,无菌取出乳鼠心脏,经分离附着组织,胰蛋白酶消化,纯化制成心肌细胞,在培养48 h后随机分为4组:正常对照(Control)组、缺氧/复氧(A/R)组、EGCG预处理组(EGCG+ A/R组)、EGCG+ LY294002+ A/R组。对以下指标进行检测:①细胞存活率(MTT法);②培养液中LDH活性;③流式细胞仪检测心肌细胞凋亡率;④Western blot法检测心肌细胞内t-Akt和p-Akt蛋白含量的表达。
结果:①细胞存活率:A/R组(45.79±3.98%)与Control组(93.62±3.01%)、EGCG+ A/R组(73.86±4.78%)相比,有显著性差异(均p<0.01);EGCG+ LY294002+ A/R组(49.62±4.83%)与A /R组相比,无显著性差异(p>0.05),但与EGCG+ A/R组相比有显著性差异(p<0.01)。②培养液中LDH活性:A/R组(31.96±3.12 U/L)与Control组(4.06±0.51 U/L)比较,有显著差异(p<0.01);EGCG + A/R组(17.46±1.91 U/L)与A/R组比较,有显著性差异(p<0.01);EGCG+ LY294002+ A/R组(29.52±2.62 U/L)与A/R组相比,无显著性差异(p>0.05);但与EGCG +A/R组相比有显著性差异(p<0.01)。③心肌细胞凋亡率:与Control组(5.24±0.86%)、EGCG+ A/R组(9.68±1.12%)相比,A/R组(19.32±1.54%)心肌细胞凋亡程度明显增加,有显著性差异(均p<0.01);EGCG+ LY294002+ A/R组(17.62±1.48%)与A/R组相比,无显著性差异(p>0.05),与EGCG+ A/R组相比有显著性差异(p<0.01)。④心肌细胞内t-Akt及p-Akt表达含量的变化:各处理组心肌细胞内t-Akt蛋白的表达无显著性差异(均p>0.05)。p-Akt蛋白的表达在EGCG +A/R组中较A/R组显著增加,两者相比有显著性差异(p<0.01);EGCG+ LY294002+ A/R组心肌细胞内p-Akt蛋白的表达较EGCG +A/R组则明显降低,两者有显著性差异(p <0.01)。
结论:从细胞水平证明了EGCG预处理对乳鼠心肌细胞缺氧/复氧损伤具有保护作用,这种预适应保护作用可能与EGCG激活了细胞内PI3K-Akt信号转导通路有关。
Objective: Establish neonatal rat myocardial cells anoxia/reoxygenation (A/R) injury model. To explore the protective effects and the mechanisms of epigallocatechin gallate (EGCG) preconditioning on neonatal rat myocardial cells A/R Injury. Methods: Cultured myocardial cells of SD neonatal rats(1～3 d), and then randomly divided into three groups after cultured 48 hours: control group, anoxia/ reoxygenation group (A / R group) , EGCE preconditionging group(EGCG+ A / R group). At the end of experiment , measured cell viability and the amount of released lactate dehydrogenase (LDH) by a colorimetric method; Apoptosis of myocardial cells was detected by Annexin-V/PI assays with flow cytometry; The expression of t-Akt and p-Akt protein in cytoplasm were measured by western blotting. Results : 1. The cell viability: the cell viability of A/R group (45.79±3.98%) significantly decreased when compared with that of control group (93.62±3.01%) and EGCG+ A/R group (73.86±4.78%)(p<0.01, respectively); The viability of EGCG+ LY294002+ A/R group(49.62±4.83%)was no significant difference compared with that of A/R group respectively(p>0.05), While the difference between that of EGCG+ A/R group was significant(p<0.01). 2. LDH activity: the activity of LDH in A/R group(31.96±3.12 U/L)significantly increased compared with that in control group(4.06±0.51 U/L()p<0.01);the activity of LDH in EGCG + A/R group (17.46±1.91 U/L) significantly decreased compared with that in A/R group(p<0.01); the activity of LDH was no significant difference between EGCG+ LY294002+ A/R group(29.52±2.62 U/L)and A/R group (p>0.05), While the difference between that of EGCG+ A/R group was significant(p<0.01). 3. Cell apoptosis levels: the apoptosis levels of A/R group(19.32±1.54%)noticeably increased compared that of control group(5.24±0.86%)and EGCG+ A/R group(9.68±1.12%)(p<0.01, respectively); the apoptosis levels of EGCG+ LY294002+ A/R group(17.62±1.48%)was no significant difference compared with A/R group, While compared with EGCG+ A/R group, the the apoptosis levels was significantly decreased(p<0.01). 4. Expression of t-Akt and p-Akt protein: the expression of t-Akt protein was no noticeably difference in all group (p>0.05,respectively). The expression of p-Akt protein in EGCG+ A/R group was significantly increased compared that of A/R group(p<0.01); while the expression of p-Akt protein in EGCG+ LY294002+ A/R group was significantly decreased compared with EGCG+ A/R group(p<0.01). Conclusion: Our research showed that EGCG could protect cardiomyocytes against A/R injury, the antagonist effect on myocardial cells injury induced by A/R was mediated by activation of intracellular PI3K-Akt signal pathway.
方法:实验用SD新生大鼠(1～3 d),雌雄不拘,无菌取出乳鼠心脏,经分离附着组织,胰蛋白酶消化,纯化制成心肌细胞,在培养48 h后随机分为4组:正常对照(Control)组、缺氧/复氧(A/R)组、EGCG预处理组(EGCG+ A/R组)、EGCG+ LY294002+ A/R组。对以下指标进行检测:①细胞存活率(MTT法);②培养液中LDH活性;③流式细胞仪检测心肌细胞凋亡率;④Western blot法检测心肌细胞内t-Akt和p-Akt蛋白含量的表达。
结果:①细胞存活率:A/R组(45.79±3.98%)与Control组(93.62±3.01%)、EGCG+ A/R组(73.86±4.78%)相比,有显著性差异(均p<0.01);EGCG+ LY294002+ A/R组(49.62±4.83%)与A /R组相比,无显著性差异(p>0.05),但与EGCG+ A/R组相比有显著性差异(p<0.01)。②培养液中LDH活性:A/R组(31.96±3.12 U/L)与Control组(4.06±0.51 U/L)比较,有显著差异(p<0.01);EGCG + A/R组(17.46±1.91 U/L)与A/R组比较,有显著性差异(p<0.01);EGCG+ LY294002+ A/R组(29.52±2.62 U/L)与A/R组相比,无显著性差异(p>0.05);但与EGCG +A/R组相比有显著性差异(p<0.01)。③心肌细胞凋亡率:与Control组(5.24±0.86%)、EGCG+ A/R组(9.68±1.12%)相比,A/R组(19.32±1.54%)心肌细胞凋亡程度明显增加,有显著性差异(均p<0.01);EGCG+ LY294002+ A/R组(17.62±1.48%)与A/R组相比,无显著性差异(p>0.05),与EGCG+ A/R组相比有显著性差异(p<0.01)。④心肌细胞内t-Akt及p-Akt表达含量的变化:各处理组心肌细胞内t-Akt蛋白的表达无显著性差异(均p>0.05)。p-Akt蛋白的表达在EGCG +A/R组中较A/R组显著增加,两者相比有显著性差异(p<0.01);EGCG+ LY294002+ A/R组心肌细胞内p-Akt蛋白的表达较EGCG +A/R组则明显降低,两者有显著性差异(p <0.01)。
结论:从细胞水平证明了EGCG预处理对乳鼠心肌细胞缺氧/复氧损伤具有保护作用,这种预适应保护作用可能与EGCG激活了细胞内PI3K-Akt信号转导通路有关。
Objective: Establish neonatal rat myocardial cells anoxia/reoxygenation (A/R) injury model. To explore the protective effects and the mechanisms of epigallocatechin gallate (EGCG) preconditioning on neonatal rat myocardial cells A/R Injury. Methods: Cultured myocardial cells of SD neonatal rats(1～3 d), and then randomly divided into three groups after cultured 48 hours: control group, anoxia/ reoxygenation group (A / R group) , EGCE preconditionging group(EGCG+ A / R group). At the end of experiment , measured cell viability and the amount of released lactate dehydrogenase (LDH) by a colorimetric method; Apoptosis of myocardial cells was detected by Annexin-V/PI assays with flow cytometry; The expression of t-Akt and p-Akt protein in cytoplasm were measured by western blotting. Results : 1. The cell viability: the cell viability of A/R group (45.79±3.98%) significantly decreased when compared with that of control group (93.62±3.01%) and EGCG+ A/R group (73.86±4.78%)(p<0.01, respectively); The viability of EGCG+ LY294002+ A/R group(49.62±4.83%)was no significant difference compared with that of A/R group respectively(p>0.05), While the difference between that of EGCG+ A/R group was significant(p<0.01). 2. LDH activity: the activity of LDH in A/R group(31.96±3.12 U/L)significantly increased compared with that in control group(4.06±0.51 U/L()p<0.01);the activity of LDH in EGCG + A/R group (17.46±1.91 U/L) significantly decreased compared with that in A/R group(p<0.01); the activity of LDH was no significant difference between EGCG+ LY294002+ A/R group(29.52±2.62 U/L)and A/R group (p>0.05), While the difference between that of EGCG+ A/R group was significant(p<0.01). 3. Cell apoptosis levels: the apoptosis levels of A/R group(19.32±1.54%)noticeably increased compared that of control group(5.24±0.86%)and EGCG+ A/R group(9.68±1.12%)(p<0.01, respectively); the apoptosis levels of EGCG+ LY294002+ A/R group(17.62±1.48%)was no significant difference compared with A/R group, While compared with EGCG+ A/R group, the the apoptosis levels was significantly decreased(p<0.01). 4. Expression of t-Akt and p-Akt protein: the expression of t-Akt protein was no noticeably difference in all group (p>0.05,respectively). The expression of p-Akt protein in EGCG+ A/R group was significantly increased compared that of A/R group(p<0.01); while the expression of p-Akt protein in EGCG+ LY294002+ A/R group was significantly decreased compared with EGCG+ A/R group(p<0.01). Conclusion: Our research showed that EGCG could protect cardiomyocytes against A/R injury, the antagonist effect on myocardial cells injury induced by A/R was mediated by activation of intracellular PI3K-Akt signal pathway.
引文
[1]Schulze CJ, Wang W, Kumafi R, et a1. Imbalante between tissue inhibitor of metallop- reteinase-4 and matrix metallopreteinases during acute myocardial correction of myocardial ischemia—reperfusion injuryl.[J].Circulation,2003,107(19):2487-2492
[2]Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. [J].Circulation,1986, 74(5):1124-1136.
[3] LAWSON CS, DOWNEY JM. Proconditioning: state of the art myocardial protection. [J].Cardiovascular Res,1993,27(4):542-550.
[4] Eisen A, Fisman EZ, Rubebfire M, et al. Ischemic preconditioning: nearly two decades of research. A comprehensive review. [J].Atherosclerosis,2004, 172(2): 201-210.
[5] Kloner RA, Jennings RB. Consequences of brief ischemia: stunning, preconditioning, and their clinical implications-part 2. [J].Circulation, 2001,104(25): 3158-3167.
[6]Kositprapa C, Ockaili RA, Kukreja BC. Bradykinin B receptor is involved in the late phase of preconditioning in rabbit heart. [J].Mol Cell Cardiol. 2001, 33(7):1355-1362.
[7]Ward SG, Finan P. Isoform-specifi c phosphoinositide 3-kinase inhibitors as therapeutic agents. Curr Opin Pharmacol,2003,3(4): 426-434
[8]Sebbagh M,Renvoize C,Hamelin J,et al. Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. [J].Nat Cell Biol,2001,3(4):346~ 352.
[9]Miki H, Yamaguchi H,Suetsugu S,etal.IRS p53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. [J].Nature,2000,408:732~735
[10]Kennedy S G,Kandel E S,Cross T K,et al. Akt/Protein Kinase B inhibits cell death by preventing the release of cytochrome from mitochondria. [J].MolCellBiol,1999,19:5800~5810
[11]Zhou H L,Li X M,Meinkoth J,et al.Akt regulates cell Survival and apoptosis at a postmitochondrial level. [J].Cell Biol,2000,151:483~494
[12]Mayo L D,Donner D B.A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the Cytoplasm to the nucleus. Proc NatlA cad Sci USA,2001,98:11598~11603
[13]Dimmeler S, Fleming I, Fisslthaler B,etal. Activation of Nitric oxidesynthase in endothelial cells by Akt-dependent phosphorylation [J]..Nature,1999,399:601~605
[14]Mocanu MM, Bell RM, Yellon DM (2002). PI3 kinase and not p42/p44 appears to be implicated in the protection conferred by ischemic preconditioning. [J].Mol Cell Cardiol 34: 661–668.
[15]Jonassen AK, Sack MN, Mjos OD, Yellon DM (2001). Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. [J].Circ Res 89: 1191–1198.
[16]Brar BK, Stephanou A, Knight R, Latchman DS (2002). Activation of protein kinase B/Akt by urocortin is essential for its ability to protect cardiac cells against hypoxia/reoxygenation- induced cell death. [J].Mol Cell Cardiol 34: 483–492.
[17]Bell RM, Yellon DM (2003b). Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K, Akt and eNOS. [J].Mol Cell Cardiol 35: 185–193.
[18]Parsa CJ, Matsumoto A, Kim J, Riel RU, Pascal LS, Walton GB et al.(2003). A novel protective effect of erythropoietin in the infracted heart. [J].Clin Invest 112: 999–1007.
[19]Hausenloy DJ, Yellon DM (2004). New directions for protecting the heart against ischaemia–reperfusion injury: targeting the reperfusion injury salvage kinase (RISK)-pathway. [J].Cardiovasc Res 61: 448–460.
[20]Nishihara M, Miura T, Miki T, Sakamoto J, Tanno M, Kobayashi H et al. (2006). Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3 beta 1. Am J Physiol Heart Circ Physiol 291:H748–H755.
[21]Jonassen AK, Sack MN, Mjos OD, Yellon DM (2001). Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. [J].Circ Res 89: 1191–1198.
[22]Townsend P A, Scarabelli T M, Pasini E, et al. Epigallocatechin-3-gallate Inhibits STAT-1 Activation and Protects Cardiac Myocytes from Ischemia/Reperfusion-induced Apoptosis[J]. The FASEB Journal, 2004,18(13): 1621–1623.
[23]Maria A. Potenza,1 Flora L. Marasciulo, et al.(2007). EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR. Am J Physiol Endocrinol Metab 292:1378-1387
[24]Higdon JV, Frei B. Tea catechins and polyphenols: health effects,metabolism, and antioxidant functions. [J].Crit Rev Food Sci Nutr 43:89–143, 2003.
[25]Elbling L, Weiss RM, Teufelhofer O, Uhl M, Knasmueller S, Schulte-Hermann R, Berger W, Micksche M. Green tea extract and (-)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidantactivities. [J]. FASEB J 19: 807–809, 2005
[26]Townsend PA, Scarabelli TM, Pasini E, Gitti G, Menegazzi M, SuzukiH, Knight RA, Latchman DS, Stephanou A. Epigallocatechin-3-gallateinhibits STAT-1 activation and protects cardiac myocytes from ischemia/reperfusion-induced apoptosis. [J].FASEB J 18: 1621–1623, 2004.
[27] Nakano A ,Baines CP , Kim SO , et al. Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart :evidence for involvement of p38 MAPK. [J].Circ Res. 2000; 86: 144-7.
[28] Mocanu MM, Baxter GF, Yue Y, et al. The p38 MAPK inhibitor, SB203580, abrogates ischaemic preconditioning in rat heart but timing of administration is critical. [J]. Basic Res Cardiol. 2003; 95: 472.
[29] Xuan R, Patel HH, Hsu AK, et al. Stress-activated protein kinase phosphorylation during cardioprotection in the ischemic myocardium. Am J Physiol Hear Circ Physiol. 2001; 281: 1184-9.
[30] Zhao ZQ,NakamuraM,Wang NP, et a1. Reperfusion induces myocardial apoptotic cell death.[ J ]. Cardiovasc Res, 2000, 45 (3) : 6512660.
[31]Shiki K,Hearse DJ.Preconditioning of ischemia myocardium:Reperfusion.in.dueed arrhythmias.[J].Am J Physiol Heart Circ Physiol,1987,253(22):1470-1476
[32]Tsang A, Hausenloy D, Mocanu MM, et al. Preconditioning the diabetic heart-The importance of Akt phosphorylation.[J].Diabetes,2005,54(8):2360-2364
[33]Kim JH, Jeong MH, Yun KH, et a1. Myocardial protective effects of nicorandil during percutaneous coronary intervention in patients with unstable angina.[J].Circulation.2005. 69(3):306-310.
[34]Ashikaga T, Nishizaki M, Arita M, et a1.Opening of KATP channel attenuateg the increase in QT dispersion produced by the first bali.on inflation during Coronary angioplasty.[J]. Circulation,2002,66(5):469-472
[35]Sommerschild HT, Kirkeboen KA Preconditioning endogenous defence mechanisms of the heart.[J].Acta Anaesthesiol Seand,2002,46(2):123-137
[36]Zhang W J, Shi ZX, Wang BB. Allitridum Mimics Effect of Ischemic Preconditioning by Activation of Protein Kinase C.[J].Act a Pharmacolsin,2001,22 (2) :132-136.
[37]刘季春,高涛,何明,等缓激肽介导的阿魏酸钠对离体大鼠心脏药理性预适应保护作用[J].中国药理学通报,2006,22(9):1100-1103.
[38]Zhu M, Feng J, Lucchinetti E, Fischer G, Xu L, Pedrazzini T et al. Ischemic postconditioning protects remodeled myocardium via the PI3K-PKB/Akt reperfusion injury salvage kinase pathway. [J].Cardiovasc Res,2006,72: 152–162.
[39]Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia– reperfusion injury: targeting the reperfusion injury salvage kinase (RISK)-pathway. [J]. Cardiovasc Res.2004, 61: 448–460.
[1]Hausenloy DJ, Yellon DM (2004). New directions for protecting the heart against ischaemia–reperfusion injury: targeting the reperfusion injury salvage kinase (RISK)-pathway . [J].Cardiovasc Res 61: 448–460
[2]Ward SG, Finan P. Isoform-specifi c phosphoinositide 3-kinase inhibitors as therapeutic agents. [J].Curr Opin Pharmacol 2003; 3: 426-434
[3]Chang F, Lee JT, Navolanic PM, et al. Involvement of P13K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation:a targetf or cancerc hemotherapy. [J].Leukemia,2003,17(3):590-603
[4]M. A. Krasilnikov. Phosphatidylinositol-3 Kinase Dependent Pathways:the Role in Control of Cell Growth, Survival,and Malignant Transformation. [J].Biochemistry, 2000, 65(1):59-67
[5]Elmendorf J S, Pessin J E. Insulin signaling regulating the trafficking and plasma membrane fusion of GLUT4-containing intracellular vesicles. [J].Exp Cell Res,1999,253:55~62
[6]Solit DB, Basso AD, Olshen AB, Scher HI, Rosen N.Inhibition of heat shock protein 90 function downregulates Akt kinase and sensitizes tumors to Taxol. [J].Cancer Res 2003; 63: 2139-2144
[7]KrieserRJ,EastmanA.Cleavageandnucleartranslocation of the caspase3 substrate RhoGDP- dissociation in hibitor,D4-GDI,duringapoptosis. [J].Cell Death Differ,1999,6(5):412~419
[8]Sebbagh M,RenvoizeC,HamelinJ,etal.Caspase-3-medi-ated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. [J].NatCellBiol,2001,3:346~352
[9]Wang X C,Wu Y C,Fadok V A,etal.Cell corpse engulfment Mediated by C.elegan sphosphatidy lserine recept or through CED-5 and CED-12. [J].Science,2003,302:1563~1566
[10]Miki H, Yamaguchi H,Suetsugu S,etal.IRS p53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. [J].Nature,2000,408:732~735
[11]Kennedy S G,Kandel E S,Cross T K,et al. Akt/Protein Kinase B inhibits cell death by preventing the release of cytochrome from mitochondria. [J].Mol Cell Biol,1999,19:5800~ 5810
[12]Zhou H L,Li X M,Meinkoth J,et al.Akt regulates cell Survival and apoptosis at apostmitochondrial level. [J].Cell Biol,2000,151:483~494
[13]Mayo L D,Donner D B.A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the Cytoplasm to the nucleus. Proc NatlA cad Sci USA,2001,98:11598~11603
[14]Dimmeler S, Fleming I, Fisslthaler B,etal. Activation of Nitric oxidesynthase in endothelial cells by Akt-dependent phosphorylation. [J].Nature,1999,399:601~605
[15]Jimenez C, Jones DR, Rodriguez-Viciana P, Gonzalez-Garcia A, Leonardo E, Wennstrom S, von Kobbe C, Toran JL, R-Borlado L, Calvo V, Copin SG, Albar JP, Gaspar ML, Diez E, Marcos MA, Downward J,Martinez-A C, Merida I, Carrera AC. Identifi cation and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase. [J].EMBO 1998, 17: 743-753
[16]Murry CE, Jennings RB, Reimer KA . Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. [J].Circulation 1986,74: 1124–1136
[17]Tong H, Chen W, Steenbergen C, Murphy E (2000). Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. [J].Circ Res 87: 309–315
[18]Mocanu MM, Bell RM, Yellon DM (2002). PI3 kinase and not p42/p44 appears to be implicated in the protection conferred by ischemic preconditioning. [J].Mol Cell Cardiol 34: 661–668
[19]Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA et al. (2003). Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemicpreconditioning. Am J Physiol Heart Circ Physiol 285: H579– H588
[20]Zhu M, Feng J, Lucchinetti E, Fischer G, Xu L, Pedrazzini T et al.(2006). Ischemic postconditioning protects remodeled myocardium via the PI3K-PKB/Akt reperfusion injury salvage kinase pathway. [J].Cardiovasc Res 72: 152–162
[21]Jonassen AK, Sack MN, Mjos OD, Yellon DM (2001). Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell- survival signaling. [J].Circ Res 89: 1191–1198
[22]Brar BK, Stephanou A, Knight R, Latchman DS (2002). Activation of protein kinase B/Akt by urocortin is essential for its ability to protect cardiac cells against hypoxia/reoxygenation- induced cell death. [J].Mol Cell Cardiol 34: 483–492
[23]Bell RM, Yellon DM (2003a). Atorvastatin, administered at the onset of reperfusion, and independent of lipid lowering, protects the myocardium by up-regulating a pro-survival pathway. [J].Am Coll Cardiol 41: 508–515
[24]Bell RM, Yellon DM (2003b). Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K, Akt and eNOS. J Mol Cell Cardiol 35: 185–193
[25]Parsa CJ, Matsumoto A, Kim J, Riel RU, Pascal LS, Walton GB et all. A novel protective effect of erythropoietin in the infracted heart. [J].Clin Invest,2003,112: 999–1007
[26]Bullard AJ, Govewalla P, Yellon DM (2005). Erythropoietin protects the myocardium against reperfusion injury in vitro and in vivo. [J].Basic Res Cardiol 100: 397–403
[27]Bose AK, Mocanu MM, Carr RD, Yellon DM (2005). Glucagon like peptide-1 is protective against myocardial ischemia/reperfusion injury when given either as a preconditioning mimetic or at reperfusion in an isolated rat heart model. [J].Cardiovasc Drugs Ther 19: 9–11.
[28]Hausenloy DJ, Yellon DM (2004). New directions for protecting the heart against ischaemia–reperfusion injury: targeting the reperfusion injury salvage kinase (RISK)-pathway. [J].Cardiovasc Res 61: 448–460
[29]Nishihara M, Miura T, Miki T, Sakamoto J, Tanno M, Kobayashi H et al. (2006). Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3 beta 1. Am J Physiol Heart Circ Physiol 291:H748–H755
[30]Jonassen AK, Sack MN, Mjos OD, Yellon DM (2001). Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. [J].Circ Res 89: 1191–1198
[31]Peso L, González-GarcáM, PageC, etal. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. [J]. Science, 1997, 278:687~689
[32]Datta S R, Dudek H, Tao X, etal. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. [J].Cell, 1997, 91:231~ 241
[33]Mocanu MM, Yellon DM (2003). p53 down-regulation: a new molecular mechanism involved in ischaemic preconditioning. [J].FEBS Lett 555: 302–306.
[2]Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. [J].Circulation,1986, 74(5):1124-1136.
[3] LAWSON CS, DOWNEY JM. Proconditioning: state of the art myocardial protection. [J].Cardiovascular Res,1993,27(4):542-550.
[4] Eisen A, Fisman EZ, Rubebfire M, et al. Ischemic preconditioning: nearly two decades of research. A comprehensive review. [J].Atherosclerosis,2004, 172(2): 201-210.
[5] Kloner RA, Jennings RB. Consequences of brief ischemia: stunning, preconditioning, and their clinical implications-part 2. [J].Circulation, 2001,104(25): 3158-3167.
[6]Kositprapa C, Ockaili RA, Kukreja BC. Bradykinin B receptor is involved in the late phase of preconditioning in rabbit heart. [J].Mol Cell Cardiol. 2001, 33(7):1355-1362.
[7]Ward SG, Finan P. Isoform-specifi c phosphoinositide 3-kinase inhibitors as therapeutic agents. Curr Opin Pharmacol,2003,3(4): 426-434
[8]Sebbagh M,Renvoize C,Hamelin J,et al. Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. [J].Nat Cell Biol,2001,3(4):346~ 352.
[9]Miki H, Yamaguchi H,Suetsugu S,etal.IRS p53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. [J].Nature,2000,408:732~735
[10]Kennedy S G,Kandel E S,Cross T K,et al. Akt/Protein Kinase B inhibits cell death by preventing the release of cytochrome from mitochondria. [J].MolCellBiol,1999,19:5800~5810
[11]Zhou H L,Li X M,Meinkoth J,et al.Akt regulates cell Survival and apoptosis at a postmitochondrial level. [J].Cell Biol,2000,151:483~494
[12]Mayo L D,Donner D B.A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the Cytoplasm to the nucleus. Proc NatlA cad Sci USA,2001,98:11598~11603
[13]Dimmeler S, Fleming I, Fisslthaler B,etal. Activation of Nitric oxidesynthase in endothelial cells by Akt-dependent phosphorylation [J]..Nature,1999,399:601~605
[14]Mocanu MM, Bell RM, Yellon DM (2002). PI3 kinase and not p42/p44 appears to be implicated in the protection conferred by ischemic preconditioning. [J].Mol Cell Cardiol 34: 661–668.
[15]Jonassen AK, Sack MN, Mjos OD, Yellon DM (2001). Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. [J].Circ Res 89: 1191–1198.
[16]Brar BK, Stephanou A, Knight R, Latchman DS (2002). Activation of protein kinase B/Akt by urocortin is essential for its ability to protect cardiac cells against hypoxia/reoxygenation- induced cell death. [J].Mol Cell Cardiol 34: 483–492.
[17]Bell RM, Yellon DM (2003b). Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K, Akt and eNOS. [J].Mol Cell Cardiol 35: 185–193.
[18]Parsa CJ, Matsumoto A, Kim J, Riel RU, Pascal LS, Walton GB et al.(2003). A novel protective effect of erythropoietin in the infracted heart. [J].Clin Invest 112: 999–1007.
[19]Hausenloy DJ, Yellon DM (2004). New directions for protecting the heart against ischaemia–reperfusion injury: targeting the reperfusion injury salvage kinase (RISK)-pathway. [J].Cardiovasc Res 61: 448–460.
[20]Nishihara M, Miura T, Miki T, Sakamoto J, Tanno M, Kobayashi H et al. (2006). Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3 beta 1. Am J Physiol Heart Circ Physiol 291:H748–H755.
[21]Jonassen AK, Sack MN, Mjos OD, Yellon DM (2001). Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. [J].Circ Res 89: 1191–1198.
[22]Townsend P A, Scarabelli T M, Pasini E, et al. Epigallocatechin-3-gallate Inhibits STAT-1 Activation and Protects Cardiac Myocytes from Ischemia/Reperfusion-induced Apoptosis[J]. The FASEB Journal, 2004,18(13): 1621–1623.
[23]Maria A. Potenza,1 Flora L. Marasciulo, et al.(2007). EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR. Am J Physiol Endocrinol Metab 292:1378-1387
[24]Higdon JV, Frei B. Tea catechins and polyphenols: health effects,metabolism, and antioxidant functions. [J].Crit Rev Food Sci Nutr 43:89–143, 2003.
[25]Elbling L, Weiss RM, Teufelhofer O, Uhl M, Knasmueller S, Schulte-Hermann R, Berger W, Micksche M. Green tea extract and (-)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidantactivities. [J]. FASEB J 19: 807–809, 2005
[26]Townsend PA, Scarabelli TM, Pasini E, Gitti G, Menegazzi M, SuzukiH, Knight RA, Latchman DS, Stephanou A. Epigallocatechin-3-gallateinhibits STAT-1 activation and protects cardiac myocytes from ischemia/reperfusion-induced apoptosis. [J].FASEB J 18: 1621–1623, 2004.
[27] Nakano A ,Baines CP , Kim SO , et al. Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart :evidence for involvement of p38 MAPK. [J].Circ Res. 2000; 86: 144-7.
[28] Mocanu MM, Baxter GF, Yue Y, et al. The p38 MAPK inhibitor, SB203580, abrogates ischaemic preconditioning in rat heart but timing of administration is critical. [J]. Basic Res Cardiol. 2003; 95: 472.
[29] Xuan R, Patel HH, Hsu AK, et al. Stress-activated protein kinase phosphorylation during cardioprotection in the ischemic myocardium. Am J Physiol Hear Circ Physiol. 2001; 281: 1184-9.
[30] Zhao ZQ,NakamuraM,Wang NP, et a1. Reperfusion induces myocardial apoptotic cell death.[ J ]. Cardiovasc Res, 2000, 45 (3) : 6512660.
[31]Shiki K,Hearse DJ.Preconditioning of ischemia myocardium:Reperfusion.in.dueed arrhythmias.[J].Am J Physiol Heart Circ Physiol,1987,253(22):1470-1476
[32]Tsang A, Hausenloy D, Mocanu MM, et al. Preconditioning the diabetic heart-The importance of Akt phosphorylation.[J].Diabetes,2005,54(8):2360-2364
[33]Kim JH, Jeong MH, Yun KH, et a1. Myocardial protective effects of nicorandil during percutaneous coronary intervention in patients with unstable angina.[J].Circulation.2005. 69(3):306-310.
[34]Ashikaga T, Nishizaki M, Arita M, et a1.Opening of KATP channel attenuateg the increase in QT dispersion produced by the first bali.on inflation during Coronary angioplasty.[J]. Circulation,2002,66(5):469-472
[35]Sommerschild HT, Kirkeboen KA Preconditioning endogenous defence mechanisms of the heart.[J].Acta Anaesthesiol Seand,2002,46(2):123-137
[36]Zhang W J, Shi ZX, Wang BB. Allitridum Mimics Effect of Ischemic Preconditioning by Activation of Protein Kinase C.[J].Act a Pharmacolsin,2001,22 (2) :132-136.
[37]刘季春,高涛,何明,等缓激肽介导的阿魏酸钠对离体大鼠心脏药理性预适应保护作用[J].中国药理学通报,2006,22(9):1100-1103.
[38]Zhu M, Feng J, Lucchinetti E, Fischer G, Xu L, Pedrazzini T et al. Ischemic postconditioning protects remodeled myocardium via the PI3K-PKB/Akt reperfusion injury salvage kinase pathway. [J].Cardiovasc Res,2006,72: 152–162.
[39]Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia– reperfusion injury: targeting the reperfusion injury salvage kinase (RISK)-pathway. [J]. Cardiovasc Res.2004, 61: 448–460.
[1]Hausenloy DJ, Yellon DM (2004). New directions for protecting the heart against ischaemia–reperfusion injury: targeting the reperfusion injury salvage kinase (RISK)-pathway . [J].Cardiovasc Res 61: 448–460
[2]Ward SG, Finan P. Isoform-specifi c phosphoinositide 3-kinase inhibitors as therapeutic agents. [J].Curr Opin Pharmacol 2003; 3: 426-434
[3]Chang F, Lee JT, Navolanic PM, et al. Involvement of P13K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation:a targetf or cancerc hemotherapy. [J].Leukemia,2003,17(3):590-603
[4]M. A. Krasilnikov. Phosphatidylinositol-3 Kinase Dependent Pathways:the Role in Control of Cell Growth, Survival,and Malignant Transformation. [J].Biochemistry, 2000, 65(1):59-67
[5]Elmendorf J S, Pessin J E. Insulin signaling regulating the trafficking and plasma membrane fusion of GLUT4-containing intracellular vesicles. [J].Exp Cell Res,1999,253:55~62
[6]Solit DB, Basso AD, Olshen AB, Scher HI, Rosen N.Inhibition of heat shock protein 90 function downregulates Akt kinase and sensitizes tumors to Taxol. [J].Cancer Res 2003; 63: 2139-2144
[7]KrieserRJ,EastmanA.Cleavageandnucleartranslocation of the caspase3 substrate RhoGDP- dissociation in hibitor,D4-GDI,duringapoptosis. [J].Cell Death Differ,1999,6(5):412~419
[8]Sebbagh M,RenvoizeC,HamelinJ,etal.Caspase-3-medi-ated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. [J].NatCellBiol,2001,3:346~352
[9]Wang X C,Wu Y C,Fadok V A,etal.Cell corpse engulfment Mediated by C.elegan sphosphatidy lserine recept or through CED-5 and CED-12. [J].Science,2003,302:1563~1566
[10]Miki H, Yamaguchi H,Suetsugu S,etal.IRS p53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. [J].Nature,2000,408:732~735
[11]Kennedy S G,Kandel E S,Cross T K,et al. Akt/Protein Kinase B inhibits cell death by preventing the release of cytochrome from mitochondria. [J].Mol Cell Biol,1999,19:5800~ 5810
[12]Zhou H L,Li X M,Meinkoth J,et al.Akt regulates cell Survival and apoptosis at apostmitochondrial level. [J].Cell Biol,2000,151:483~494
[13]Mayo L D,Donner D B.A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the Cytoplasm to the nucleus. Proc NatlA cad Sci USA,2001,98:11598~11603
[14]Dimmeler S, Fleming I, Fisslthaler B,etal. Activation of Nitric oxidesynthase in endothelial cells by Akt-dependent phosphorylation. [J].Nature,1999,399:601~605
[15]Jimenez C, Jones DR, Rodriguez-Viciana P, Gonzalez-Garcia A, Leonardo E, Wennstrom S, von Kobbe C, Toran JL, R-Borlado L, Calvo V, Copin SG, Albar JP, Gaspar ML, Diez E, Marcos MA, Downward J,Martinez-A C, Merida I, Carrera AC. Identifi cation and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase. [J].EMBO 1998, 17: 743-753
[16]Murry CE, Jennings RB, Reimer KA . Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. [J].Circulation 1986,74: 1124–1136
[17]Tong H, Chen W, Steenbergen C, Murphy E (2000). Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. [J].Circ Res 87: 309–315
[18]Mocanu MM, Bell RM, Yellon DM (2002). PI3 kinase and not p42/p44 appears to be implicated in the protection conferred by ischemic preconditioning. [J].Mol Cell Cardiol 34: 661–668
[19]Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA et al. (2003). Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemicpreconditioning. Am J Physiol Heart Circ Physiol 285: H579– H588
[20]Zhu M, Feng J, Lucchinetti E, Fischer G, Xu L, Pedrazzini T et al.(2006). Ischemic postconditioning protects remodeled myocardium via the PI3K-PKB/Akt reperfusion injury salvage kinase pathway. [J].Cardiovasc Res 72: 152–162
[21]Jonassen AK, Sack MN, Mjos OD, Yellon DM (2001). Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell- survival signaling. [J].Circ Res 89: 1191–1198
[22]Brar BK, Stephanou A, Knight R, Latchman DS (2002). Activation of protein kinase B/Akt by urocortin is essential for its ability to protect cardiac cells against hypoxia/reoxygenation- induced cell death. [J].Mol Cell Cardiol 34: 483–492
[23]Bell RM, Yellon DM (2003a). Atorvastatin, administered at the onset of reperfusion, and independent of lipid lowering, protects the myocardium by up-regulating a pro-survival pathway. [J].Am Coll Cardiol 41: 508–515
[24]Bell RM, Yellon DM (2003b). Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K, Akt and eNOS. J Mol Cell Cardiol 35: 185–193
[25]Parsa CJ, Matsumoto A, Kim J, Riel RU, Pascal LS, Walton GB et all. A novel protective effect of erythropoietin in the infracted heart. [J].Clin Invest,2003,112: 999–1007
[26]Bullard AJ, Govewalla P, Yellon DM (2005). Erythropoietin protects the myocardium against reperfusion injury in vitro and in vivo. [J].Basic Res Cardiol 100: 397–403
[27]Bose AK, Mocanu MM, Carr RD, Yellon DM (2005). Glucagon like peptide-1 is protective against myocardial ischemia/reperfusion injury when given either as a preconditioning mimetic or at reperfusion in an isolated rat heart model. [J].Cardiovasc Drugs Ther 19: 9–11.
[28]Hausenloy DJ, Yellon DM (2004). New directions for protecting the heart against ischaemia–reperfusion injury: targeting the reperfusion injury salvage kinase (RISK)-pathway. [J].Cardiovasc Res 61: 448–460
[29]Nishihara M, Miura T, Miki T, Sakamoto J, Tanno M, Kobayashi H et al. (2006). Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3 beta 1. Am J Physiol Heart Circ Physiol 291:H748–H755
[30]Jonassen AK, Sack MN, Mjos OD, Yellon DM (2001). Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. [J].Circ Res 89: 1191–1198
[31]Peso L, González-GarcáM, PageC, etal. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. [J]. Science, 1997, 278:687~689
[32]Datta S R, Dudek H, Tao X, etal. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. [J].Cell, 1997, 91:231~ 241
[33]Mocanu MM, Yellon DM (2003). p53 down-regulation: a new molecular mechanism involved in ischaemic preconditioning. [J].FEBS Lett 555: 302–306.