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

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 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 in 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 desorption/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 anastomosis 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, signal 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. Conclusion: 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 ischemia 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 the 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.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 activation 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 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 distinguished 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 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 mechanism 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 Kupffer 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 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 related 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 ischemia 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 biopsied65from 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. 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 maintained cold (4°C) 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 anastomosis 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 takes 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 male 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, 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 treated 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 liver specimens used for histological assessment were fixed in formalin for less than24 hours and then paraffin embedded histological slides (4-µm thick) were stained with hematoxylin and eosin. Sinusoidal leucocytes were observed under the ten high-power fields (field diameter 0.50, Zeiss Photo microscope). 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. Frozen 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) 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 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, sonicated for 30 seconds, and centrifuged at 150,000 g for 1 hour (15°C). Supernatants were collected and stored at -80°C 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 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 (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 dimension 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/TOF 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 bicarbonate for 15 hours at 34°C. The supernatants were collected, and the tryptic peptides were extracted from the gel consecutively with 5% TFA at 40°C for 1 hour and with 2.5% TFA, 50% ACN at 30°C for 1 hour. Peptides were mixed with MALDI matrix (7 mg/mL CHCA and 0.1% TFA and 50% ACN) and spotted on 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 cleavage 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<0.05) protein score (basedon combined mass and mass/mass spectra) and best ion score (based on mass/mass spectra). If more than one protein was identified in identical spot, the only protein ID with the highest score (top rank) was selected.2.7Western blotting analysisTo ensure our 2-DE and MS/MS measurements could be confirmed using other methods, we used western blotting analysis to compare protein expression in normal, ischemic and reperfusion donor liver biopsies. Proapolipoprotein, carbonic anhydrase I, peroxiredoxin 6, catalase, and heat shock protein 90kDa beta, member 1 (HSP90b1) were chosen for western blotting analysis. Equal amounts of protein extracts were subjected to SDSPAGE on a 12% polyacrylamide gel and transferred to a PVDF membrane (Millipore Corp, MA, USA). The membrane was incubated overnight at 4°C in primary antibody diluted according to the manufacturers protocol. Then, the membrane was washed three times with TBS-T for 15 minutes and incubated at room temperature for 1 hour with diluted (1:2000) secondary antibody (Santa Cruz, CA, USA). Protein bands were visualized with horseradish peroxidase substrate peroxide solution (Millipore, Billerica, MA, USA).2.8Statistical analysisAnalysis of 2D DIGE was done using DeCyder 5.0 software (GE Healthcare). Briefly the DeCyder biological variation analysis module was used to detect spots and simultaneously match all 34 protein spots maps from eight gels. All matches were also confirmed manually 13. The paired t test was used for statistical analysis of the data. Protein spots that were differentiallyexpressed in T1, T2, and T3 time points (ratio> 1.5, P< 0.01) 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 as 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, and 295 min