缺血后处理对大鼠肝脏缺血再灌注损伤的保护作用研究
摘要
目的:探讨缺血后处理对大鼠肝脏缺血再灌注损伤的保护作用和eNOS表达的影响。
方法:建立大鼠肝脏局部缺血再灌注(IR)模型,将75只健康雄性SD大鼠随机分为假手术组、缺血再灌注组及缺血后处理组,缺血后处理组于缺血60min恢复血液再灌注前,给予六个循环的灌注10s/阻断10s的缺血后处理,观察血清天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、肝组织中丙二醛(MDA)、超氧化物歧化酶(SOD)的含量变化,并行肝组织电镜、病理学检查及免疫组化法检测肝细胞eNOS蛋白的表达。
结果:与缺血再灌注组相比,缺血后处理组肝酶的漏出及肝组织MDA明显降低(P＜0.01),而SOD含量则显著升高(P＜0.01),同时肝组织病理形态学损伤亦明显减轻,缺血7min后eNOS蛋白表达更明显(P＜0.01)。
结论:缺血后处理可通过抑止再灌注后氧自由基的过量生成、促进抗氧化剂的生成及通过激活再灌注损伤补救激酶(RISK)通路,减轻肝细胞损伤,发挥保护效应。
Objective:To investigate the protective effect of ischemic postconditioning (IPo)on hepatic ischemia-reperfusion injury and the expressions of eNOS in rats.
Methods:With a rat model of acute liver ischemia-reperfusion,75 healthy male Sprague-Dawley rats were randomly divided into shamoperated(S), ischemia-reperfusion(IR)and IPo group.Before persistent reperfusion,animals were given six brief reperfusion-ischemia in IPo group.The sample of blood and hepatic tissue of all groups were taken after experiment.
Results:Compared with IR group,the concentrations of both aspartate aminotransferase(AST)and alanine aminotransferase(ALT),the concentrations of malondialdehyde(MDA)in hepatic tissue in IPo group decreased markedly(P＜0.01), while the concentrations of superoxide dismutase(SOD)increased significantly(P＜0.01),the hepatic congestion and pathological damage also relieved obviously.The expression of eNOS protein in Ipo group was significantly higher than the two remaining groups(p＜0.01).
Conclusion:IPo can attenuate hepatic ischemia-reperfusion injury by activating the prosurvival kinases eNOS in accordance with the RISK pathway and reducing the concentrations of oxygen free radical.
方法:建立大鼠肝脏局部缺血再灌注(IR)模型,将75只健康雄性SD大鼠随机分为假手术组、缺血再灌注组及缺血后处理组,缺血后处理组于缺血60min恢复血液再灌注前,给予六个循环的灌注10s/阻断10s的缺血后处理,观察血清天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、肝组织中丙二醛(MDA)、超氧化物歧化酶(SOD)的含量变化,并行肝组织电镜、病理学检查及免疫组化法检测肝细胞eNOS蛋白的表达。
结果:与缺血再灌注组相比,缺血后处理组肝酶的漏出及肝组织MDA明显降低(P＜0.01),而SOD含量则显著升高(P＜0.01),同时肝组织病理形态学损伤亦明显减轻,缺血7min后eNOS蛋白表达更明显(P＜0.01)。
结论:缺血后处理可通过抑止再灌注后氧自由基的过量生成、促进抗氧化剂的生成及通过激活再灌注损伤补救激酶(RISK)通路,减轻肝细胞损伤,发挥保护效应。
Objective:To investigate the protective effect of ischemic postconditioning (IPo)on hepatic ischemia-reperfusion injury and the expressions of eNOS in rats.
Methods:With a rat model of acute liver ischemia-reperfusion,75 healthy male Sprague-Dawley rats were randomly divided into shamoperated(S), ischemia-reperfusion(IR)and IPo group.Before persistent reperfusion,animals were given six brief reperfusion-ischemia in IPo group.The sample of blood and hepatic tissue of all groups were taken after experiment.
Results:Compared with IR group,the concentrations of both aspartate aminotransferase(AST)and alanine aminotransferase(ALT),the concentrations of malondialdehyde(MDA)in hepatic tissue in IPo group decreased markedly(P＜0.01), while the concentrations of superoxide dismutase(SOD)increased significantly(P＜0.01),the hepatic congestion and pathological damage also relieved obviously.The expression of eNOS protein in Ipo group was significantly higher than the two remaining groups(p＜0.01).
Conclusion:IPo can attenuate hepatic ischemia-reperfusion injury by activating the prosurvival kinases eNOS in accordance with the RISK pathway and reducing the concentrations of oxygen free radical.
引文
[1]Young CS,Palma JM,Mosher BD,et al.Hepatic ischemia/reperfusion injury in P-selectin and intercellular adhesion molecule-1 double-mutant mice.Am Surg,2001,67(8):737-744.
[2]Wu RQ,Xu YX,Song XH,et al.Adhesion molecule and proinflammatory cytokine gene expression in hepatic sinusoidal endothelial cells following cecal ligation and puncture.World J Gastroenterol,2001,7(1):128-130.
[3]Yoshiji H,Kuriyama S,Yoshii J,et al.Angiotensin-Ⅱ type 1 receptor interaction is a Major regulator for liver fibrosis development in rats.Hepatology,2001,34(4):745 -750.
[4]Murry CE,Jennings RB,Reimer KA.Preconditioning with ischemia in a delay of lethal cell injury in ischemia myocardium.Circulation 1986;74(5):1124-1136.
[5]高峰,Yan WL,Geng YJ,et al.“缺氧后处理”对大鼠缺氧一复氧心室肌细胞的保护作用.心功能杂志,1999,11(4):241-242.
[6]陶凌,李源,高峰等。缺血后处理对急性心肌缺血再灌注兔心脏的保护作用。第四军医大学学报,2000,21(6):116-118.
[7]李源,陶凌,减益民等.缺血后处理对缺血再灌注大鼠心肌梗死和心肌细胞凋亡的作用.第四军医大学学报,2002,23(18):1690-1693。
[8]Zhao ZQ,Corvera JS,Halkos ME,et al.Inhibition of myocardial injuryby ischemic Postconditioning during reperfusion:comparision with ischemic preconditioning.Am J Physiol,2003,285(2):579-588.
[9]Sun HY,Wang NP,Kerendi F,et al.Hypoxic Postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca~(2+)overload.Am J Physiol Heart Circ Physiol,2005,288(4):1900-1908.
[10]Halkos ME,Kerendi F,Corvera JS,et al.Myocardial protection with Postconditioning is not enhanced by ischemic preconditioning.Ann Thorac Surg,2004,78(3):961-969.
[11]Kin H,Zhao ZQ,Sun HY,et al.Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion.Cardiovasc Res,2004,62(1):74-85.
[12]Yang XM,Proctor JB,Cui L,et al.Multiple,brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways.J Am Coil Cardiol,2004,44(5):1103-1110.
[13]Darling CE,Jiang R,Maynard M,et al.'Postconditioning'via stuttering reperfusion limits myocardial infarct size in rabbit hearts:role of ERK1/2.Am J Physiol Heart Circ Physiol,2005,289(4):H1618-1626.
[14]Tsang A,Hausenloy DJ,Macanu MM,et al.Postconditioning:A form of"modified reperfusion"protects the myocardium by activating the PI3K- Akt pathway.Circ Res,2004,95(3):230-232.
[15]王迪浔主编 病理生理学 北京:人民卫生出版社 第一版,1994;8:366-379.
[16]Kloner RA,Przyklenk K.Understanding the jargon:a glossary of termsused in the study of ischemia and reperfusion.Cardiovas Res.1993;27:162-166.
[17]Kloner RA.Does reperfusion injury exist in human.J Am Coll Cardio 1993;21(2):537.
[18]Becker LC.Schaper J,Jeremy R.et al.Severity of ischemia determines the occurance of myocardial reperfusion injury.Circulation.1991;84(Suppi Ⅱ):Ⅱ -254.
[19]Jennings RB.Reimer KA.Factors involved in Salvging ischemic myocardium:effects of reperfusion of the arteral blood.Circulation 1989,68(supp Ⅰ):Ⅰ -25-26.
[20]Hearse DJ.Kusama Y.Rapid electrophysiological changes leads of arrhythmia in the aerobic rat hearts:photosensitization studies with rose Bengal derived reactive oxygen intermediate.Circ Res.1989;65:146.
[21]Stahl LD,Weiss HR.Myocardial oxgen supply demond heterogeneity and Microvascular patency in regionally stunned myocardium.Circulation 1988;77:965-972.
[22]Mehta JL,Nichols WW.Neutrophils as potential Participants in acute myocardial ischemia:relevance to reperfusion.J Am Coll Cardiol.1988;11(6):1309 - 1316.
[23]Ciafo HA,Quyan P,Becker LC,et al.Reduction of Sympathetic in tropic response after isehemia in dogs Contribute to Stunned myocardium.J Clin Invest 1985;75:1504-1509.
[24]Stewart JR.Blackwell WH.Grute SL,et al.Inhibition of Surgically induced ischemia reperfusion injury by oxygen free radicals Scarengers:Thorac Cardiovasc Surg 1983;86:262-272.
[25]Kukreja RC,Hess ML.The oxygen free radical System:From equation through membrane protein interactions to cardiovascular injury and protection.Cardiovasc Res.1991;26:641.
[26]Fronk L,Massaro DD.Oxygen toxicity.Am J Med 1980;19:117-11.
[27]Mccord JM.Oxygen derived free radicals in postischemic tissue injury.N Eng J Med 1985;312:159.
[28]Werns SW,Shea MJ.Free radicals and myocardial injury:pharmacologic implications.Circulation,1986;74:5-7.
[29]Hess ML.Manson NH,Molecular oxygen:Friend and foe:The role of the oxygen free radical system in the calcium paradox the oxygen paradox and ischemia reperfusion injury.I Mol Cell Cardiol.1984;16:969-985.
[30]陈波.心肌缺血再灌注损伤与钙通道阻滞剂的保护作用.心血管病学进展,1991:12(2):77.
[31]Rowe GT,Manson NH,Caplan M.etal.Hydrogen peroxide and Hydroxyl radical Mediation of activated leukocyte depression of Cardiac Sarcoplasmic reticulum.Circ Res 1983;53(5):584-591.
[32]Walker PR,Sikorska M.Endonuclease activities,chromatin structure,and DNA degradation in apoptosis.Biochem Cell Biol.1994;72:615-623.
[33]Kajstura J,Cheng W,Reiss K,et al.Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats.Lab Invest.1996;74:86-107.
[34]高峰,减益民。缺氧与心肌细胞凋亡,中国病理生理杂志,1998;14(7):811.
[35]Barbosa A,Sievers RE,Zaugg CE,et al.Preconditioning ischemia time determines the degree of glycogen depletion and infarct size reduction in rat hearts.Am Heart J.1996;131:224-230.
[36]Wolfe CL,Sievers RE,Visseren FL,et al.Loss of myocardial protection after preconditioning correlates with the time course of glycogen recovery within the preconditioned segment.Circulation.1993;87:881-892.
[37]谢志泉,刘伊丽.细胞凋亡与缺血性心脏病心血管病学进展,1999;20(5):279.
[38]Fliss H.Accelerated apoptosis in reperfused myocardium:Frieng or foe? Basic Res Cardiol,1998;93:90.
[39]Zhao ZQ,Vinten-Johansen J.Postconditioning:Reduction of reperfusion-induced injury.Cardiovasc Res,2006,70(2):200-211.
[40]Vinten-Johansen J,Zhao ZQ,Jiang Rong,et al.Myocardial protection in reperfusion with Postconditioning.Expert Rev Cardiovasc Ther,2005,3(6):1035-1045.
[41]Dosenko VE,Nagibin VS,Tumanovskaya LV,et al.Postconditioning prevents apoptotic necrotic and autophagic cardiomyocyte cell death in culture.Fiziol Zh,2005,51(3):12-17.
[42]Yang XM,Philipp S,Downey JM,et al.Postconditioning's protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3-kinase and guanylyl cyclase activation.Bas Res Cardiol,2005,100(1):57-63.
[43]Kin H,Zatta AJ,Lofye MT,et al.Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine[J].Cardiovasc Res,2005,67(1):124-133.
[44]Krolikowski JG,Weihrauch D,Bienengraeber M,et al.Role of Erk1/2,p70s6K,and eNOS in isoflurane-induced cardioprotection during early reperfusion in vivo.Can J Anaesth,2006,53(2):174-282.
[45]Philipp SD,Yang XM,Cui L,et al.Postconditioning protects rabbit hearts through a protein kinase C-adenosine A(2b)receptor cascade.Cardiovasc Res,2006,70(2):308-314.
[46]Zatta AJ,Kin H,Lee G,et al.Infarct-sparing effect of myocardial Postconditioning is dependent on protein kinase C signalling.Cardiovasc Res, 2006, 70(2): 315-324.
[47] Argaud L,Gateau-Roesch O, Raisky O, et al. Post2conditioning inhibits mitochondrial permeability transition [J]. Circulation, 2005, 111 (2):194-197.
[48] Yellon DM, Hausenloy DJ.Realizing the clinical potential of ischemic preconditioning and Postconditioning. Nat Clin Pract Cardiovasc Med, 2005,2 (11): 568-575.
[49] Iliodromitis EK, Zoga A, Vrettou A, et al. The effectiveness of Postconditioning and preconditioning on infarct size in hypercholesterolemic and normal anesthetized rabbits [OB/OL] .Atherosclerosis, Http://www.sciencedirect.com/science/journal/00219150,2005-12-27.
[50] Kohy IV, Selzner M, Madden JF, et al. Endothelial cell and hepatocyte deaths occur by apoptosis after ischemia - reperfusion injury in the rat liver [J] .Transplantation, 1999, 67(8): 1099.
[51] Staat P, Rioufol G, Piot C, et al.Postconditioning the human heart. Circulation, 2005,112(14): 2143-2148.
[52] Loukoqeorqakis SP, Panaqiotidou AT, Yellon DM, et al.Postconditioning protects against endothelial ischemia reperfusion injury in the human forearm [J]. Circulation, 2006,113(7):1015-1019.
[2]Wu RQ,Xu YX,Song XH,et al.Adhesion molecule and proinflammatory cytokine gene expression in hepatic sinusoidal endothelial cells following cecal ligation and puncture.World J Gastroenterol,2001,7(1):128-130.
[3]Yoshiji H,Kuriyama S,Yoshii J,et al.Angiotensin-Ⅱ type 1 receptor interaction is a Major regulator for liver fibrosis development in rats.Hepatology,2001,34(4):745 -750.
[4]Murry CE,Jennings RB,Reimer KA.Preconditioning with ischemia in a delay of lethal cell injury in ischemia myocardium.Circulation 1986;74(5):1124-1136.
[5]高峰,Yan WL,Geng YJ,et al.“缺氧后处理”对大鼠缺氧一复氧心室肌细胞的保护作用.心功能杂志,1999,11(4):241-242.
[6]陶凌,李源,高峰等。缺血后处理对急性心肌缺血再灌注兔心脏的保护作用。第四军医大学学报,2000,21(6):116-118.
[7]李源,陶凌,减益民等.缺血后处理对缺血再灌注大鼠心肌梗死和心肌细胞凋亡的作用.第四军医大学学报,2002,23(18):1690-1693。
[8]Zhao ZQ,Corvera JS,Halkos ME,et al.Inhibition of myocardial injuryby ischemic Postconditioning during reperfusion:comparision with ischemic preconditioning.Am J Physiol,2003,285(2):579-588.
[9]Sun HY,Wang NP,Kerendi F,et al.Hypoxic Postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca~(2+)overload.Am J Physiol Heart Circ Physiol,2005,288(4):1900-1908.
[10]Halkos ME,Kerendi F,Corvera JS,et al.Myocardial protection with Postconditioning is not enhanced by ischemic preconditioning.Ann Thorac Surg,2004,78(3):961-969.
[11]Kin H,Zhao ZQ,Sun HY,et al.Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion.Cardiovasc Res,2004,62(1):74-85.
[12]Yang XM,Proctor JB,Cui L,et al.Multiple,brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways.J Am Coil Cardiol,2004,44(5):1103-1110.
[13]Darling CE,Jiang R,Maynard M,et al.'Postconditioning'via stuttering reperfusion limits myocardial infarct size in rabbit hearts:role of ERK1/2.Am J Physiol Heart Circ Physiol,2005,289(4):H1618-1626.
[14]Tsang A,Hausenloy DJ,Macanu MM,et al.Postconditioning:A form of"modified reperfusion"protects the myocardium by activating the PI3K- Akt pathway.Circ Res,2004,95(3):230-232.
[15]王迪浔主编 病理生理学 北京:人民卫生出版社 第一版,1994;8:366-379.
[16]Kloner RA,Przyklenk K.Understanding the jargon:a glossary of termsused in the study of ischemia and reperfusion.Cardiovas Res.1993;27:162-166.
[17]Kloner RA.Does reperfusion injury exist in human.J Am Coll Cardio 1993;21(2):537.
[18]Becker LC.Schaper J,Jeremy R.et al.Severity of ischemia determines the occurance of myocardial reperfusion injury.Circulation.1991;84(Suppi Ⅱ):Ⅱ -254.
[19]Jennings RB.Reimer KA.Factors involved in Salvging ischemic myocardium:effects of reperfusion of the arteral blood.Circulation 1989,68(supp Ⅰ):Ⅰ -25-26.
[20]Hearse DJ.Kusama Y.Rapid electrophysiological changes leads of arrhythmia in the aerobic rat hearts:photosensitization studies with rose Bengal derived reactive oxygen intermediate.Circ Res.1989;65:146.
[21]Stahl LD,Weiss HR.Myocardial oxgen supply demond heterogeneity and Microvascular patency in regionally stunned myocardium.Circulation 1988;77:965-972.
[22]Mehta JL,Nichols WW.Neutrophils as potential Participants in acute myocardial ischemia:relevance to reperfusion.J Am Coll Cardiol.1988;11(6):1309 - 1316.
[23]Ciafo HA,Quyan P,Becker LC,et al.Reduction of Sympathetic in tropic response after isehemia in dogs Contribute to Stunned myocardium.J Clin Invest 1985;75:1504-1509.
[24]Stewart JR.Blackwell WH.Grute SL,et al.Inhibition of Surgically induced ischemia reperfusion injury by oxygen free radicals Scarengers:Thorac Cardiovasc Surg 1983;86:262-272.
[25]Kukreja RC,Hess ML.The oxygen free radical System:From equation through membrane protein interactions to cardiovascular injury and protection.Cardiovasc Res.1991;26:641.
[26]Fronk L,Massaro DD.Oxygen toxicity.Am J Med 1980;19:117-11.
[27]Mccord JM.Oxygen derived free radicals in postischemic tissue injury.N Eng J Med 1985;312:159.
[28]Werns SW,Shea MJ.Free radicals and myocardial injury:pharmacologic implications.Circulation,1986;74:5-7.
[29]Hess ML.Manson NH,Molecular oxygen:Friend and foe:The role of the oxygen free radical system in the calcium paradox the oxygen paradox and ischemia reperfusion injury.I Mol Cell Cardiol.1984;16:969-985.
[30]陈波.心肌缺血再灌注损伤与钙通道阻滞剂的保护作用.心血管病学进展,1991:12(2):77.
[31]Rowe GT,Manson NH,Caplan M.etal.Hydrogen peroxide and Hydroxyl radical Mediation of activated leukocyte depression of Cardiac Sarcoplasmic reticulum.Circ Res 1983;53(5):584-591.
[32]Walker PR,Sikorska M.Endonuclease activities,chromatin structure,and DNA degradation in apoptosis.Biochem Cell Biol.1994;72:615-623.
[33]Kajstura J,Cheng W,Reiss K,et al.Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats.Lab Invest.1996;74:86-107.
[34]高峰,减益民。缺氧与心肌细胞凋亡,中国病理生理杂志,1998;14(7):811.
[35]Barbosa A,Sievers RE,Zaugg CE,et al.Preconditioning ischemia time determines the degree of glycogen depletion and infarct size reduction in rat hearts.Am Heart J.1996;131:224-230.
[36]Wolfe CL,Sievers RE,Visseren FL,et al.Loss of myocardial protection after preconditioning correlates with the time course of glycogen recovery within the preconditioned segment.Circulation.1993;87:881-892.
[37]谢志泉,刘伊丽.细胞凋亡与缺血性心脏病心血管病学进展,1999;20(5):279.
[38]Fliss H.Accelerated apoptosis in reperfused myocardium:Frieng or foe? Basic Res Cardiol,1998;93:90.
[39]Zhao ZQ,Vinten-Johansen J.Postconditioning:Reduction of reperfusion-induced injury.Cardiovasc Res,2006,70(2):200-211.
[40]Vinten-Johansen J,Zhao ZQ,Jiang Rong,et al.Myocardial protection in reperfusion with Postconditioning.Expert Rev Cardiovasc Ther,2005,3(6):1035-1045.
[41]Dosenko VE,Nagibin VS,Tumanovskaya LV,et al.Postconditioning prevents apoptotic necrotic and autophagic cardiomyocyte cell death in culture.Fiziol Zh,2005,51(3):12-17.
[42]Yang XM,Philipp S,Downey JM,et al.Postconditioning's protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3-kinase and guanylyl cyclase activation.Bas Res Cardiol,2005,100(1):57-63.
[43]Kin H,Zatta AJ,Lofye MT,et al.Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine[J].Cardiovasc Res,2005,67(1):124-133.
[44]Krolikowski JG,Weihrauch D,Bienengraeber M,et al.Role of Erk1/2,p70s6K,and eNOS in isoflurane-induced cardioprotection during early reperfusion in vivo.Can J Anaesth,2006,53(2):174-282.
[45]Philipp SD,Yang XM,Cui L,et al.Postconditioning protects rabbit hearts through a protein kinase C-adenosine A(2b)receptor cascade.Cardiovasc Res,2006,70(2):308-314.
[46]Zatta AJ,Kin H,Lee G,et al.Infarct-sparing effect of myocardial Postconditioning is dependent on protein kinase C signalling.Cardiovasc Res, 2006, 70(2): 315-324.
[47] Argaud L,Gateau-Roesch O, Raisky O, et al. Post2conditioning inhibits mitochondrial permeability transition [J]. Circulation, 2005, 111 (2):194-197.
[48] Yellon DM, Hausenloy DJ.Realizing the clinical potential of ischemic preconditioning and Postconditioning. Nat Clin Pract Cardiovasc Med, 2005,2 (11): 568-575.
[49] Iliodromitis EK, Zoga A, Vrettou A, et al. The effectiveness of Postconditioning and preconditioning on infarct size in hypercholesterolemic and normal anesthetized rabbits [OB/OL] .Atherosclerosis, Http://www.sciencedirect.com/science/journal/00219150,2005-12-27.
[50] Kohy IV, Selzner M, Madden JF, et al. Endothelial cell and hepatocyte deaths occur by apoptosis after ischemia - reperfusion injury in the rat liver [J] .Transplantation, 1999, 67(8): 1099.
[51] Staat P, Rioufol G, Piot C, et al.Postconditioning the human heart. Circulation, 2005,112(14): 2143-2148.
[52] Loukoqeorqakis SP, Panaqiotidou AT, Yellon DM, et al.Postconditioning protects against endothelial ischemia reperfusion injury in the human forearm [J]. Circulation, 2006,113(7):1015-1019.