[精品论文]Proteomic analysis of human donor liver tissues subjected to ischemiareperfusion injury during liver transplantation

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1、精品论文Proteomic analysis of human donor liver tissues subjected to ischemia/reperfusion injury during liver transplantationWU Bin1, WU Hongli2, CHEN Jianning3, HUA Xuefeng1, LI Ning1, LU Minqiang15(1. Department of liver transplantation, the 3rd affiliated hospital of Sun-Yat-Sen University, GuangZhou

2、 510630;2. Department of veterinary and biomedical sciences, 120 VBS, University of Nebraska-Lincoln,Lincon, USA, 68583-0905;3. Department of pathology, Tianhe road 600, The 3rd affiliated hospital of Sun-Yat- Sen10University, GuangZhou 510630)Abstract: Purpose: to explore the specific alterations i

3、n protein profiles that took place during ischemia/reperfusion injury (I/RI) and find novel therapeutic strategies to reduce I/RI during orthotopic liver transplantation (OLT). Method: we used the comparative proteomic approach of two-dimensional electrophoresis (2-DE) and matrix-assisted laser deso

4、rption/ionization time-of-flight mass spectrometry15(MALDI-TOF MS) to compare the proteomic profiles of the same donor liver at three different time points: T1, immediately after cardiac arrest of donors (normal control). T2, before portal vein was recirculated (ischemia). T3, two hours after the an

5、astomosis of the hepatic artery (reperfusion). Result: we identified 34 proteins that were significantly altered during I/RI. These differentially expressedproteins were functionally classified into seven categories: metabolic enzyme, molecular chaperone,20antioxidant enzyme, cytoskeleton protein, s

6、ignal transduction protein, cyclin, and binding protein.Among the 34 proteins, nine proteins changed only during ischemia (from T1 to T2) period, eleven proteins changed only during reperfusion (from T2 to T3) period, others changed during both ischemia and reperfusion (from T1 to T3) periods. Concl

7、usion: ischemia and reperfusion during LT may lead to different modifications of the liver proteins. Most metabolic enzymes and antioxidant enzymes were25up-regulated during ischemia period, indicated that lipidic metabolic disorder and oxidative stress were closely related to the development of isc

8、hemia injury. ER chaperones may play a vital role in mediatingI/RI and prevention of ER stress caused by I/RI may be used as a key therapeutic target to improve theoutcome of LT.Keywords: liver transportation; ischemia reperfusion injury; protemics301IntroductionLiver transplantation (LT) remains th

9、e only feasible intervention for patients with end-stage liver disease. Almost all donor livers experience some degree of preservation damage related to I/RI. I/RI causes up to 10% of early organ graft failures 1. Primary graft dysfunction related to35I/RI may also hamper the long-term outcome of LT

10、.Different mechanisms participate in the development of I/RI during LT. Adenosine triphosphate (ATP) depletion during prolonged hypoxia can lead to I/RI 2. ATP-depletion of the hepatic cells results in swelling of mitochondria and cytochrome c release from the mitochondria. And cytochrome c activati

11、on triggers the apoptotic signaling cascade 3. The destructive effects of40I/RI are inflicted by the generation of superoxide and other forms of reactive oxygen species (ROS) after reoxygenation during reperfusion period 4. This may cause impairment of mitochondrial respiratory chain and eventually

12、lead to mitochondrial dysfunction. Sustained ROS/reactive nitrogen species (RNS) generation activates important stress signaling (e.g., p38MAPK, JNK) and proinflammatory pathways (e.g., NF-B 5) in various cell types, resulting in inflammatory and45cell death processes. Two periods of injury are dist

13、inguished in liver transplantation procedure:Foundations: Doctoral science foundation of Chinese educational ministry, (No. 20090171110071)，Guangzhoutechnology scheming project funding, (No. 2009Z1-E211)Brief author introduction: Wu Bin,（1972-），Man，Deputy chief physician，Clinical and basic research

14、of livertransportation.Correspondance author: Lu Minqiang，Man，professor，clinical and basic research of liver transportation. E-mail:minqianglu- 15 -ischemia injury and reperfusion injury. During ischemia period a donor liver is cooled, transported, prepared to be reconnected to a blood supply. The m

15、echanism of the ischemia injury is related to the conversion of the enzyme xanthine dehydrogenase to xanthine oxidase 6. The reperfusion starts after all vessels are connected between donor organ and recipient vasculature. Reperfusion50injury is mainly due to polymorphonuclear neutrophils and Kupffe

16、r cells, which produce inflammatory cytokines and oxygen-derived free radicals 7. The injury mechanisms are different between ischemia injury and reperfusion injury, implies that different proteins may be involved in mediating ischemia and reperfusion injury. Up to now, only few studies investigated

17、 the ratio of ischemia to reperfusion in protein abundances, the results have been inconclusive 8.55To better understand the molecular bases of I/RI and perform therapeutic interventions to optimize LT strategies, it is important to identify proteins whose expression and functions are closely relate

18、d to I/RI. In this present study, we used 2-DE and MS approaches to identify the proteins that have been changed significantly during I/RI. For the first time, we explored protein variations during ischemia and reperfusion periods respectively. This study unveils independent60protein profiles of isc

19、hemia injury and reperfusion injury in donor livers. Such a finding will help us find novel therapeutic strategies to reduce I/RI and improve clinical outcomes.2Materials and methods2.1Liver tissue biopsies and time recordingFrom November 23，2008 to March 3，2009, 15 wedge liver tissue samples were b

20、iopsied65from five donor livers using sterile scissors at three different time points. Biopsies were retrospectively analyzed. Written informed consents were obtained from the recipients relatives. The study protocol was approved by the Clinical Research (Ethics) Committee of Sun-Yat-Sen University.

21、 Donor livers were obtained according to standard multiorgan harvesting procedure. A first scissors cut biopsy (1 g) was obtained from each donor liver immediately after cardiac arrest70of donor, this time point was recorded as T1. During transportation and back-table preparation, donor livers were

22、maintained cold (4C) in University of Wisconsin solution. Then the donor livers were reconnected to the recipients, the time point before recirculation of portal vein was recorded as T2. The time period from T1 to T2 represented ischemia period. After the anastomosis of the portal vein, the anastomo

23、sis of the hepatic artery was performed. A third scissors cut liver75biopsy was performed about two hours after the anastomosis of the hepatic artery. This biopsied time point was recorded as T3. The period from T2 to T3 represented the early reperfusion stage, during which the reperfusion injury ta

24、kes place. All liver tissue samples were cut into two equal parts (0.5 g each). Part A was frozen in liquid nitrogen for proteomic research, and part B was saved for histological assessment, immunohistochemistry analysis, and western blot detection.802.2Donor and recipient profilesAll donors were ma

25、le adults with glutamic pyruvic transaminase (ALT) values within the normal range (1041 IU). The average age of these donors was 28 years old (range 21-31). All of the donors were free from viral hepatitis, autoimmune liver disease or cystic liver disease. Histology was normal in five donor livers,

26、but only one case showed fatty infiltration about 10%.85Hepatocellular carcinoma and cirrhosis were the indications of the five recipients (four males and one female) who received OLT. OLT was performed in all cases using a modified piggyback technique by the same surgeon. All recipients were treate

27、d by standard immunosuppressive therapywithtacrolimus(Novartis,Basel,Switzerland)andprednisolone(Shanghai Pharmaceuticals Corporation, China).90951001051101151201251302.3 Histological scoring, malondialdehyde (MDA) content, superoxide dismutase(SOD) and myeloperoxidase (MPO) activity assayBiopsied l

28、iver specimens used for histological assessment were fixed in formalin for less than24 hours and then paraffin embedded histological slides (4-m thick) were stained with hematoxylin and eosin. Sinusoidal leucocytes were observed under the ten high-power fields (field diameter 0.50, Zeiss Photo micro

29、scope). According to Ishak et al 9, the micromorphological changes in hepatic tissues occurring during I/RI were blindly selected by one single observer whois experienced in liver pathology. Variable score grading in different regions of the same slide with the highest injury grade were assigned. Fr

30、ozen liver tissue was homogenized on ice in five volumes of normal saline. Homogenates were centrifuged at 1,200 g for 10 minutes. The SOD activity, MPO activity, and Malondialdehyde (MDA) content of the supernatant were determined using the assay kits (Nanjing Jiancheng Corporation, Nanjing, China)

31、 according to the manufacturers recommendations.2.4 Preparation of total tissue extracts and CyDye difference gel electrophoresis(DIGE) fluor labelingBiopsied liver tissues saved for proteomic research were ground into a fine powder in liquid nitrogen and solubilized in a cocktail of 20 mM Tris, 7 M

32、 urea, 2 M thiourea, 4%3-(3-cholamidopropyl) dimethylammonio-1-propanesulfonate, 10 mM dithiothreitol, 1 mM EDTA, 1 mM PMSF, and one tablet Complete Protease Inhibitors (Roche Diagnostics, Mannheim, Germany). The suspension was homogenized with a dounce homogenizer for approximately 5 minutes, sonic

33、ated for 30 seconds, and centrifuged at 150,000 g for 1 hour (15C). Supernatants were collected and stored at -80C for later use. Protein concentrations weredetermined using a commercial Bradford assay kit (BioRad, Hercules, USA) 10. Liver tissuelysates were labeled with Cy2, Cy3, and Cy5 following

34、protocols described in the Ettan DIGEuser manual (18-1164-40 Edition AA, GE Healthcare).2.52-D DIGE and imagingThe labeled protein mixture of each gel was applied to Immobiline DryStrip strips (24-cm, pH 3-10, Bio-Rad). The Immobilized pH gradient (IPG) strips were treated with equilibration buffer

35、(6 M urea, 2% SDS, 50 mM Tris-Cl (pH 8.8) 30% glycerol) supplemented with 0.5%dithiothreitol for 15 minutes at room temperature followed by 4.5% iodoacetamide in equilibration buffer for another 15 minutes incubation. IPG strips were placed on top of 12% homogeneous polyacrylamide gels. The second d

36、imension SDS-PAGE was carried out using a Protein Plus system (Bio-Rad). For the pooled sample in each group, 2-DE runs were repeated under the same conditions for at least five times. After 2-DE, gels were scanned using a technique as described bySun, W11.2.6In-gel tryptic digestion and MALDI-TOF/T

37、OF analysisTwo-dimensional electrophoresis was carried out as described under “2D DIGE and Imaging”. Gels were stained with Coomassie brilliant Blue. Protein spots of interest were excised and washed. In-gel digestion was performed with 0.01g/l trypsin solution (Promega, WI, USA) in 25 mM ammonium b

38、icarbonate for 15 hours at 34C. The supernatants were collected, and the tryptic peptides were extracted from the gel consecutively with 5% TFA at 40C for 1 hour and with 2.5% TFA, 50% ACN at 30C for 1 hour. Peptides were mixed with MALDI matrix (7 mg/mL CHCA and 0.1% TFA and 50% ACN) and spotted on

39、 to the MALDI target plates, then analyzed using an ABI 4700 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied135140145150155160165170175Biosystems, MA, USA). MS/MS analyses were conducted, database searching were performed using a technique as described by Deng, X et al 12. One missing c

40、leavage was allowed. Precursor error tolerance was set to 0.1 Da and MS/MS fragment error tolerance 0.2 Da. All the proteins identified should have protein scores greater than 63. Known contaminant ions (keratin) were excluded. The confident identification had a statistically significant (P 1.5, P 0

41、.01) were marked. Only spots altered consistently in at least four of the five donor livers were selected for identification.Value of SOD and MPO activities, and content of MDA were expressed as the mean S. D. Statistical significance was evaluated by the paired students t test with P0.05 regarded a

42、s significant.3Results3.1Histological assessment of I/RI and immunohistochemical analysisThe ischemia time period (from T1 to T2) of five donor livers were 377, 85, 303, 325, and280 minutes, respectively. The reperfusion time period (from T2 to T3) of the five donor livers were 285, 342, 246, 265, a

43、nd 295 minutes, respectively. As seen in Figure 1A, only mild hepatocellular injury, such as hepatocellular swelling was observed (hematoxylineosin, magnification, 200). However, after ischemia, liver tissue damage was aggravated compared with T1. As shown in figure 1B, focal hepatocyte fatty degene

44、ration, spotty hepatocyte necrosis, and mild endothelial cell swelling were observed. Mild to moderate cytoplasmic swelling of hepatocytes and neutrophil infiltration were also displayed. In T3 (Fig. 1C) slides, liver injury was significantly aggravated compared with T1 and T2 slides. Conspicuous cy

45、toplasmic swelling of hepatocytes and endothelial cell swelling were seen. Focal hepatocyte necrosis, fatty degeneration and hyperemia of hepatic sinusoids were obvious. More marked and diffuse neutrophil infiltration180185190195also appeared. Under higher power lens (Fig. 1D, hematoxylineosin, magn

46、ification 400), there were marked hepatocytic swelling and neutrophil infiltration. In addition, apoptosis (eosinophilic) and fatty degeneration with big and small lipid droplets were present in the cytosol of hepatocytes. Hyperemia and distended hepatic sinusoids packed with erythrocytes were also

47、observed. The detailed morphological changes occurring during I/RI were shown in Figure 1E.3.2Biochemical assays for SOD, MDA, and MPODetection of malondialdehyde (MDA) was used as a biomarker of free radical mediated lipid peroxidation in the liver. As shown in Figure 1F, MDA level was significantl

48、y increased after reperfusion (T2, 215.3319.2 vs T3, 439.42 39.4nmol/mg, P0.05).Oxidative stress and the increased production of ROS are among leading destructive consequences of I/RI during LT. Superoxide dismutase (SOD) is one of the most important antioxidant enzymes to detoxify the excess ROS. A

49、s shown in Figure 1G, SOD activity was markedly decreased during ischemia period compared with normal control (T1,10.721.1 vs T2, 6.560.8 U/mg, P0.05) decrease in the activity of SOD (T3, 5.8450.6 vs T2, 6.560.8 U/mg).Myeloperoxidase (MPO) was used as an index of liver neutrophil accumulation. In Fi

50、gure 1G, MPO activity was significantly increased during ischemia period, (T2, 3.660.6 vs T1, 1.180.3 U/g, P0.01) and reperfusion period (T2, 3.660.6 vs T3, 7.1981.2 U/g, P0.05).200205210Figure 1. Light microscopy examination and enzmatic changes of a liver graft during the I/RI period.(A) T1 sample

51、s (normal control), only mild hepatocellular injury, such as hepatocellular swelling was observed (hematoxylineosin, magnification: AC 200). (B) T2 samples (ischemia), injury was aggravated afterischemia period. Focal hepatocyte fatty degeneration, spotty hepatocyte necrosis, and mild endothelial ce

52、ll swelling were observed. Mild to moderate cytoplasmic swelling of hepatocytes and few neutrophil infiltrationwere also displayed，hyperemia of hepatic sinusoids was not remarkable. (C) T3 samples (ischemia andreperfusion), after reperfusion period, injury was markedly aggravated, conspicuous cytopl

53、asmic swelling of hepatocytes and endothelial cell swelling were seen. Focal hepatocyte necrosis and fatty degeneration were remarkable. Compared with T1 and T2 slides, more marked and diffuse neutrophil infiltration appeared. Hyperemia of hepatic sinusoids was remarkable. (D) T3 samples (ischemia a

54、nd reperfusion, hematoxylineosin, magnification 400). Under higher power lens, there were marked hepatocytic swelling and neutrophil infiltration. In addition, apoptosis (eosinophilic) and fatty degeneration with big and small lipid droplets were present in the cytosol of hepatocytes. (E) Morphologi

55、cal changes of hepatocyte occured during I/RI. According to Ishak et al 9, variable score grading in different regions of the same slide with the highest injury grade were assigned. Liver tissues collected from each time point was homogenized with normal saline. MDA content (F), MPO and SOD levels (

56、G) were measured as described as under “Materials and methods”. Error bars indicate S.D., n=5, *P0.05vs. T1 (normal control). #P0.05 vs. T2 (ischemia).2152202253.3Profiles of liver proteome variation upon ischemia/reperfusion injuryA total of 1580 proteins were revealed by using 2-DE gels. Then 34 p

57、roteins that contributed to I/RI were eventually identified. In general these proteins were functionally classified into seven categories: metabolic enzyme (15 proteins, 44.1%), molecular chaperone (2 proteins, 5.8%), antioxidant enzyme (10 proteins, 29.4%), cytoskeleton protein (3 proteins, 8.8%), signal transduction (1 protein, 2.9%), cyclin