蛋白激酶C的活化对大鼠供肺的保护
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摘要
研究背景
目前,肺移植已经成为终末期肺病的标准治疗手段之一。尽管临床肺移植已取得成功,但许多资料表明,肺移植术后仍有15%—20%患者表现严重肺损伤,不可避免的缺血再灌注损伤增加了供肺急性排斥反应和远期肺功能衰竭的发生概率。此外,目前等待肺移植的患者不断增加,但供肺只有20%—30%可供使用。
如何提高肺保存的效果,找到更适合临床应用的新的肺保存方法,以提高供肺的可利用率,减少供肺的缺血再灌注损伤,降低移植肺术后的近期和远期并发症是当前肺保存中的重要课题之一。
随着缺血预适应概念的提出和随后近20年的针对性研究,发现此现象不仅广泛存在于不同种属的哺乳动物,而且对体内的多种组织和器官具有保护作用,是一种效果肯定的内源性保护机制。
然而,到目前为止,缺血预适应的器官保护方法因其自身特有的缺陷并未在临床上,特别是外科临床,得到广泛的应用。一些学者便在缺血预适应的基础上提出了药物预适应,这一新的器官保护方法。
研究发现,短暂缺血可引起内源性物质释放增加。这些物质分别与相应的G
Background: Lung transplantation (LT) has recently become one of thestandard therapies to prolong life and lung capacity for the patients with end-stage pulmonary diseases. Despite of advances in lung transplantation, there is 15%-20% post-transplantation patients suffered from severe lung injury, inevitable injury of ischemic/reperfusion, increased acute rejection and long term dysfunction of donor lung. Moreover, the patients who are waiting for transplantation are increasing, but 20%-30% of donor lungs only are available.The purpose of the lung preservation is to find a new method to preserve and increase the availability of donor lungs, to prevent the injury of ischemic/reperfusion and to decrease the short or long term complications.Ischemic preconditioning (IP) is a phenomenon by which a brief episode(s) of ischemia increases the ability of the ischemic organ or tissue to tolerate a subsequent prolonged period of ischemic injury. After two decades of extensive research, it has been demonstrated that IP not only exist in different mammalians, but can also protect many organs and tissues. The effect of this phenomenon is affirmative, and served as an endogenous protective mechanism.
Nevertheless, this method of organ-protection has not been widely used in the clinic, especially in the surgical field, because of its own shortfalls. To circumvent this, some researchers have used a brand-new organ-protective method - pharmacological preconditioning, on the base of ischemic preconditioning.It has been demonstrated that transient ischemic can release the endogenous triggers, these triggers bind to the G protein coupled receptors on the myocyte surface, activate phospholipase c (PLC), which hydrolyzes phosphatidylinositol 4,5-diphosphate and phosphatidylcholine to produce 1,2-diacylglycerol and inositol-l,4,5-triphosphate. Increased levels of diacylglycerol then activate protein kinase c (PKC) and the translocation of PKC from cytosol to the cell membrane. Activated PKC open the ATP-sensitive potassium channel (KAtp channel) and phosphorylates other effector proteins. This is the early phase of ischemic preconditioning (first window of protection). PKC can also translocate into nucleus and activate transcriptional regulator nuclear factor-? B (NF-?B), and increases the transcription of protective proteins, some of which have been identified so far. For example, superoxide dismutase, which is an important antioxidant enzyme, and heat shock proteins, are upregulated and are important in delayed preconditioning. This is the late phase of ischemic preconditioning (second window of protection.). Most researchers agree that translocation of the PKC plays an important role in triggering initial intracellular signaling cascade and ultimately increased transcription of cardioprotective genes. In some animal models inhibition of PKC alone during the index ischaemia aborts preconditioning, suggesting that PKC is a common step of the ischemic preconditioning but not a trigger.The purpose of this project is to directly test this hypothesis in the lung transplantation using protein kinase c activator. The activator will directly stimulate PKC translocation, trigger intracellular signaling cascade, and ultimately increases transcription of protective proteins. We will determine whether this will protect the donor lung or not.Phorbol myristate acetate (PMA) is an analogue of DAG and is a specific agonist of PKC. The molecular weight of PMA is 616. It has been demonstrated that PKC is the main
receptor and target of PMA;In contrast to the short-lived DAG, PMA is stable and has the same active group just as DAG.The experimental design is to develop an animal model for the left lung transplantation on rats, perfusing PMA via the pulmonary artery before retrieving the donor lung, preserve the lung in modified EC solution, and then observe the effect of PMA.Part onePurpose: To improve the former cuff techniques and establish a new simplified effective rat lung orthotopic transplantation model.Methods: Preparation of the donor lung: after perfusing the donor lung, wecut the pulmonary artery, pulmonary vein and bronchus in the proximal end, and then pick out the most proper cuff tubes and stent tube. The vessels were drawn through the cuff tubes and everted over the cuff, then tied by 5-0 sutures. Half length of the stent tube was inserted into the donor bronchus and tied by 5-0 sutures.Operation of the recipient: Cut the distal part of the left pulmonary artery ^ vein and bronchus. Anastomose the vessels using traditional cuff technique and anastomose bronchus using stent technique.Results: The transplanted lungs have good and satisfied results. They meetthe criteria of a standard successful transplantation.Conclusion: This model (two-cuff-and-one-stent technique) is much easier and more economical to use on studying lung preservation/transplantation.Part twoPurpose: Based on the ischemic preconditioning mechanism, in which activation of PKC is the common step of the ischemic preconditioning, we perfuse the
donor lung with PMA, the exogenous activator of PKC, and assess the lung preservative effects by analyzing blood gas, SOD, HSP70, apoptotic index, the expression of Bcl-2, and the morphological change of alveolar, and to observe whether PMA can mimic the protective effects of ischemic preconditioning.Methods: (1) SD rats were divided into four groups:Group C (control group, n=10 pairs): Modified EC solution was perfused into donor's pulmonary artery directly after anesthesia, orotracheal intubation and thoracotomy, and then the donor lung was retrieved and the two-cuff-and-one-stent technique was used. After stored in cold modified EC solution for 12 hrs, the donor lung was transplanted into recipient. 30 min after the transplantation was finished;blood was drawn from left pulmonary vein for blood gas analysis and the specimen was collected for later analysis. Group P (preconditioning group, n=10 pairs): The protocol was similar to Group C, except left lung was ischemic preconditioned before perfusing modified EC solution from pulmonary artery. Group B (blockade group, n=10 pairs): The protocol was just like Group P,except polymyxin B was perfused after ischemic preconditioning. Group A (PMA group, n=10): The protocol was just like Group C, except perfusing PMA before perfusing modified EC solution from pulmonary artery.(2)Indicators tested: 1) Blood gas analysis: Blood was drawn from left pulmonary vein after transplantation and records the PO2. 2) Transmission electron microscopy: Morphological and ultrastructural changes of lung tissue were observed. 3) Level of SOD, HSP70 mRNA: To measure the expression of SOD and HSP70 mRNA in the lung tissue. 4) Apoptotic index and the expression of Bcl-2: Apoptotic index was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The expression of Bcl-2 in lung tissue was measured by quantitative immunohistochemistry.
(3)Statistical analysis: All data were represented as Mean ± Standard Deviation. Statistical differences between groups were evaluated by one-way ANOVA. A value of p<0.05 was considered significant. All the statistical analysis was carried on using SPSS 11.5 program.Results: (1) PO2 of left pulmonary vein: PO2 in Group P and A weresignificantly higher than that of Group C (P<0.05). In contrast, Group P was not significantly different compared to Group A (P>0.05), but Group B was significantly lower (P<0.05). (2) SOD activity: Compared to Group C, Group P and A were significantly higher (p<0.05). Compared to Group A, all other groups have significantly difference (P<0.05). (3) The expression of HSP70 mRNA: Compared to Group C, Group P and Group A were significantly higher (P<0.05). Compared to Group A, Group B> C were significantly lower (p<0.05). (4) Apoptotic index: Compared to Group C, Group A and C were significantly difference (P<0.05). Compared to Group A, Group C and B had significantly difference (P<0.05). (5) The expression of Bcl-2: Compared to Group C, Group P and A were significantly difference (P<0.05). Compared to Group A, Group C and B had significantly difference (P<0.05). (6) H & E stain: the lung tissues in Group P and A showed a little edema and swelling (both in interstitial and alveolar space), and a little congestion in interstitial space. These changes became more prominent in Group C and B. (7)Transmission electron microscopy: In Group C and B, the cells of alveolar were smaller, and the nuclear chromatin was condensed, appeared as "semi-lunar" shape. There were less organelles were found, including mitochondria and lamellar body. Fewer changes were found in Group P and A.Conclusions:1. Ischemic preconditioning has lung protective effect.2. Activating PKC by perfusing PMA can mimic the protective effect of ischemic preconditioning. In the aspect of SOD activity, pharmacological preconditioning is even better than ischemic preconditioning.3. PKC is the mediator of the ischemic preconditioning. The ischemic
preconditioning can be blocked if PKC is inactivated;even the preconditioning has been triggered.4. Once the pharmacological preconditioning started, preconditioning can continue to have effect on this process even though the prime trigger disappeared.5. The activity of SOD in Group A are higher than that of Group P. It was demonstrated that SOD might be the direct effector of PKC.6. The underlying mechanism of the pharmacological preconditioning might be that the triggers activate PKC, up-regulate SOD, HSP and Bcl-2, ameliorate the injury of ischemic/reperfusion and inhibit apoptosis, which will protect the construction and the function of the lung.
目前,肺移植已经成为终末期肺病的标准治疗手段之一。尽管临床肺移植已取得成功,但许多资料表明,肺移植术后仍有15%—20%患者表现严重肺损伤,不可避免的缺血再灌注损伤增加了供肺急性排斥反应和远期肺功能衰竭的发生概率。此外,目前等待肺移植的患者不断增加,但供肺只有20%—30%可供使用。
如何提高肺保存的效果,找到更适合临床应用的新的肺保存方法,以提高供肺的可利用率,减少供肺的缺血再灌注损伤,降低移植肺术后的近期和远期并发症是当前肺保存中的重要课题之一。
随着缺血预适应概念的提出和随后近20年的针对性研究,发现此现象不仅广泛存在于不同种属的哺乳动物,而且对体内的多种组织和器官具有保护作用,是一种效果肯定的内源性保护机制。
然而,到目前为止,缺血预适应的器官保护方法因其自身特有的缺陷并未在临床上,特别是外科临床,得到广泛的应用。一些学者便在缺血预适应的基础上提出了药物预适应,这一新的器官保护方法。
研究发现,短暂缺血可引起内源性物质释放增加。这些物质分别与相应的G
Background: Lung transplantation (LT) has recently become one of thestandard therapies to prolong life and lung capacity for the patients with end-stage pulmonary diseases. Despite of advances in lung transplantation, there is 15%-20% post-transplantation patients suffered from severe lung injury, inevitable injury of ischemic/reperfusion, increased acute rejection and long term dysfunction of donor lung. Moreover, the patients who are waiting for transplantation are increasing, but 20%-30% of donor lungs only are available.The purpose of the lung preservation is to find a new method to preserve and increase the availability of donor lungs, to prevent the injury of ischemic/reperfusion and to decrease the short or long term complications.Ischemic preconditioning (IP) is a phenomenon by which a brief episode(s) of ischemia increases the ability of the ischemic organ or tissue to tolerate a subsequent prolonged period of ischemic injury. After two decades of extensive research, it has been demonstrated that IP not only exist in different mammalians, but can also protect many organs and tissues. The effect of this phenomenon is affirmative, and served as an endogenous protective mechanism.
Nevertheless, this method of organ-protection has not been widely used in the clinic, especially in the surgical field, because of its own shortfalls. To circumvent this, some researchers have used a brand-new organ-protective method - pharmacological preconditioning, on the base of ischemic preconditioning.It has been demonstrated that transient ischemic can release the endogenous triggers, these triggers bind to the G protein coupled receptors on the myocyte surface, activate phospholipase c (PLC), which hydrolyzes phosphatidylinositol 4,5-diphosphate and phosphatidylcholine to produce 1,2-diacylglycerol and inositol-l,4,5-triphosphate. Increased levels of diacylglycerol then activate protein kinase c (PKC) and the translocation of PKC from cytosol to the cell membrane. Activated PKC open the ATP-sensitive potassium channel (KAtp channel) and phosphorylates other effector proteins. This is the early phase of ischemic preconditioning (first window of protection). PKC can also translocate into nucleus and activate transcriptional regulator nuclear factor-? B (NF-?B), and increases the transcription of protective proteins, some of which have been identified so far. For example, superoxide dismutase, which is an important antioxidant enzyme, and heat shock proteins, are upregulated and are important in delayed preconditioning. This is the late phase of ischemic preconditioning (second window of protection.). Most researchers agree that translocation of the PKC plays an important role in triggering initial intracellular signaling cascade and ultimately increased transcription of cardioprotective genes. In some animal models inhibition of PKC alone during the index ischaemia aborts preconditioning, suggesting that PKC is a common step of the ischemic preconditioning but not a trigger.The purpose of this project is to directly test this hypothesis in the lung transplantation using protein kinase c activator. The activator will directly stimulate PKC translocation, trigger intracellular signaling cascade, and ultimately increases transcription of protective proteins. We will determine whether this will protect the donor lung or not.Phorbol myristate acetate (PMA) is an analogue of DAG and is a specific agonist of PKC. The molecular weight of PMA is 616. It has been demonstrated that PKC is the main
receptor and target of PMA;In contrast to the short-lived DAG, PMA is stable and has the same active group just as DAG.The experimental design is to develop an animal model for the left lung transplantation on rats, perfusing PMA via the pulmonary artery before retrieving the donor lung, preserve the lung in modified EC solution, and then observe the effect of PMA.Part onePurpose: To improve the former cuff techniques and establish a new simplified effective rat lung orthotopic transplantation model.Methods: Preparation of the donor lung: after perfusing the donor lung, wecut the pulmonary artery, pulmonary vein and bronchus in the proximal end, and then pick out the most proper cuff tubes and stent tube. The vessels were drawn through the cuff tubes and everted over the cuff, then tied by 5-0 sutures. Half length of the stent tube was inserted into the donor bronchus and tied by 5-0 sutures.Operation of the recipient: Cut the distal part of the left pulmonary artery ^ vein and bronchus. Anastomose the vessels using traditional cuff technique and anastomose bronchus using stent technique.Results: The transplanted lungs have good and satisfied results. They meetthe criteria of a standard successful transplantation.Conclusion: This model (two-cuff-and-one-stent technique) is much easier and more economical to use on studying lung preservation/transplantation.Part twoPurpose: Based on the ischemic preconditioning mechanism, in which activation of PKC is the common step of the ischemic preconditioning, we perfuse the
donor lung with PMA, the exogenous activator of PKC, and assess the lung preservative effects by analyzing blood gas, SOD, HSP70, apoptotic index, the expression of Bcl-2, and the morphological change of alveolar, and to observe whether PMA can mimic the protective effects of ischemic preconditioning.Methods: (1) SD rats were divided into four groups:Group C (control group, n=10 pairs): Modified EC solution was perfused into donor's pulmonary artery directly after anesthesia, orotracheal intubation and thoracotomy, and then the donor lung was retrieved and the two-cuff-and-one-stent technique was used. After stored in cold modified EC solution for 12 hrs, the donor lung was transplanted into recipient. 30 min after the transplantation was finished;blood was drawn from left pulmonary vein for blood gas analysis and the specimen was collected for later analysis. Group P (preconditioning group, n=10 pairs): The protocol was similar to Group C, except left lung was ischemic preconditioned before perfusing modified EC solution from pulmonary artery. Group B (blockade group, n=10 pairs): The protocol was just like Group P,except polymyxin B was perfused after ischemic preconditioning. Group A (PMA group, n=10): The protocol was just like Group C, except perfusing PMA before perfusing modified EC solution from pulmonary artery.(2)Indicators tested: 1) Blood gas analysis: Blood was drawn from left pulmonary vein after transplantation and records the PO2. 2) Transmission electron microscopy: Morphological and ultrastructural changes of lung tissue were observed. 3) Level of SOD, HSP70 mRNA: To measure the expression of SOD and HSP70 mRNA in the lung tissue. 4) Apoptotic index and the expression of Bcl-2: Apoptotic index was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The expression of Bcl-2 in lung tissue was measured by quantitative immunohistochemistry.
(3)Statistical analysis: All data were represented as Mean ± Standard Deviation. Statistical differences between groups were evaluated by one-way ANOVA. A value of p<0.05 was considered significant. All the statistical analysis was carried on using SPSS 11.5 program.Results: (1) PO2 of left pulmonary vein: PO2 in Group P and A weresignificantly higher than that of Group C (P<0.05). In contrast, Group P was not significantly different compared to Group A (P>0.05), but Group B was significantly lower (P<0.05). (2) SOD activity: Compared to Group C, Group P and A were significantly higher (p<0.05). Compared to Group A, all other groups have significantly difference (P<0.05). (3) The expression of HSP70 mRNA: Compared to Group C, Group P and Group A were significantly higher (P<0.05). Compared to Group A, Group B> C were significantly lower (p<0.05). (4) Apoptotic index: Compared to Group C, Group A and C were significantly difference (P<0.05). Compared to Group A, Group C and B had significantly difference (P<0.05). (5) The expression of Bcl-2: Compared to Group C, Group P and A were significantly difference (P<0.05). Compared to Group A, Group C and B had significantly difference (P<0.05). (6) H & E stain: the lung tissues in Group P and A showed a little edema and swelling (both in interstitial and alveolar space), and a little congestion in interstitial space. These changes became more prominent in Group C and B. (7)Transmission electron microscopy: In Group C and B, the cells of alveolar were smaller, and the nuclear chromatin was condensed, appeared as "semi-lunar" shape. There were less organelles were found, including mitochondria and lamellar body. Fewer changes were found in Group P and A.Conclusions:1. Ischemic preconditioning has lung protective effect.2. Activating PKC by perfusing PMA can mimic the protective effect of ischemic preconditioning. In the aspect of SOD activity, pharmacological preconditioning is even better than ischemic preconditioning.3. PKC is the mediator of the ischemic preconditioning. The ischemic
preconditioning can be blocked if PKC is inactivated;even the preconditioning has been triggered.4. Once the pharmacological preconditioning started, preconditioning can continue to have effect on this process even though the prime trigger disappeared.5. The activity of SOD in Group A are higher than that of Group P. It was demonstrated that SOD might be the direct effector of PKC.6. The underlying mechanism of the pharmacological preconditioning might be that the triggers activate PKC, up-regulate SOD, HSP and Bcl-2, ameliorate the injury of ischemic/reperfusion and inhibit apoptosis, which will protect the construction and the function of the lung.
引文
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51. Gottlieb RA, Gruol DL, Zhu JY. Preconditioning rabbit cardiomyocytes: role of pH, vacuolar proton ATPase, and apoptosis. J Clin Invest. 1996;97(10): 2391-2398.
52.刘兴德,陈运贞.蛋白激酶C活化对大鼠缺血/再灌注心肌细胞凋亡和bcl-2表达的影响.中国病理生理杂志.2002;18(8):978—980.
53. Giffard RG, Yenari MA. Many mechanisms for hsp70 protection from cerebral ischemia. J Neurosurg Anesthesiol. 2004;16(1): 53-61.
54. Sasaki M, Muraoka R, Chiba Y, et al. Influence of pulmonary arterial pressure during flushing on lung preservation. Transplantation. 1996;61(1): 22-27.
55. Tanaka H, Chiba Y, Sasaki M, et al. Relationship between flushing pressure and nitric oxide production in preserved lungs. Transplantation. 1998;65(4): 460-464.
56. Fukuse T, Hirata T, Nakamura T, et al. Influence of deflated and anaerobic conditions during cold storage on rat lungs. Am J Respir Crit Care Med. 1999;160(2): 621-627.
57. DeCampos KN, Keshavjee S, Liu M, et al. Optimal inflation volume for hypothermic preservation of rat lungs. J Heart Lung Transplant. 1998;17(6): 599-607.
58. Baretti R, Bitu-Moreno J, Beyersdorf F, et al. Distribution of lung preservation solutions in parenchyma and airways: influence of atelectasis and route of delivery. J Heart Lung Transplant. 1995;14(1 pt 1): 80-91.
59. Haniuda M, Hasegawa S, Shiraishi T, et al. Effects of inflation volume during lung preservation on pulmonary capillary permeability. J Thorac Cardiovasc Surg. 1996;112(1): 85-93.
60. Date H, Lima O, Matsumura A, et al. In a canine model, lung preservation at 10 degrees C is superior to that at 4 degrees C. A comparison of two preservation temperatures on lung function and on adenosine triphosphate level measured by phosphorus 31-nuclear magnetic resonance. J Thorac Cardiovasc Surg. 1992;103(4): 773-780.
61. Kirk AJ, Colquhoun IW, JH. D. Lung preservation: a review of current practice and future directions. Ann Thorac Surg. 1993;56(4): 990-1000.
62. Fehrenbach H, Riemann D, Wahlers T, et al. Scanning and transmission electron microscopy of human donor lungs: fine structure of the pulmonary parenchyma following preservation and ischemia. Acta Anat(Basel). 1994;151(4): 220-231.
63. Muller C, Hoffmann H, Bittmann I, et al. Hypothermic storage alone in lung preservation for transplantation: a metabolic, light microscopic, and functional analysis after 18 hours of preservation. Transplantation. 1997;63(5):625-630.
64. Steen S, Sjoberg T, Ingemansson R, et al. Efficacy of topical cooling in lung preservation: is a reappraisal due? Ann Thorac Surg. 1994;58(6): 1657-1663.
65. Hopkinson DN, Bhabra MS, TL. H. Pulmonary graft preservation: a worldwide survey of current clinical practice. J Heart Lung Transplant. 1998;17(5):525-531.
66. Struber M, Hohlfeld JM, Kofidis T, et al. Surfactant function in lung transplantation after 24 hours of ischemia: advantage of retrograde flush perfusion for preservation. J Thorac Cardiovasc Surg. 2002;123(l):98-103.
67. Al-Mehdi AB, Zhao G, Dodia C, et al. Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+. Circ Res. 1998;83(7):730-737.
68. Keshavjee SH, Yamazaki F, Yokomise H, et al. The role of dextran 40 and potassium in extended hypothermic lung preservation for transplantation. J Thorac Cardiovasc Surg. 1992;103(2):314-325.
69. Schneuwly OD, Licker M, Pastor CM, et al. Beneficial effects of leukocyte-depleted blood and low-potassium dextran solutions on microvascular permeability in preserved porcine lung. Am J Respir Crit Care Med. 1999;160(2):689-697.
70. Sakamaki F, Hoffmann H, Munzing S, et al. Effects of lung preservation solutions on PMN activation in vitro. Transpl Int. 1999;12(2):113-121.
71. Maccherini M, Keshavjee SH, Slutsky AS, et al. The effect of low-potassium-dextran versus Euro-Collins solution for preservation of isolated type II pneumocytes. Transplantation. 1991;52(4):621-626.