乙酰半胱氨酸对脑死亡大鼠肝脏保护作用的实验研究
摘要
肝脏移植(Liver transplantation，LTx)已成为治疗肝脏终末期疾病的主要方法，而供肝来源短缺是制约肝移植发展的主要原因之一。利用脑死亡供体(Brain-dead donor，BDD)器官进行移植可有效缓解器官来源短缺这一问题。脑死亡(Brain-death)是指包括脑干在内的全脑功能丧失不可逆转的状态，即死亡。
然而，临床研究表明活体供体器官无论是在短期还是长期移植效果方面均较脑死亡供体器官优越。说明脑死亡供体器官在切取以前即已发生了损伤性改变。目前，脑死亡导致外周器官发生损伤的确切机制尚不明确。因此，研究脑死亡供体肝脏的损伤机制及脑死亡供体肝脏的保护对肝移植学科的发展具有重要的意义。研究发现，乙酰半胱氨酸(NAC)具有清除自由基、抑制核转录因子(NF-κB)活化、降低炎症介质水平的作用。NAC已被广泛应用于器官保存、脏器缺血再灌注损伤的保护中。那么NAC对脑死亡供体肝脏是否具有保护作用?国内外尚未见报道。
目的
本实验的目的在于：1、探讨脑死亡状态下肝脏功能与形态的改变及其机制。2、探讨NAC对脑死亡大鼠肝脏是否具有保护作用及其可能机制。
方法
Wistar大鼠60只，雌雄不限，随机分为三组，每组20只，即对照组(C组)、脑死亡组(B组)、NAC保护组(N组)。B、N组建立脑死亡模型；C组除不行
郑州大学加04届硕士毕业论文
乙酞半肤氨酸对脑死亡大鼠肝脏保护作用的实验研究
硬脑膜外导管加压外，其余处理同脑死亡组;N组为NAC处理的脑死亡组，于
脑死亡模型建立成功后立即经尾静脉给予乙酞半肤氨酸(NAC)巧0m眺g。术后
各组于对照2h或脑死亡2h时随机取10只大鼠取材，剩余10只大鼠于对照4h
或脑死亡4h时取材。于肝上下腔静脉抽取血液标本3ml，血清自动生化分析仪检
测丙氨酸氨基转移酶(al耐ne alninotransferase，ALT)、天冬氨酸氨基转移酶
(aspartate aminotransferase，A ST)，放射免疫法检测肿瘤坏死因子一。(tUmor
neerosis factor一a，TNF一)、白细胞介素一6(interleukin一6，IL一6)、透明质酸
(hyaluronicac记，HA)水平;切取肝脏组织标本包埋固定，用于HE染色及免疫组化
法检测NF一KB的表达程度;各组4h点肝脏组织用于透射电镜观察肝细胞及肝窦
内皮细胞的损伤情况。应用SPSS 10.0软件分析，采用单因素方差分析、成组设计
资料t检验进行统计学处理，显著性检验水准取a司.05。
结果
1、MAP变化:加压前各组间无显著性差异(尸)0.05);与C相比，加压过
程中B组与N组MAP显著升高(尸<0.05)，脑死亡状态下B组与N组MAP显
著降低(尸<0 .0.5);在加压过程中与脑死亡状态，B组与N组间M今P均无显著性
差异(尸>0.05)。
2、HR变化:加压前与脑死亡状态各组间无显著性差异(P>0.05);加压过
程中，B组、N组与C组相比均显著升高(尸<0.05)，但B组与N组间无显著性
差异(P>.05)。
3、ALI，、AST、HA、TNF一a、IL一6水平、NF一KB表达水平:在相同时间点
B、N组上述指标水平均显著高于C组(尸<0.05)，但N组明显低于B组(P<0.05);
在同组不同时间点之间，B组4h点明显高于B组Zh点(尸<0.05)，N组4h点明
显高于N组Zh点(尸<0.05)，C组4h点与C组Zh点间无显著性差异(P>.05)。
4、肝脏形态学变化:
光镜下:C组肝脏组织结构正常;B组与N组Zh点肝脏组织结构基本正常;
B组4h点肝脏组织结构紊乱、肝细胞水肿、空泡样改变;N组4h点肝脏组织结
郑州大学2004届硕士毕业论文
乙酞半胧氨酸对脑死亡大鼠肝脏保护作用的实验研究
构稍紊乱、肝细胞水肿、空泡样改变但较B组4h点减轻。
电镜下:C组4h点肝细胞结构正常;B组4h点SEC内线粒体肿胀、线粒体
婿断裂、消失;N组4h点SEC内仍可见线粒体靖断裂、消失，但较B组4h点损
伤明显减轻;B组4h点肝细胞可见明显空泡样改变;N组4h点肝细胞仍可见空
泡样改变，但较B组4h点减轻。
结论
1、脑死亡可导致大鼠肝脏发生损伤。
2、脑死亡可导致炎症介质TNF一Q、IL一6释放增加，肝细胞NF一KB活化程
度增强。
3、NAC对脑死亡大鼠肝脏具有保护作用，其机制可能与抑制肝脏细胞NF-
KB活化，减少TNF一Q、IL一6的释放有关。
Liver transplantation (LTx) has become the most effective treatment for the end-stage liver diseases. However, the shortage of donor livers restricts the development of clinical liver transplantation. Brain-dead donor (BDD) is the effective way to solve the problem. Brain death is the loss of whole cerebral functions, which means death.
However, some clinical experiments conclude that the organs from living donors consistently demonstrate superior results to those from cadaver sources over both the short- and long-term. It means that the organs from the brain-dead donors have been injuried before procurement.
Now, the mechanism of the BDD liver injury is not clear. So, the experimental studies, which focus on the mechanism of the BDD liver injury and the prevention of it, have of great significance to the development of the liver transplantation.lt has been found that N-acetylcysteine(NAC) has the function of cleaning the free radical , inhibiting the activation of NF- K B and deminishing the levels of the inflammatory mediators. NAC has been widely used into the organ preservation and the prevention of the organ injury of ischemic reperfution. Whether NAC has the protection on the hepatic injury of brain-dead rats remains unknown.
Objective
Our research is mainly aimed to: (1). demonstrate the functional and morphological change of the hepatic in the state of brain death and the mechanism of it. (2). investigate whether N-acetylcysteine(NAC) have the protection on the liver of the brain-dead rats and the possible mechanism.
Methods
Sixty Wistar rats were randomly allocated into 3 groups, 20 rats in each group: control group (group C), brain-dead group (group B) and NAC protection group (group N). Brain-dead model was established in group B and N. There was no inflation of Fogarty balloon in group C, the other operation was same with the group B. NAC at 150mg/kg was injected via the vein of the tail in group N immediately after the establishment of the brain-death model. 1 0 rats were randomly selected for acquiring specimen at 2 hour hi each group, the other 10 rats were used for acquiring the specimen at 4 hour in each group. 3ml blood from the suprahepatic vena cava were abtained at the different time points. The serum levels of ALT and AST were detected by biochemical analyzer. The serum levels of TNF- a , HA and IL-6 were detected by radioimmunoassay. Liver tissues were abtained for HE staining and detecting the activity of NF- B by immunohistochemistry. At 4 hour of each group, the morphological changes of hepatocytes and SEC were observed under electron microscope. All data were analyzed with statistic software SPSS 10.0. Results
1. MAP: There was no significant difference among groupC, B, and N before inflation of the Fogarty balloon(P>0.05); in contrast to group C , the MAP in group B and N was significantly higher during inflation of Fogarty balloon(P<0.05), and was significantly lower in the state of brain-death(P<0.05); there was no significantly difference between group B and N during inflation of Fogarty balloon and in the state of brain-death(P>0.05).
2. HR: There was no significant difference among group C, B, and N before the inflation of Fogarty balloon and in the state of brain-death(P>0.05); in contrast to group C, during the inflation of Fogarty balloon, HR in group B and N was significantly higher(P<0.05), and there was no significantly difference between group BandN(P>0.05).
3. ALT, AST, HA, TNF- a , IL-6 and NF- K B: At the same time points, the levels of these parameters in group B and N was significantly higher than those in group C(/*<0.05), and those in group N was significantly lower than those in group B(P<0.05); At the different time points for the same group, the levels of the parameters at 4 hour hi group B was signicantly higher than those at 2 hour hi group B(P<0.05), the levels of those at 4 hour in group N was significantly higher than those at 2 hour in group N(P<0.05), there was no significantly difference between those at 2 hour in group C and those at 4 hour in
然而，临床研究表明活体供体器官无论是在短期还是长期移植效果方面均较脑死亡供体器官优越。说明脑死亡供体器官在切取以前即已发生了损伤性改变。目前，脑死亡导致外周器官发生损伤的确切机制尚不明确。因此，研究脑死亡供体肝脏的损伤机制及脑死亡供体肝脏的保护对肝移植学科的发展具有重要的意义。研究发现，乙酰半胱氨酸(NAC)具有清除自由基、抑制核转录因子(NF-κB)活化、降低炎症介质水平的作用。NAC已被广泛应用于器官保存、脏器缺血再灌注损伤的保护中。那么NAC对脑死亡供体肝脏是否具有保护作用?国内外尚未见报道。
目的
本实验的目的在于：1、探讨脑死亡状态下肝脏功能与形态的改变及其机制。2、探讨NAC对脑死亡大鼠肝脏是否具有保护作用及其可能机制。
方法
Wistar大鼠60只，雌雄不限，随机分为三组，每组20只，即对照组(C组)、脑死亡组(B组)、NAC保护组(N组)。B、N组建立脑死亡模型；C组除不行
郑州大学加04届硕士毕业论文
乙酞半肤氨酸对脑死亡大鼠肝脏保护作用的实验研究
硬脑膜外导管加压外，其余处理同脑死亡组;N组为NAC处理的脑死亡组，于
脑死亡模型建立成功后立即经尾静脉给予乙酞半肤氨酸(NAC)巧0m眺g。术后
各组于对照2h或脑死亡2h时随机取10只大鼠取材，剩余10只大鼠于对照4h
或脑死亡4h时取材。于肝上下腔静脉抽取血液标本3ml，血清自动生化分析仪检
测丙氨酸氨基转移酶(al耐ne alninotransferase，ALT)、天冬氨酸氨基转移酶
(aspartate aminotransferase，A ST)，放射免疫法检测肿瘤坏死因子一。(tUmor
neerosis factor一a，TNF一)、白细胞介素一6(interleukin一6，IL一6)、透明质酸
(hyaluronicac记，HA)水平;切取肝脏组织标本包埋固定，用于HE染色及免疫组化
法检测NF一KB的表达程度;各组4h点肝脏组织用于透射电镜观察肝细胞及肝窦
内皮细胞的损伤情况。应用SPSS 10.0软件分析，采用单因素方差分析、成组设计
资料t检验进行统计学处理，显著性检验水准取a司.05。
结果
1、MAP变化:加压前各组间无显著性差异(尸)0.05);与C相比，加压过
程中B组与N组MAP显著升高(尸<0.05)，脑死亡状态下B组与N组MAP显
著降低(尸<0 .0.5);在加压过程中与脑死亡状态，B组与N组间M今P均无显著性
差异(尸>0.05)。
2、HR变化:加压前与脑死亡状态各组间无显著性差异(P>0.05);加压过
程中，B组、N组与C组相比均显著升高(尸<0.05)，但B组与N组间无显著性
差异(P>.05)。
3、ALI，、AST、HA、TNF一a、IL一6水平、NF一KB表达水平:在相同时间点
B、N组上述指标水平均显著高于C组(尸<0.05)，但N组明显低于B组(P<0.05);
在同组不同时间点之间，B组4h点明显高于B组Zh点(尸<0.05)，N组4h点明
显高于N组Zh点(尸<0.05)，C组4h点与C组Zh点间无显著性差异(P>.05)。
4、肝脏形态学变化:
光镜下:C组肝脏组织结构正常;B组与N组Zh点肝脏组织结构基本正常;
B组4h点肝脏组织结构紊乱、肝细胞水肿、空泡样改变;N组4h点肝脏组织结
郑州大学2004届硕士毕业论文
乙酞半胧氨酸对脑死亡大鼠肝脏保护作用的实验研究
构稍紊乱、肝细胞水肿、空泡样改变但较B组4h点减轻。
电镜下:C组4h点肝细胞结构正常;B组4h点SEC内线粒体肿胀、线粒体
婿断裂、消失;N组4h点SEC内仍可见线粒体靖断裂、消失，但较B组4h点损
伤明显减轻;B组4h点肝细胞可见明显空泡样改变;N组4h点肝细胞仍可见空
泡样改变，但较B组4h点减轻。
结论
1、脑死亡可导致大鼠肝脏发生损伤。
2、脑死亡可导致炎症介质TNF一Q、IL一6释放增加，肝细胞NF一KB活化程
度增强。
3、NAC对脑死亡大鼠肝脏具有保护作用，其机制可能与抑制肝脏细胞NF-
KB活化，减少TNF一Q、IL一6的释放有关。
Liver transplantation (LTx) has become the most effective treatment for the end-stage liver diseases. However, the shortage of donor livers restricts the development of clinical liver transplantation. Brain-dead donor (BDD) is the effective way to solve the problem. Brain death is the loss of whole cerebral functions, which means death.
However, some clinical experiments conclude that the organs from living donors consistently demonstrate superior results to those from cadaver sources over both the short- and long-term. It means that the organs from the brain-dead donors have been injuried before procurement.
Now, the mechanism of the BDD liver injury is not clear. So, the experimental studies, which focus on the mechanism of the BDD liver injury and the prevention of it, have of great significance to the development of the liver transplantation.lt has been found that N-acetylcysteine(NAC) has the function of cleaning the free radical , inhibiting the activation of NF- K B and deminishing the levels of the inflammatory mediators. NAC has been widely used into the organ preservation and the prevention of the organ injury of ischemic reperfution. Whether NAC has the protection on the hepatic injury of brain-dead rats remains unknown.
Objective
Our research is mainly aimed to: (1). demonstrate the functional and morphological change of the hepatic in the state of brain death and the mechanism of it. (2). investigate whether N-acetylcysteine(NAC) have the protection on the liver of the brain-dead rats and the possible mechanism.
Methods
Sixty Wistar rats were randomly allocated into 3 groups, 20 rats in each group: control group (group C), brain-dead group (group B) and NAC protection group (group N). Brain-dead model was established in group B and N. There was no inflation of Fogarty balloon in group C, the other operation was same with the group B. NAC at 150mg/kg was injected via the vein of the tail in group N immediately after the establishment of the brain-death model. 1 0 rats were randomly selected for acquiring specimen at 2 hour hi each group, the other 10 rats were used for acquiring the specimen at 4 hour in each group. 3ml blood from the suprahepatic vena cava were abtained at the different time points. The serum levels of ALT and AST were detected by biochemical analyzer. The serum levels of TNF- a , HA and IL-6 were detected by radioimmunoassay. Liver tissues were abtained for HE staining and detecting the activity of NF- B by immunohistochemistry. At 4 hour of each group, the morphological changes of hepatocytes and SEC were observed under electron microscope. All data were analyzed with statistic software SPSS 10.0. Results
1. MAP: There was no significant difference among groupC, B, and N before inflation of the Fogarty balloon(P>0.05); in contrast to group C , the MAP in group B and N was significantly higher during inflation of Fogarty balloon(P<0.05), and was significantly lower in the state of brain-death(P<0.05); there was no significantly difference between group B and N during inflation of Fogarty balloon and in the state of brain-death(P>0.05).
2. HR: There was no significant difference among group C, B, and N before the inflation of Fogarty balloon and in the state of brain-death(P>0.05); in contrast to group C, during the inflation of Fogarty balloon, HR in group B and N was significantly higher(P<0.05), and there was no significantly difference between group BandN(P>0.05).
3. ALT, AST, HA, TNF- a , IL-6 and NF- K B: At the same time points, the levels of these parameters in group B and N was significantly higher than those in group C(/*<0.05), and those in group N was significantly lower than those in group B(P<0.05); At the different time points for the same group, the levels of the parameters at 4 hour hi group B was signicantly higher than those at 2 hour hi group B(P<0.05), the levels of those at 4 hour in group N was significantly higher than those at 2 hour in group N(P<0.05), there was no significantly difference between those at 2 hour in group C and those at 4 hour in
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34.Ruth.ford RA, Mccarthy A, Sullivan MH, et al.Nitricoxide synthase in human plac- eta and umbilical cord from normal, intrauterine growth-retarded and pre-eclamptic pregnancies.Br J pharma, 1995, 166:1846-53
35.Weiss SJ.Tissue destruction by neutrophils.N Engl J Med, 1989, 320:365-376
36.Golling M, Mehrabi A, Blumk, et al.Effect of hemodynamic instability on brain death-induced preservation liver damage.Transplantation, 2003, 8:1154～1159
37.Kosieradzki M, Kuczynska J, Piwowarska J, et al.Prognostic significance free radicals: mediated injury occurring in the kidney donor.Transplantation, 2003, 8: 1221-7
38.Marzi I, et al.Reduction by superoxide dismutase of leukocyte endothelial adherence after liver transplantation.Surgery, 1992, 1:90
39.Kubes P, et al.Role of platelate activating factor in ischemia reperfution induced leukocyte adherence.Am J physoil, 1990, 259:G300
40.Tran K, Merika M, Thanos D, Distinct functional properties of Iк appaB alpha and Iк appaB.Mol cell Biol, 1997, 9:5386-99
41.Whiteside ST, Israel A.Iк appaB protein: structure, function and regulation.Semin Cancer Biol, 1997, 2:75-82
42.Flohe L, Brigelins Flohe R, Salion C, et al.Packor Lredox regulation of NF-кB
activation.Free Radic Biol Med, 1997, 6:1115-26
43.May MJ, Ghosh S.Signal transduction through NF-к appaB.Immunoi Today,1998, 2:80-8
44.Collart MA, Baeuerle P, Vassali P.Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four к appaB-like motifs and of constitutive and inducible formes ofNF-к appaB.Mol Cell Biol, 1990, 4:1498-1506
45.Hiscott J, Marois J, Garoufalis J, et al.Characterization of a functional NF-к appaB site in the human interleukin L beta promoter: Evidence for a positive autoregulatory loop.Mol Cell Biol, 1993, 10:6231-6240
46.Konishi K, Tanaka Y, Yamamoto K, et al.Structure of the gene encoding rat neutrophil chemoattmctant.Gene, 1993, 2:285-286
47.Collins T, Read MA, Neish MZ, et al.Transcriptional regulation of endothelical cell adhesion molecules: NF-к appaB and cytokine-inducible enhancers.FASEB J,1995, 10:899-909
48.Ueland PM, Mansoor ma, Guttormsen AB, et al.Reduced, oxidized and protein-bound forms of homocysteine and other aminothiol in plasma comprise the redox thiol status-apossible element of the extracelluar antioxidant defense system.J Nutr, 1996, 126(4 suppl): 1281s-4s
49.Zimmerman BJ, Granger DN.Mechanisms of reperfusion injury.Am J Med Sci,1994, 307:284
50.Paterson RL, Galley HF, Webster NR.The effect of N-acetylcysteine on nuclear factor-к appaB activation,interleukin-6, interleukin-8, and intercellular adhesion molecule-1 expression in patients with sepsis.Crit Care Med, 2003, 11: 2574-8
51.Li J, Quan N, Bray TM.Supplementation of N-acetylcysteine normalizes lipopolysaccharide-induced nuclear factorк appaB activation and proinflammatory cytokine production during early rehabilitation of protein malnourished mice.J Nutr, 2002, 11:3286-92
52.Kim H, Seo JY, Roh KH, et al.Suppression of NF-к appaB activation and cytokine production by N-acetylcysteine in pancreatic acinar cells.Free Radic Biol Med, 2000, 7:674-83
53.Blinma TA, Gukosky I, Gukovskaya A, et al.Intrapancreatic expression of cytokines IL-6 and KC increased in ceruleine pancreatitis regulation by NF-кB.Gastroenterology, 1998, 114(4pt 2): A443G1801
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28.Wilhelm MJ, Pratschke J, Beato F, et al.Activation of the heart by donor brain death accelerate acute rejection after transplantation.Circulation, 2000, 19:2426-33
29.Pratschke J, Wilhelm MJ, Kusaka M, et al.Accelerate rejection of renal allografts from brain-dead donors.Ann Surg, 2000, 2:263-71
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31.Bussolino F, Camussi G, Baglioni, et al.Synthesis and release ofplatelet-activating factor by human vascular endothelial cells treated with tumor necrosis factor or interleukin-lalpha.J Biol Chem, 1998, 24:1185-93
32.Pohlman TH, Wirm RK, Callahem, et al.A glycolipid presuror of bacterial lipidx lacks activity against endothelial cells in vitro and is not toxic in vivo.J Surg Res, 1988, 2:228-32
33.Salyer JL, Bohnsack JF, K.nape WA, et al.Mechanisms of tumor necrosis-alpha alteration of pMN adhesion and migration.Am J phathol, 1990, 4:83-89
34.Ruth.ford RA, Mccarthy A, Sullivan MH, et al.Nitricoxide synthase in human plac- eta and umbilical cord from normal, intrauterine growth-retarded and pre-eclamptic pregnancies.Br J pharma, 1995, 166:1846-53
35.Weiss SJ.Tissue destruction by neutrophils.N Engl J Med, 1989, 320:365-376
36.Golling M, Mehrabi A, Blumk, et al.Effect of hemodynamic instability on brain death-induced preservation liver damage.Transplantation, 2003, 8:1154～1159
37.Kosieradzki M, Kuczynska J, Piwowarska J, et al.Prognostic significance free radicals: mediated injury occurring in the kidney donor.Transplantation, 2003, 8: 1221-7
38.Marzi I, et al.Reduction by superoxide dismutase of leukocyte endothelial adherence after liver transplantation.Surgery, 1992, 1:90
39.Kubes P, et al.Role of platelate activating factor in ischemia reperfution induced leukocyte adherence.Am J physoil, 1990, 259:G300
40.Tran K, Merika M, Thanos D, Distinct functional properties of Iк appaB alpha and Iк appaB.Mol cell Biol, 1997, 9:5386-99
41.Whiteside ST, Israel A.Iк appaB protein: structure, function and regulation.Semin Cancer Biol, 1997, 2:75-82
42.Flohe L, Brigelins Flohe R, Salion C, et al.Packor Lredox regulation of NF-кB
activation.Free Radic Biol Med, 1997, 6:1115-26
43.May MJ, Ghosh S.Signal transduction through NF-к appaB.Immunoi Today,1998, 2:80-8
44.Collart MA, Baeuerle P, Vassali P.Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four к appaB-like motifs and of constitutive and inducible formes ofNF-к appaB.Mol Cell Biol, 1990, 4:1498-1506
45.Hiscott J, Marois J, Garoufalis J, et al.Characterization of a functional NF-к appaB site in the human interleukin L beta promoter: Evidence for a positive autoregulatory loop.Mol Cell Biol, 1993, 10:6231-6240
46.Konishi K, Tanaka Y, Yamamoto K, et al.Structure of the gene encoding rat neutrophil chemoattmctant.Gene, 1993, 2:285-286
47.Collins T, Read MA, Neish MZ, et al.Transcriptional regulation of endothelical cell adhesion molecules: NF-к appaB and cytokine-inducible enhancers.FASEB J,1995, 10:899-909
48.Ueland PM, Mansoor ma, Guttormsen AB, et al.Reduced, oxidized and protein-bound forms of homocysteine and other aminothiol in plasma comprise the redox thiol status-apossible element of the extracelluar antioxidant defense system.J Nutr, 1996, 126(4 suppl): 1281s-4s
49.Zimmerman BJ, Granger DN.Mechanisms of reperfusion injury.Am J Med Sci,1994, 307:284
50.Paterson RL, Galley HF, Webster NR.The effect of N-acetylcysteine on nuclear factor-к appaB activation,interleukin-6, interleukin-8, and intercellular adhesion molecule-1 expression in patients with sepsis.Crit Care Med, 2003, 11: 2574-8
51.Li J, Quan N, Bray TM.Supplementation of N-acetylcysteine normalizes lipopolysaccharide-induced nuclear factorк appaB activation and proinflammatory cytokine production during early rehabilitation of protein malnourished mice.J Nutr, 2002, 11:3286-92
52.Kim H, Seo JY, Roh KH, et al.Suppression of NF-к appaB activation and cytokine production by N-acetylcysteine in pancreatic acinar cells.Free Radic Biol Med, 2000, 7:674-83
53.Blinma TA, Gukosky I, Gukovskaya A, et al.Intrapancreatic expression of cytokines IL-6 and KC increased in ceruleine pancreatitis regulation by NF-кB.Gastroenterology, 1998, 114(4pt 2): A443G1801