大鼠肝脏缺血预处理中一氧化氮和内皮素对微循环的调节作用
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摘要
缺血再灌注损伤是一个重要的临床课题,它与许多临床病理过程相关,例如外伤、烧伤、休克、外科血管重建、肝移植及肝脏外科手术等。再灌注后肝脏肝血窦内皮细胞易受到损害而发生微循环障碍。再灌注后微循环障碍是一个复杂的病理过程，有一系列的细胞及细胞因子参与其中，包括中性粒细胞、内皮细胞、Kupffer细胞、细胞素、粘附蛋白、活性氧、一氧化氮及内皮素等。补体系统的激活也是引起肝脏微循环功能障碍的原因之一。虽然，缺血再灌注所引起的微循环功能障碍的确切机制不完全清楚，但至少包括以下三个方面：内皮素与一氧化氮的比例失调；一氧化氮与超氧负离子的比例改变；中性粒细胞的粘附和激活。
近年来，缺血预处理已经成为减轻再灌注损伤的一个有效手段，它是指器官经过短暂几次缺血再灌注后对较长时间的缺血再灌注可以产生显著的耐受性。缺血预处理的保护作用最初发现于心脏，现已在其它许多器官都发现这种现象，它是机体对短暂阻断血流后的一种应激反应，从时间与机制上可分为早期的经典保护作用和延迟的保护作用。在早期的保护作用中，腺苷起主要作用，腺苷能抑制中性粒细胞的氧化过程，减轻其与内皮细胞的粘附，促进葡萄糖的转运，进而增强细胞膜的稳定性，增加能量的产生，另外，腺苷可以通过激活ATP依赖性钾离子通道减轻钙超载。缺血预处理延迟的保护作用与早的保护作用机制不同，它主要通过基因的表达和蛋白质的合成或对已合成蛋白的修饰，来发挥保护作用。在此过程中一氧化氮及内皮素的比例关系倍受重视，被认为是调节肝脏微循环的重要因素。一氧化氮和内皮素是近年来发现的血管活性肽和低分子，它们是具
有细胞信使样作用的生物活性物质，血管内皮细胞均可合成，可通过自分泌、旁分泌和循环内分泌的形式而发挥其广泛的生物学效应。内皮素具有收缩血管、促进血小板聚集、血管平滑肌增生和细胞间粘连的作用，而一氧化氮则具有与此相反的作用，即舒张血管、抗血小板聚集、抗平滑肌增生和防止细胞之间及其与血管内皮之间的粘连，从而有利于血液的流动。故一氧化氮及内皮素就形成基本作用相反的血管内在的自身平衡调节系统，对血管的舒缩功能、血液的流动性和维持肝脏微循环功能有着重要的生理意义。本研究的目的就是在建立大鼠缺血再灌注模型的基础上观察肝脏在缺血预处理前后一氧化氮和内皮素的水平及微循环变化，探讨缺血预处理对再灌注后肝脏微循环的保护作用。本研究对预处理和肝脏微循环的关系进行探讨，发现预处理可减轻再灌注损伤后的微循环衰竭，其机理由一氧化氮、内皮素等调控肝脏微循环变化的血管活性介质有关。缺血预处理可增加再灌注后血液中一氧化氮的产生，减少内皮素水平，而同时可减轻红细胞和中性粒细胞在肝血窦中的积聚，减轻肝血窦内皮细胞的肿胀，改善微循环。这说明缺血预处理的保护作用与诱导一氧化氮产生，减少内皮素 产生有关。缺血预处理诱导一氧化氮产生的机理尚不完全清楚，可能是通过腺苷-一氧化氮途径，缺血预处理可增加腺苷的产生，腺苷可通过腺苷受体诱导内皮细胞中一氧化氮的合成。实验中我们通过门静脉系统给予L-精氨酸，在促进肝脏一氧化氮产生的同时，可减轻肝细胞损伤，减少肝血窦内皮细胞损伤，达到类似缺血预处理的保护作用，但在降低肝功酶和内皮素 等效果上尚不如预处理组，说明影响肝功能和微循环的因素较多，一氧化氮及内皮素只是其中之一，而且应用一氧化氮的剂量、方法等尚需进一步研究。
总之，缺血再灌注损伤是许多因子参与复杂的病理生理过程，缺血可导致细胞的致命性损伤，而再灌注过程中微循环功能障可以加重这种损伤。所有的因子在不同程度上参与这一全部病理生理过程。因此，针对某一个调节因子或某一种机制都不能发挥有效的保护作用。相比之下，缺血预处理是最有前途的预防缺血再灌注损伤的方法，它能增加肝细胞对缺血再灌注损伤的抵抗力，对临床进行肝脏外科手术及肝移植具有极其重要的意义。
Hepatic injury secondary to ischemia and reperfusion is an important clinical issue. It has been implicated in the pathogenesis of a variety of clinical conditions including trauma, thermal injury, and shock, reconstructive vascular surgery, liver transplantation, and liver resectional surgery. The hepatic microvasculature, i.e., the sinusoidal space with the lining endothelium is vulnerable to the deleterious consequences. Microvascular dysfunction results from a series of events that involve the interaction of intravascular blood cells, i.e. neutrophil, with nonparenchymal cells, endothelial and Kupffer cells, and are mediated by the synthesis and release of adhesion proteins, cytokines, reactive oxygen species, nitric oxide, and endothelins. Another mechanism involved in the pathogenesis of the microvascular dysfunction is the local complement activation. Although the mechanism is not clear, ischemia-reperfusion-induced microvascular dysfunction cannot be expained by means of : (i) imbalance between the rate of NO production and the release of endothelin;(ii)alteration of the ratio between NO and superoxide anion;(iii)degree of neutrophil adhesion and activation. Over recent years,a surgical strategy known as ischemic-perconditioning has been developed to reduce ischemia-reperfusion injury.By this procedure,an organ is made resistant to the deleterious effects of sustained ischemia and reperfusion by previous exposure to repeated short periods of ischemia,seperated by intermitent reperfusion.The protective effect of ischemic-preconditioning was first described in the heart,and has since been demonstrated in many other organs.While originally described as an immediate adaptation to bridf vascular occlusion,ischemia preconditioning actually affords two types of protection,which differ in time frame and mechanisms. For this reason ,distinction is made between early and
delayed preconditioning.With regard to the liver,adenosine has thus far been consierd the major player in triggering early preconditioning,which has been shown to inhibit neutrophil oxidative metabolism and adhesion to endothelial cells,to increase membrane stability and energy production by promoting glucose transport ,and to reduce Ca2+ influx through the acivation of ATP-dependent K+ channels.While the triggering and amplification of signals for procection seem to be as in early preconditioning, the effectors of this second window are probably different.The time frame within which the second wimdow confers protection lends itself to the theory that altered gene expression,with the consequent synthesis of new proteins,is the protection method. Several proteins have been proposed as possible effectors, including nitric oxide synthase, cyclooxygenase-2, antioxidant enzymes, and heat shock proteins.During delayed preconditioning the rate of NO production and the release of endothelin is considered as significant factor to modulate hepatic microvascular dysfunction. Nitric oxide and endothelin are low-molecular-weight bioactive peptide discoveried by recent decades, which can comprehensively exert various biological effects as cellular messages by means of autocrine, paracrine and circulation. Endothelin is a potent vasoconstrictor, which promotes hematoblastic conglomeration, hyperplasia of vascular smooth muscle and cellular adhesion, but NO exerts adverse action and ameliorate blood flow.Therefore,a endogenous endoself-balanced system is constructed by NO and endothelin ,which play a important role in regulating vasoconstriction and vasodialation , maintaining circulation and hepatic microcirculation.The aim of this research is to observe the level of NO and endothelin and change of hepaeic microcirculation during ischemic preconditioning in ischemia-reperfusion models,and investigate protection of microcirculation after preconditioning .Data indicate that
preconditioning can meliorate microvascular dysfunction which is involved with increase of NO and decrease of endothelin.The mechanisms ,by which NO is relesed during ischemic p
近年来，缺血预处理已经成为减轻再灌注损伤的一个有效手段，它是指器官经过短暂几次缺血再灌注后对较长时间的缺血再灌注可以产生显著的耐受性。缺血预处理的保护作用最初发现于心脏，现已在其它许多器官都发现这种现象，它是机体对短暂阻断血流后的一种应激反应，从时间与机制上可分为早期的经典保护作用和延迟的保护作用。在早期的保护作用中，腺苷起主要作用，腺苷能抑制中性粒细胞的氧化过程，减轻其与内皮细胞的粘附，促进葡萄糖的转运，进而增强细胞膜的稳定性，增加能量的产生，另外，腺苷可以通过激活ATP依赖性钾离子通道减轻钙超载。缺血预处理延迟的保护作用与早的保护作用机制不同，它主要通过基因的表达和蛋白质的合成或对已合成蛋白的修饰，来发挥保护作用。在此过程中一氧化氮及内皮素的比例关系倍受重视，被认为是调节肝脏微循环的重要因素。一氧化氮和内皮素是近年来发现的血管活性肽和低分子，它们是具
有细胞信使样作用的生物活性物质，血管内皮细胞均可合成，可通过自分泌、旁分泌和循环内分泌的形式而发挥其广泛的生物学效应。内皮素具有收缩血管、促进血小板聚集、血管平滑肌增生和细胞间粘连的作用，而一氧化氮则具有与此相反的作用，即舒张血管、抗血小板聚集、抗平滑肌增生和防止细胞之间及其与血管内皮之间的粘连，从而有利于血液的流动。故一氧化氮及内皮素就形成基本作用相反的血管内在的自身平衡调节系统，对血管的舒缩功能、血液的流动性和维持肝脏微循环功能有着重要的生理意义。本研究的目的就是在建立大鼠缺血再灌注模型的基础上观察肝脏在缺血预处理前后一氧化氮和内皮素的水平及微循环变化，探讨缺血预处理对再灌注后肝脏微循环的保护作用。本研究对预处理和肝脏微循环的关系进行探讨，发现预处理可减轻再灌注损伤后的微循环衰竭，其机理由一氧化氮、内皮素等调控肝脏微循环变化的血管活性介质有关。缺血预处理可增加再灌注后血液中一氧化氮的产生，减少内皮素水平，而同时可减轻红细胞和中性粒细胞在肝血窦中的积聚，减轻肝血窦内皮细胞的肿胀，改善微循环。这说明缺血预处理的保护作用与诱导一氧化氮产生，减少内皮素 产生有关。缺血预处理诱导一氧化氮产生的机理尚不完全清楚，可能是通过腺苷-一氧化氮途径，缺血预处理可增加腺苷的产生，腺苷可通过腺苷受体诱导内皮细胞中一氧化氮的合成。实验中我们通过门静脉系统给予L-精氨酸，在促进肝脏一氧化氮产生的同时，可减轻肝细胞损伤，减少肝血窦内皮细胞损伤，达到类似缺血预处理的保护作用，但在降低肝功酶和内皮素 等效果上尚不如预处理组，说明影响肝功能和微循环的因素较多，一氧化氮及内皮素只是其中之一，而且应用一氧化氮的剂量、方法等尚需进一步研究。
总之，缺血再灌注损伤是许多因子参与复杂的病理生理过程，缺血可导致细胞的致命性损伤，而再灌注过程中微循环功能障可以加重这种损伤。所有的因子在不同程度上参与这一全部病理生理过程。因此，针对某一个调节因子或某一种机制都不能发挥有效的保护作用。相比之下，缺血预处理是最有前途的预防缺血再灌注损伤的方法，它能增加肝细胞对缺血再灌注损伤的抵抗力，对临床进行肝脏外科手术及肝移植具有极其重要的意义。
Hepatic injury secondary to ischemia and reperfusion is an important clinical issue. It has been implicated in the pathogenesis of a variety of clinical conditions including trauma, thermal injury, and shock, reconstructive vascular surgery, liver transplantation, and liver resectional surgery. The hepatic microvasculature, i.e., the sinusoidal space with the lining endothelium is vulnerable to the deleterious consequences. Microvascular dysfunction results from a series of events that involve the interaction of intravascular blood cells, i.e. neutrophil, with nonparenchymal cells, endothelial and Kupffer cells, and are mediated by the synthesis and release of adhesion proteins, cytokines, reactive oxygen species, nitric oxide, and endothelins. Another mechanism involved in the pathogenesis of the microvascular dysfunction is the local complement activation. Although the mechanism is not clear, ischemia-reperfusion-induced microvascular dysfunction cannot be expained by means of : (i) imbalance between the rate of NO production and the release of endothelin;(ii)alteration of the ratio between NO and superoxide anion;(iii)degree of neutrophil adhesion and activation. Over recent years,a surgical strategy known as ischemic-perconditioning has been developed to reduce ischemia-reperfusion injury.By this procedure,an organ is made resistant to the deleterious effects of sustained ischemia and reperfusion by previous exposure to repeated short periods of ischemia,seperated by intermitent reperfusion.The protective effect of ischemic-preconditioning was first described in the heart,and has since been demonstrated in many other organs.While originally described as an immediate adaptation to bridf vascular occlusion,ischemia preconditioning actually affords two types of protection,which differ in time frame and mechanisms. For this reason ,distinction is made between early and
delayed preconditioning.With regard to the liver,adenosine has thus far been consierd the major player in triggering early preconditioning,which has been shown to inhibit neutrophil oxidative metabolism and adhesion to endothelial cells,to increase membrane stability and energy production by promoting glucose transport ,and to reduce Ca2+ influx through the acivation of ATP-dependent K+ channels.While the triggering and amplification of signals for procection seem to be as in early preconditioning, the effectors of this second window are probably different.The time frame within which the second wimdow confers protection lends itself to the theory that altered gene expression,with the consequent synthesis of new proteins,is the protection method. Several proteins have been proposed as possible effectors, including nitric oxide synthase, cyclooxygenase-2, antioxidant enzymes, and heat shock proteins.During delayed preconditioning the rate of NO production and the release of endothelin is considered as significant factor to modulate hepatic microvascular dysfunction. Nitric oxide and endothelin are low-molecular-weight bioactive peptide discoveried by recent decades, which can comprehensively exert various biological effects as cellular messages by means of autocrine, paracrine and circulation. Endothelin is a potent vasoconstrictor, which promotes hematoblastic conglomeration, hyperplasia of vascular smooth muscle and cellular adhesion, but NO exerts adverse action and ameliorate blood flow.Therefore,a endogenous endoself-balanced system is constructed by NO and endothelin ,which play a important role in regulating vasoconstriction and vasodialation , maintaining circulation and hepatic microcirculation.The aim of this research is to observe the level of NO and endothelin and change of hepaeic microcirculation during ischemic preconditioning in ischemia-reperfusion models,and investigate protection of microcirculation after preconditioning .Data indicate that
preconditioning can meliorate microvascular dysfunction which is involved with increase of NO and decrease of endothelin.The mechanisms ,by which NO is relesed during ischemic p
引文
1. Yellon DM, Alkhualaifi AM, Pugsley WB.Preconditioning the human myocardium. Lancet, 1993, 31:276-277.
2．王吉耀，朱无难.透明质酸的代谢与肝病［J］.中华消化杂志，1991，11(1)：41-42.
Jaeschke H. Molecular mechanism of hepatic ischemia-reperfusion injury and priconditioning. Am J Physiol, 2003,284:G15-G26.
Serra-Inglott F, Habib N A, et al. Hepatic ischemia-reperfusion injury. Am J Surg, 2001, 181:160-166.
5．Juan C, Maria-Giulia P, et al, Microvascular dysfunction induced by reperfusion injury and protection effect of ischemia preconditioning. Free Radic Biol Med, 2002, 33:1200-1208.
6. Kauamura E,Yamakana N,Okamoto E,et al.Response lf plasma and tissue endothelin-1 to liver ischemia and its implication in ischemia-reperfusion in ischemia-reperfusion inyury [J]. Hepatolohy, 1999, 29(1):65-70.
7. Isobe M, Katasuramadi T, Kimura H, et al.Correlation between nitric oxide and endothelin after prolonged warm ischemic- reperfusion in pig liver [J]. Transplant Proc, 1998, 30: 3750-3753.
8. Scommotaus S, Uhlmann D, Loffler BM, et al. Involvement of endothelin/nitric oxide balance in heaptic ischemia-
reperfusion injury [J]. Langenbecks Arch Surg, 1999, 348(1): 65-70.
9．Murry C, Jenning R, et al.Preconditioning with ischimia:a delay in lethal cell injury in ischemic myocardium. Circulation 1986, 74:1124-1136.
10. 涂兵，严律南等.缺血预处理对大鼠肝脏微循环的保护作用.中华器官移植杂志,2002,23(6):327-329.
11．Carini R, Albano E. Recent insights on the mechanisms of liver preconditioning. Gastroenterology，2003 Nov;125(5):1480-91.
Nilsson B, Friman S, et al. The liver protective effect of ischemia preconditioning may be mediated by adenosine. Transp Int, 2000, 13(Suppl.1):s558-s561.
Howell J G, Brown M F, et al. Both ischemic and pharmacological preconditioning decrease hepatic leukocyte/endothelial cell interactions. Transplantation, 2000, 69:300-314.
Ricciardi R, Meyers W, et al. Protein Kinase C inhibition abrogates hepatic ischemic preconditioning responses. J Surg res. 2001, 97:144-149.
Eisen A, Fisman EZ, et al. Ischemic preconditioning: nearly two decades of research. A comprehensive review. Atheroscle- rosis. 2004 ,172(2):201-210.
2．王吉耀，朱无难.透明质酸的代谢与肝病［J］.中华消化杂志，1991，11(1)：41-42.
Jaeschke H. Molecular mechanism of hepatic ischemia-reperfusion injury and priconditioning. Am J Physiol, 2003,284:G15-G26.
Serra-Inglott F, Habib N A, et al. Hepatic ischemia-reperfusion injury. Am J Surg, 2001, 181:160-166.
5．Juan C, Maria-Giulia P, et al, Microvascular dysfunction induced by reperfusion injury and protection effect of ischemia preconditioning. Free Radic Biol Med, 2002, 33:1200-1208.
6. Kauamura E,Yamakana N,Okamoto E,et al.Response lf plasma and tissue endothelin-1 to liver ischemia and its implication in ischemia-reperfusion in ischemia-reperfusion inyury [J]. Hepatolohy, 1999, 29(1):65-70.
7. Isobe M, Katasuramadi T, Kimura H, et al.Correlation between nitric oxide and endothelin after prolonged warm ischemic- reperfusion in pig liver [J]. Transplant Proc, 1998, 30: 3750-3753.
8. Scommotaus S, Uhlmann D, Loffler BM, et al. Involvement of endothelin/nitric oxide balance in heaptic ischemia-
reperfusion injury [J]. Langenbecks Arch Surg, 1999, 348(1): 65-70.
9．Murry C, Jenning R, et al.Preconditioning with ischimia:a delay in lethal cell injury in ischemic myocardium. Circulation 1986, 74:1124-1136.
10. 涂兵，严律南等.缺血预处理对大鼠肝脏微循环的保护作用.中华器官移植杂志,2002,23(6):327-329.
11．Carini R, Albano E. Recent insights on the mechanisms of liver preconditioning. Gastroenterology，2003 Nov;125(5):1480-91.
Nilsson B, Friman S, et al. The liver protective effect of ischemia preconditioning may be mediated by adenosine. Transp Int, 2000, 13(Suppl.1):s558-s561.
Howell J G, Brown M F, et al. Both ischemic and pharmacological preconditioning decrease hepatic leukocyte/endothelial cell interactions. Transplantation, 2000, 69:300-314.
Ricciardi R, Meyers W, et al. Protein Kinase C inhibition abrogates hepatic ischemic preconditioning responses. J Surg res. 2001, 97:144-149.
Eisen A, Fisman EZ, et al. Ischemic preconditioning: nearly two decades of research. A comprehensive review. Atheroscle- rosis. 2004 ,172(2):201-210.