- Email: [email protected]
Reperfusion Injury With or Without Octreotide Preconditioning in a Rabbit Model
Hepatocellular Protein Profiles After Hepatic Ischemia/Reperfusion Injury With or Without Octreotide Preconditioning in a Rabbit Model J. Yang, H. Sun, R. Guan, W. Liu, Y. Xia, J. Zhao, and J. Liu* Department of Anesthesiology, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, China
ABSTRACT Hepatic ischemic/reperfusion injury (HIRI) is a major complication of liver resection and transplantation. Octreotide, a somatostatin analogue, has been used to treat hepatic fibrosis and portal hypertension; however, its function against HIRI remains unclear. To elucidate the effect of octreotide in HIRI, we investigated the hepatocellular protein profiles in response to octreotide preconditioning in a rabbit model by using proteomic analysis. Twenty-four rabbits were divided into 3 groups: the sham operative group (control), the ischemia/reperfusion group (IR), and the ischemia/reperfusion þ octreotide group (IRþOct). They were subjected to 30 minutes of normothermic ischemia followed by 120 minutes of reperfusion by using Pringle’s maneuver method. Proteomic studies were then performed to compare the protein profiles of their left liver lobe. A total of 16 differential proteins were successfully identified. These findings suggest that octreotide might exert an effect against HIRI through up-regulating the expression of the anti-injury substances, such as heat-shock proteins 70 and 27 (confirmed by using Western blot analysis); significantly raising the phosphatidylethanolamine-binding protein that alleviates IR-related apoptosis; and down-regulating mitochondrial metabolic enzymes such as NADH2 dehydrogenase and triosephosphate isomerase.
H
EPATIC ischemic/reperfusion injury (HIRI) is a major cause of morbidity and mortality after vascular exclusion in liver and liver transplantation surgery. Ischemic preconditioning, which could improve clinical outcomes after HIRI [1], is an adaptional phenomenon in which tissues are rendered resistant to the deleterious effects of ischemia/reperfusion (IR), either by previous exposure to brief periods of vascular occlusion or by the administration of certain chemical agents. Ischemic preconditioning might exert its protective effects through transient nitric oxide production, diminution of toxic reactive species, preservation of energy metabolism during ischemia, and involvement of nuclear transcription factor and others [2e5]. Octreotide, a synthetic analogue of somatostatin, was approved for use by FDA in 1988. It was primarily used to improve the management of carcinoid and VIPomas [6e8]. Since then, research studies and clinical experience have accumulated that suggest a potentially much broader therapeutic role for octreotide. Octreotide is now used for treating pituitary tumors, gastroenteropancreatic tumors, diabetes mellitus, pancreatitis, complications of pancreatic
surgery and transplantation, and short bowel syndrome [9e11]. Other indications for its use include hypercalcemia, cancer-related pain, polycystic ovary syndrome, and certain cancers. Octreotide inhibits hepatic fibrosis, bile duct proliferation, and bacterial translocation in obstructive jaundice [12]. It could cause strong hepatic glucose release even under hypoxic-ischemic conditions in rat livers with filled glycogen stores [13]. It may also protect the liver against HIRI [14]. In our previous studies, we showed that octreotide could protect the liver against HIRI in a rabbit model. Liver function can be protected and hepatocellular apoptosis decreased
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.06.071
ª 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-SA license (http:// creativecommons.org/licenses/by-nc-sa/3.0/). 360 Park Avenue South, New York, NY 10010-1710
3282
This study was funded by Hunan Provincial Science and Technology Bureau of China (06SK3079, 2007FJ3081). *Address correspondence to Jingshi Liu, MD, PhD, Department of Anesthesiology, Perioperative Medicine and Pain Management, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Tongzipo Road 283#, Yuelu District, Changsha, Hunan 410013. E-mail: [email protected]
Transplantation Proceedings, 46, 3282e3288 (2014)
HEPATOCELLULAR PROTEIN PROFILES
by reducing endotoxin production and down-regulating the inflammatory cytokines tumor necrosis factorea and interleukin-1b [15]. However, those results are not enough to clarify octreotide’s mechanism of action regarding HIRI. Proteomic studies of IR samples represent an important step toward a better understanding of the disease and may lead to identification of clinically useful markers [16]. Methodologic advances in proteomics allowed the comparison of the protein content in different IR and octreotide-treated IR samples, using gel or liquid-based approaches coupled with mass spectrometry [17]. Proteomic studies have the advantage of being able to discern changes in expression levels and help elucidate the underlying mechanism [18]. The goal of the present study was to investigate the mechanisms of HIRI and protective mechanisms of octreotide on HIRI. We evaluated the hepatocellular protein profiles after HIRI with or without octreotide preconditioning in a rabbit model and identified some potential proteins by using a proteomic-based approach.
MATERIALS AND METHODS Animals Preparation All institutional and national guidelines for the care and use of laboratory animals were followed. Twenty-four New Zealand rabbits of both sexes, weighing 1.6 to 1.9 kg, were used in this study. All animals were obtained from the Laboratory Animal Center of Xiangya Medical School of Central South University. All experimental procedures and study design were reviewed and approved by the Institutional Animal Care Committee of Xiangya Medical School of Central South University, China.
HIRI Model All rabbits were fasted for 12 hours before the experiment but were given free access to water. Twenty-four rabbits were randomly divided into 3 equal groups: the sham-operated group (control), the IR group (IR), and the IR þ octreotide pretreatment group (IRþOct). Each rabbit was anesthetized with an intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg). After the induction of anesthesia, an intravenous line was established in an ear vein and a 0.9% saline solution infusion was begun at 10 mL/kg per hour. The animal then underwent a tracheotomy and was mechanically ventilated (DH-150 Animal Ventilator, Zhejiang University Medical Apparatus Ltd., Zhejiang, China) on 100% oxygen with a tidal volume of 10 mL/kg. Anesthesia was maintained by a continuous infusion of fentanyl at 10 mg/kg per hour and intermittent administrations of 3% pentobarbital (10 mg/kg). Vecuronium was infused at 0.1 mg/kg per hour to maintain relaxation. Thirty minutes before laparotomy, the IRþOct group rabbits received an injection of octreotide (Jilin Province A-Think Pharmaceutical Co., Ltd., Jilin, China) 20 ug/kg intraperitoneally and 30 ug/kg subcutaneously. Control and IR group rabbits received the same volume isotonic saline, and all rabbits underwent a laparotomy. HIRI models were set up in the IR and IRþOct groups by using Pringle’s method [19], which consisted of ischemia for 30 minutes followed by reperfusion for 120 minutes. All animals were killed, and the left liver lobe of each rabbit was removed and fixed in liquid nitrogen and then stored at 80 C for protein profiles analysis.
3283
Two-Dimensional Electrophoresis Eighty milligrams of rabbit liver left lobe tissues of each group were lysed in 400 uL of lysis buffer (urea 7 mol/L, thiourea 2 mol/L, DTT 100 mmol/L, 4% CHAPS, Tris 40 mmol/L, 2% Pharmalyte, 1 mg/mL DNase I) at 37 C for 1 hour and then centrifuged at 15,000 rpm for 30 minutes at 4 C. The supernatant was transferred and the concentration of the total proteins was determined by using a 2-dimensional quantification kit (Amersham Biosciences, Sweden). Two-dimensional electrophoresis (2-DE) was performed to separate 800-mg protein samples, as described by Feng et al [19]. After sodium dodecyl sulfateepolyacrylamide gel electrophoresis, the protein spots were visualized by staining with colloidal Coomassie (Bio-Rad Laboratories, Minneapolis, Minn, United States). Triplicate gels were made for each cell line.
Image Analysis To minimize the contribution of experimental variations, 3 separate 2-dimensional gels were prepared for each tissue. The stained 2-dimensional gels were scanned with the MagicScan software on ImageScanner (Amersham Biosciences, Uppsala, Sweden) and were analyzed by using the PDQuest system (Bio-Rad Laboratories, California, United States). Spot intensities were quantified by calculating the spot volume after normalization of the image using the total spot volume normalization method multiplied by the total area of all the spots. An average 2-DE map for each group was constructed with the PDQuest image analysis software after spot matching, and the expression level of a protein spot was determined by its average intensity volume of 3 gels. The average 2-DE maps were used to perform the various expression analysis. The differences of the matched protein spots between the tissues was calculated by the ratio of their average expression levels, and proteins were classified as being differentially expressed between the tissues when average spot intensity was showed a difference 2-fold variation. Significant differences in protein expression levels were determined by the Student t test with a set value of P < .05.
Mass Spectrometry Analysis All the differential protein spots exhibiting consistent differences between the tissues in triplicate experiments were excised from stained gels using punch, in-gel trypsin digested. They were analyzed with a Voyager System DE-STR 4307 matrix-assisted laser desorption/ionizationetime-of-flight (MALDI-TOF) mass spectrometry (MS) to produce a peptide mass fingerprint as previously described by Feng et al [19]. The protein spots identified according to MALDI-TOF MS were also subjected to analysis of electrospray ionization quadrupole time-of-flight MS (Micromass, Waters Corporation, Milford, Mass, United States). Briefly, the samples were loaded onto a precolumn (320 mm 50 mm, 5-mm C18 silica beads; Waters Corporation) at 30 mL/min flow rates for concentrations and fast desalting through a Waters CapLC autosampler and then eluted to the reversed-phase column (75 mm 150 mm, 5 mm, 100Å; LC Packing) at a flow rate of 200 nL/min after flow splitting for separation. MS/MS spectra were performed in data-depended mode in which up to 4 precursor ions above an intensity threshold of 7 counts per second were selected for MS/MS analysis from each survey “scan.”
Western Blot Analysis Western blot analysis was performed to detect the expression of heat-shock protein (HSP) 70 and HSP27 in the frozen liver tissues, as previously described by Feng et al [19]. Briefly, 40 mg of lysates
3284 were separated by 10% sodium dodecyl sulfateepolyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Blots were blocked with 5% nonfat dry milk for 2 hours at room temperature and then incubated with 1:1000 dilution of monoclonal mouse antieHSP70 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif, United States), 1:3000 dilution of monoclonal mouse anti-HSP27 antibody (Santa Cruz Biotechnology, Santa Cruz, California, United States) for 2 hours at room temperature, and then by incubation with 1:3000 dilution of horseradish peroxidaseeconjugated secondary antibody for 1 hour at room temperature. The signal was visualized with an enhanced chemiluminescence detection reagent. b-actin was detected simultaneously by using 1:3000 dilution of monoclonal mouse antieb-actin antibody (Santa Cruz Biotechnology, Santa Cruz, California, United States), as a loading control. Three independent experiments were conducted.
Database Search In peptide mass fingerprint map database searching, Mascot Distiller was used to obtain the monoisotopic peak list from the raw mass spectrometry files. Peptide matching and protein searches against the Swiss-Prot database were performed by using the Mascot search engine (http://www.matrixscience.com/) with a mass tolerance of 50 ppm. Protein scores of >64 (threshold) indicate identity or extensive homology (P < .05) and were considered significant. In the tandem mass spectrometry data database query, the MS/MS data (PKL format file) was imported into the Mascot search engine with a MS/MS tolerance of 0.3 Da to search the Swiss-Prot database. For the protein matching of 2 unique peptides, individual ion scores of >28 (threshold) indicate identity or extensive homology (P < .05) and were considered significant. For the protein matching only 1 unique peptide, individual ion scores of >35 (threshold) indicate identity or extensive homology (P < .05) and were considered significant.
RESULTS Screening for HIRI-Octreotide Associated Proteins by 2-DE
A comparative proteomic study of the IR, IRþOct, and control rabbits was conducted to identify proteins involved in the octreotide-associated HIRI process. Two representative, high-resolution 2-dimensional maps (pH 3e10 gradient) from liver tissues of 3 groups were demonstrated in Fig 1. In total, 1012 29, 1007 34, and 1019 26 protein spots were separated and visualized respectively. The match rates were 92.8%, 94.2%, and 91.6%. We analyzed and compared quantitatively 2-DE using PDQuest image analysis
Fig 1. Representative 2-dimensional electrophoresis (2-DE) maps (pH 3e10 gradient) in 3 groups: (A) sham operative group (control); (B) ischemia/reperfusion group (IR); and (C) the ischemia/ reperfusion þ octreotide group (IRþOct).
YANG, SUN, GUAN ET AL
and found 31 differentially expressed proteins, including the expression level of 16 proteins such as HSP70. Phosphatidylethanolamine-binding proteins (PEBPs) were up-regulated and 11 proteins were down-regulated in the IRþOct group compared with the control group. Six proteins were elevated and 4 proteins were decreased in the IRþOct group compared with the IR group. Representative protein spots identified by MALDI-TOF-MS are shown in Fig 2. Spots 5, 6, 15, 16, 20 were up-regulatedin the ischemia/ reperfusion+octreotide group than in the ischemia/reperfusion group and the control group. Proteins Identified by Using MALDI-TOF-MS
After comparing the average 2-DE maps, differential protein spots (2-fold) were detected and subjected to the analysis of MALDI-TOF MS. Then, peptide matching and protein searches against the Swiss-Prot database were performed by using the Mascot search engine. 16 differential proteins were successfully identified. The MS/MS results of spot 5 are shown in Fig 3. The database search results showed that the access number of spot 5 in Swiss-Prot database is BAB18615 and its molecular weight is 53,484. Thus, the protein spot 5 was identified as HSP70. Table 1 summarizes the additional information for all the proteins identified. These proteins were metabolism enzymes involving mitochondrial, molecular chaperone, antiapoptotic proteins and other proteins, or initiation factors involving cellular structures, signal transduction and transcript translation. Validation of HSP27 and HSP70 by Western Blot Analysis
To verify the expression of differential proteins screened by proteomic analysis, we selected 2 proteins, HSP70 (protein spot 5) and HSP27 (protein spot 30), whose expression exhibited significant differences between the IRþOct group and the control group and detected their expression level according to Western blot analysis. As demonstrated in Fig 4, the expression levels of HSP70 were significantly elevated (4.5-fold higher in the IR group and 9.3-fold higher in the IRþOct group than that in the control group). Meanwhile, the expression of HSP27 was up-regulated 7.2-fold higher in the IR group and 10.9-fold higher in the IRþOct group than in the control group. More importantly, the expression levels of HSP27 and HSP70 were promoted in the IRþOct group by
HEPATOCELLULAR PROTEIN PROFILES
3285
Fig 2. Representative discrepancy protein spots identified by matrix-assisted laser desorption/ionizationetime-of-flight (MALDI-TOF) mass spectrometer (MS). Proteins spots were zoomed and showed that the expression level of (A) spots 5, 15, 16, and (B) spots 6 and 20 were up-regulated more in the ischemia/ reperfusion þ octreotide group (IRþOct) than in the ischemia/reperfusion (IR) group and the control group.
more than 2.0- and 1.5-fold, respectively, than that in the IR group, which is consistent with the results from proteomic analysis. These findings imply that HSP27 and HSP70 may play an important role in the protection of octreotide against IR. DISCUSSION
HIRI is a complicated pathophysiological process. It could result in excessive intracellular calcium [20], increasing free oxygen radicals in activated neutrophils and Kupffer cells and apoptosis in liver parenchymal cells [21,22]. Using proteomics technology, Hirsch et al [23] identified 1559
Fig 3. Database search results of spot 5 heat-shock protein 70. (A) Peptide matching and protein searches against the Swiss-Prot database were performed by using the Mascot search engine, producing results of its peptide mass fingerprint. (B) The top score of spot 5 was 142, serial number was BAB18615; it was identified as HSP70.
proteins in an HIRI model and found that IR caused certain inflammation-related proteins, such as calgranulin, C3, myeloperoxidase, copper/zinc superoxide dismutase, and hydrogen peroxide, to be up-regulated. We also confirmed that HIRI causes hepatocyte damage, and that octreotide, a synthetic somatostatin octapeptide, may be a promising pharmacologic agent for preconditioning against HIRI [7]. In the present study, we investigated the mechanism of HIRI and the protection mechanism of octreotide by using a proteomics model. We obtained high-resolution, reproducible 2-DE maps (pH 3e10 gradient) (Fig 1) of liver tissues from the 3 groups (control, IR, and IRþOct
3286
YANG, SUN, GUAN ET AL Table 1. Differential Expression Proteins Identified by Mass Spectrometry Between the IR, IRDOct, and Control Groups
No.
Swiss-Prot Accession No.
2 3 5 6 8 9 10 13 20 21 22 23 25 29
AAH22368 Q5VWY1 BAB18615 Q8MK67_RA-BIT A29821 AAA67526 AAB58347 Q96E67 gij999892 gij21730859 gij31543380 FIHUA Q6GMY0 Q6IBM4
30 31
HHHU27 A33370
Expression Ratio
Protein Name
Molecular Weight (Da)
Scores
Function
IR:Control
IRþOct:Control
NADH-ubiquinone oxidoreductase Mitochondrial short-chain enoyl-coenzyme A Heat-shock protein 70 Phosphatidylethanolamine-binding protein HSPA5 precursor Heat-shock protein 9B (mortalin-2) Envelope glycoprotein ACTB protein Triosephosphate isomerase Superoxide dismutase RNA-binding protein Translation initiation factor eIF-5A Keratin 8 HNRPH1 protein
79,465 31,367 70 kD 21,097 72,187 70,000 70,895 40,194 26,807 22,295 21,142 16,821 53,717 49,099
161 80 128 104 139 98 130 134 106 72 79 64 97 85
Y0.4 [2.6 [2.1 [3.3 [[4.2 [2.0 Y0.4 [[4.1 [2.5 [2.2 Y0.48 Y0.43 Y0.36 Y0.45
[3.5 Y0.3 [[5.3 [[7.4 [3.6 [[5.0 Y0.3 [2.3 [[4.6 [2.8 1.6 YY0.22 1.3 YY0.22
Heat shock protein 27 Hþ-transporting 2-sector ATPase b-chain precursor
22,768 56,525
98 99
Metabolism Fatty acid b-oxidation Chaperone Anti-apoptosis Chaperone Chaperone Signal transduction Metabolism Metabolism Signal transduction Signal transduction Transcript Anti-apoptotic Ubiquinone-binding protein Molecular chaperone Hydrogen ion transporting ATP synthase
[2.1 YY0.24
[[7.2 Y0.42
Abbreviations: [, protein spots that are up-regulated >2-fold compared with the control group; [[, protein spots that are up-regulated >4-fold compared with the control group; ATP, adenosine triphosphate; Control, sham operative group; IR, ischemia/reperfusion group; IRþOct, ischemia/reperfusion þ octreotide group; RNA, ribonucleic acid.
groups) and compared 2-DE data by using PDQuest image analysis. We found 31 differentially expressed proteins, 20 up-regulated proteins in the IR group, 17 up-regulated proteins in the IRþOct group, 7 down-regulated proteins
in the IR group, and 11 down-regulated proteins in the IRþOct group. These proteins were then identified according to MALDI-TOF-MS, and a total of 16 differential expression proteins such as HSP27 and HSP70 were
Fig 4. Up-regulation of heat-shock protein (HSP) 70 and HSP27 were confirmed by Western blot analysis in the ischemia/ reperfusion þ octreotide group (IRþOct) group. (A) Western blot analysis was performed to confirm the up-regulation of HSP70 and HSP27 in response to octreotide treatment. Representative results are shown from 1 of 3 independent experiments. (B) Summary of mean band intensity of HSP70 and HSP27 from Western blot images was plotted on the right. Error bars indicate SE. Upper *P ¼ .0134, lower *P ¼ .0117 upper **P ¼ .0037, lower **P ¼ .0019, *P < .05, **P < .05. Abbreviations: Ctrl, sham operative group; IR, ischemia/reperfusion group.
HEPATOCELLULAR PROTEIN PROFILES
successfully identified. These proteins were metabolism enzymes involving mitochondria, antiapoptotic proteins, molecular chaperone, and other proteins or initiation factors involving cellular structures, signal transduction, and transcript translation (Table 1). In this study, the verified protein spots 2, 3, 13, 20, and 31 were related to material and energy metabolism. Among them, spot 2 was identified as NADH-ubiquinone oxidoreductase, which is also referred to mitochondrial precursor or NADH2 dehydrogenase (ubiquinone). It is one of the main components of the mitochondrial inner membrane respiratory chain. NADH-ubiquinone oxidoreductase is also involved in ATP synthesis by participating oxidative phosphorylation of cells as a hydrogen carrier. In addition, protein spot 20 was triosephosphate isomerase, which could catalyze glucose to 1,6 diphosphate in glycolysis. Our results showed that the expression of NADH2 dehydrogenase was significantly down-regulated in the IR group; furthermore, the extent of the down-regulation was bigger in the IRþOct group compared with the IR group. The expression of triosephosphate isomerase in the IR group was significantly upregulated; interestingly, the up-regulated magnitude was bigger in the IRþOct group relative to the IR group. This may be because, after IR injury, the ATP demand increased, and NADH2 dehydrogenase consumption also increased when liver cells made up ATP. In addition, the energy supply of liver cells was mainly produced from aerobic oxidation of tricarboxylic acid cycle in the IRþOct group, which compensated with more ATP compared with the IR group according to glycolysis. The voltage-dependent anion channels located in the outer mitochondrial membrane may control the transport of some ions and metabolites. They regulate mitochondrial-mediated cell death in 2 main ways: one, as the main components of the mitochondrial permeability transition pore, and the other through the interaction with the Bcl-2 family of proteins [24e26]. Our previous study found that serious mitochondria damage occurred in the hepatic ischemia-reperfusion process, and it was associated with expression of apoptosisrelated proteins such as Bcl-2/Bax [15]. In this study, our results showed that PEBP from spot 6 was up-regulated in the IR and IRþOct groups, and that expression of PEBP in the IRþOct group was much higher by 2.2-fold than that in the IR group (Table 1). It has been reported by Zhang et al [27] that PEBP4 could promote mouse cells transposition by inhibiting the activities of ERK1/2 and JNK and by upregulating the express of cyclooxygenase-2; thus, it could decrease cell apoptosis induced by epirubicin. Based on the function of PEBP against apoptosis, our findings suggest that the apoptosis of liver cells resulting from HIRI might be related to PEBP. More importantly, the results imply that octreotide might exert a protective effect against liver cells apoptosis from HIRI by up-regulating PEBP. We also showed that the expression levels of HSP70 and HSP27 were up-regulated in the IR group and the IRþOct group and were especially higher in the IRþOct group compared with the control group; this was according to
3287
proteomics analysis and was also verified by Western blot analysis (Fig 4). HSP is a nonsecreted protein, which is called a “molecular chaperone” involved in important physiological activities of the living cell (eg, protein translocation, folding and assembly, maintaining cell homeostasis). Consistent with our findings, HSP70 has undergone extensive research as an anti-cell damage marker in HIRI [28,29] and pharmacologic preconditioning models [30,31]. HSP 27 has also been considered as one of the anti-cell damage markers in cardiac IRI [32,33] and the kidney IRI model [34]. The elevation of HSP70 and HSP27 suggest that both might be critical modulates in response to octreotide preconditioning against HIRI for anti-cell damage. Further investigation regarding their detailed mechanisms will help us elucidate the role of octreotide against HIRI. In conclusion, the mechanism of HIRI and the protection mechanism of octreotide on HIRI are complicated, involving the influence of multiple factors. Our study found that octreotide may play an important role in the protection of HIRI through up-regulation of the expression of the antiinjury substances such as HSP70 and HSP27, significantly raising the PEBP which alleviates IR-related apoptosis, and down-regulating mitochondrial metabolic enzymes such as NADH2 dehydrogenase and triosephosphate isomerase. These findings strongly support the promise of octreotide preconditioning for liver and liver transplantation surgery and also shed some light on the protective mechanism of octreotide against HIRI. REFERENCES [1] Jaeschke H. Molecular mechanisms of hepatic ischemiareperfusion injury and preconditioning. Am J Physiol Gastrointest Liver Physiol 2003;284:G15e26. [2] Fernandez L, Heredia N, Peralta C, Xaus C, RoselloCatafau J, Rimola A, et al. Role of ischemic preconditioning and the portosystemic shunt in the prevention of liver and lung damage after rat liver transplantation. Transplantation 2003;76: 282e9. [3] Montalvo-Jave EE, Pina E, Montalvo-Arenas C, Urrutia R, Benavente-Chenhalls L, Pena-Sanchez J, et al. Role of ischemic preconditioning in liver surgery and hepatic transplantation. J Gastrointest Surg 2009;13:2074e83. [4] Niemann CU, Hirose R, Liu T, Behrends M, Brown JL, Kominsky DF, et al. Ischemic preconditioning improves energy state and transplantation survival in obese Zucker rat livers. Anesth Analg 2005;101:1577e83. [5] Fernandez L, Carrasco-Chaumel E, Serafin A, Xaus C, Grande L, Rimola A, et al. Is ischemic preconditioning a useful strategy in steatotic liver transplantation? Am J Transplant 2004;4: 888e99. [6] Oberg K, Norheim I, Theodorsson E, Ahlman H, Lundqvist G, Wide L. The effects of octreotide on basal and stimulated hormone levels in patients with carcinoid syndrome. J Clin Endocrinol Metab 1989;68:796e800. [7] McCrirrick A, Hickman J. Octreotide for carcinoid syndrome. Can J Anaesth 1991;38:539e40. [8] Chieffo C, Cook D, Xiang Q, Frohman LA. Efficacy and safety of an octreotide implant in the treatment of patients with acromegaly. J Clin Endocrinol Metab 2013;98:4047e54. [9] Glatstein M, Scolnik D, Bentur Y. Octreotide for the treatment of sulfonylurea poisoning. Clin Toxicol (Phila) 2012;50: 795e804.
3288 [10] Li J, Wang R, Tang C. Somatostatin and octreotide on the treatment of acute pancreatitisdbasic and clinical studies for three decades. Curr Pharm Des 2011;17:1594e601. [11] Sharkey AJ, Rao JN. The successful use of octreotide in the treatment of traumatic chylothorax. Tex Heart Inst J 2012;39:428e30. [12] Turkcapar N, Bayar S, Koyuncu A, Ceyhan K. Octreotide inhibits hepatic fibrosis, bile duct proliferation and bacterial translocation in obstructive jaundice. Hepatogastroenterology 2003;50: 680e3. [13] Bloechle C, Kusterer K, Konrad T, Hosch SB, Izbicki JR, Knoefel WT, et al. Rat liver injury induced by hypoxic ischemia and reperfusion: protective action by somatostatins is independent from changes in glucose metabolism. Horm Metab Res 1994;26:270e5. [14] Li JQ, Qi HZ, He ZJ, Hu W, Si ZZ, Li YN. Protective effect of octreotide on liver warm ischemia reperfusion injury. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2006;31:792e6 [in Chinese]. [15] Yang J, Sun H, Takacs P, Zhang Y, Liu J, Chang Y, et al. The effect of octreotide on hepatic ischemia-reperfusion injury in a rabbit model. Transplant Proc 2013;45:2433e8. [16] Uto H, Kanmura S, Takami Y, Tsubouchi H. Clinical proteomics for liver disease: a promising approach for discovery of novel biomarkers. Proteome Sci 2010;8:70. [17] Josic D, Hixson DC. Liver proteomics. Methods and protocols. Methods Mol Biol 2012;909:vevi. [18] Beretta L. Liver proteomics applied to translational research in liver disease and cancer. Proteomics Clin Appl 2012;4:359e61. [19] Feng XP, Yi H, Li MY, Li XH, Yi B, Zhang PF, et al. Identification of biomarkers for predicting nasopharyngeal carcinoma response to radiotherapy by proteomics. Cancer Res 2010;70: 3450e62. [20] Takemoto Y, Uchida M, Nagasue N, Ohiwa K, Kimoto T, Dhar DK, et al. Changes in calcium content of the liver during hepatic ischemia-reperfusion in dogs. J Hepatol 1994;21:743e7. [21] Zhou W, Zhang Y, Hosch MS, Lang A, Zwacka RM, Engelhardt JF. Subcellular site of superoxide dismutase expression differentially controls AP-1 activity and injury in mouse liver following ischemia/reperfusion. Hepatology 2001;33:902e14. [22] Oikawa K, Ohkohchi N, Sato M, Masamume A, Satomi S. Kupffer cells play an important role in the cytokine production and activation of nuclear factors of liver grafts from non-heart-beating donors. Transpl Int 2002;15:397e405.
YANG, SUN, GUAN ET AL [23] Hirsch J, Hansen KC, Choi S, Noh J, Hirose R, Roberts JP, et al. Warm ischemia-induced alterations in oxidative and inflammatory proteins in hepatic Kupffer cells in rats. Mol Cell Proteomics 2006;5:979e86. [24] Walz W. Chloride/anion channels in glial cell membranes. Glia 2002;40:1e10. [25] Lang F, Foller M, Lang KS, Lang PA, Ritter M, Gulbins E, et al. Ion channels in cell proliferation and apoptotic cell death. J Membr Biol 2005;205:147e57. [26] Murphy E, Imahashi K, Steenbergen C. Bcl-2 regulation of mitochondrial energetics. Trends Cardiovasc Med 2005;15:283e90. [27] Zhang Y, Wang X, Xiang Z, Li H, Qiu J, Sun Q, et al. Promotion of cellular migration and apoptosis resistance by a mouse eye-specific phosphatidylethanolamine-binding protein. Int J Mol Med 2007;19:55e63. [28] Xu X, Ye Q, Xu N, He X, Luo G, Zhang X, et al. Effects of ischemia-reperfusion injury on apolipoprotein M expression in the liver. Transplant Proc 2006;38:2769e73. [29] Dai CL, Kume M, Yamamoto Y, Yamagami K, Yamamoto H, Nakayama H, et al. Heat shock protein 72 production in liver tissue after experimental total hepatic inflow occlusion. Br J Surg 1998;85:1061e5. [30] Matsuo K, Togo S, Sekido H, Morita T, Kamiyama M, Morioka D, et al. Pharmacologic preconditioning effects: prostaglandin E1 induces heat-shock proteins immediately after ischemia/ reperfusion of the mouse liver. J Gastrointest Surg 2005;9:758e68. [31] Bedirli A, Sakrak O, Muhtaroglu S, Soyuer I, Guler I, Riza Erdogan A, et al. Ergothioneine pretreatment protects the liver from ischemia-reperfusion injury caused by increasing hepatic heat shock protein 70. J Surg Res 2004;122:96e102. [32] Chen H, Wu XJ, Lu XY, Zhu L, Wang LP, Yang HT, et al. Phosphorylated heat shock protein 27 is involved in enhanced heart tolerance to ischemia in short-term type 1 diabetic rats. Acta Pharmacol Sin 2005;26:806e12. [33] Efthymiou CA, Mocanu MM, de Belleroche J, Wells DJ, Latchmann DS, Yellon DM. Heat shock protein 27 protects the heart against myocardial infarction. Basic Res Cardiol 2004;99: 392e4. [34] Park KM, Cho HJ, Bonventre JV. Orchiectomy reduces susceptibility to renal ischemic injury: a role for heat shock proteins. Biochem Biophys Res Commun 2005;328:312e7.
ABSTRACT Hepatic ischemic/reperfusion injury (HIRI) is a major complication of liver resection and transplantation. Octreotide, a somatostatin analogue, has been used to treat hepatic fibrosis and portal hypertension; however, its function against HIRI remains unclear. To elucidate the effect of octreotide in HIRI, we investigated the hepatocellular protein profiles in response to octreotide preconditioning in a rabbit model by using proteomic analysis. Twenty-four rabbits were divided into 3 groups: the sham operative group (control), the ischemia/reperfusion group (IR), and the ischemia/reperfusion þ octreotide group (IRþOct). They were subjected to 30 minutes of normothermic ischemia followed by 120 minutes of reperfusion by using Pringle’s maneuver method. Proteomic studies were then performed to compare the protein profiles of their left liver lobe. A total of 16 differential proteins were successfully identified. These findings suggest that octreotide might exert an effect against HIRI through up-regulating the expression of the anti-injury substances, such as heat-shock proteins 70 and 27 (confirmed by using Western blot analysis); significantly raising the phosphatidylethanolamine-binding protein that alleviates IR-related apoptosis; and down-regulating mitochondrial metabolic enzymes such as NADH2 dehydrogenase and triosephosphate isomerase.
H
EPATIC ischemic/reperfusion injury (HIRI) is a major cause of morbidity and mortality after vascular exclusion in liver and liver transplantation surgery. Ischemic preconditioning, which could improve clinical outcomes after HIRI [1], is an adaptional phenomenon in which tissues are rendered resistant to the deleterious effects of ischemia/reperfusion (IR), either by previous exposure to brief periods of vascular occlusion or by the administration of certain chemical agents. Ischemic preconditioning might exert its protective effects through transient nitric oxide production, diminution of toxic reactive species, preservation of energy metabolism during ischemia, and involvement of nuclear transcription factor and others [2e5]. Octreotide, a synthetic analogue of somatostatin, was approved for use by FDA in 1988. It was primarily used to improve the management of carcinoid and VIPomas [6e8]. Since then, research studies and clinical experience have accumulated that suggest a potentially much broader therapeutic role for octreotide. Octreotide is now used for treating pituitary tumors, gastroenteropancreatic tumors, diabetes mellitus, pancreatitis, complications of pancreatic
surgery and transplantation, and short bowel syndrome [9e11]. Other indications for its use include hypercalcemia, cancer-related pain, polycystic ovary syndrome, and certain cancers. Octreotide inhibits hepatic fibrosis, bile duct proliferation, and bacterial translocation in obstructive jaundice [12]. It could cause strong hepatic glucose release even under hypoxic-ischemic conditions in rat livers with filled glycogen stores [13]. It may also protect the liver against HIRI [14]. In our previous studies, we showed that octreotide could protect the liver against HIRI in a rabbit model. Liver function can be protected and hepatocellular apoptosis decreased
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.06.071
ª 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-SA license (http:// creativecommons.org/licenses/by-nc-sa/3.0/). 360 Park Avenue South, New York, NY 10010-1710
3282
This study was funded by Hunan Provincial Science and Technology Bureau of China (06SK3079, 2007FJ3081). *Address correspondence to Jingshi Liu, MD, PhD, Department of Anesthesiology, Perioperative Medicine and Pain Management, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Tongzipo Road 283#, Yuelu District, Changsha, Hunan 410013. E-mail: [email protected]
Transplantation Proceedings, 46, 3282e3288 (2014)
HEPATOCELLULAR PROTEIN PROFILES
by reducing endotoxin production and down-regulating the inflammatory cytokines tumor necrosis factorea and interleukin-1b [15]. However, those results are not enough to clarify octreotide’s mechanism of action regarding HIRI. Proteomic studies of IR samples represent an important step toward a better understanding of the disease and may lead to identification of clinically useful markers [16]. Methodologic advances in proteomics allowed the comparison of the protein content in different IR and octreotide-treated IR samples, using gel or liquid-based approaches coupled with mass spectrometry [17]. Proteomic studies have the advantage of being able to discern changes in expression levels and help elucidate the underlying mechanism [18]. The goal of the present study was to investigate the mechanisms of HIRI and protective mechanisms of octreotide on HIRI. We evaluated the hepatocellular protein profiles after HIRI with or without octreotide preconditioning in a rabbit model and identified some potential proteins by using a proteomic-based approach.
MATERIALS AND METHODS Animals Preparation All institutional and national guidelines for the care and use of laboratory animals were followed. Twenty-four New Zealand rabbits of both sexes, weighing 1.6 to 1.9 kg, were used in this study. All animals were obtained from the Laboratory Animal Center of Xiangya Medical School of Central South University. All experimental procedures and study design were reviewed and approved by the Institutional Animal Care Committee of Xiangya Medical School of Central South University, China.
HIRI Model All rabbits were fasted for 12 hours before the experiment but were given free access to water. Twenty-four rabbits were randomly divided into 3 equal groups: the sham-operated group (control), the IR group (IR), and the IR þ octreotide pretreatment group (IRþOct). Each rabbit was anesthetized with an intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg). After the induction of anesthesia, an intravenous line was established in an ear vein and a 0.9% saline solution infusion was begun at 10 mL/kg per hour. The animal then underwent a tracheotomy and was mechanically ventilated (DH-150 Animal Ventilator, Zhejiang University Medical Apparatus Ltd., Zhejiang, China) on 100% oxygen with a tidal volume of 10 mL/kg. Anesthesia was maintained by a continuous infusion of fentanyl at 10 mg/kg per hour and intermittent administrations of 3% pentobarbital (10 mg/kg). Vecuronium was infused at 0.1 mg/kg per hour to maintain relaxation. Thirty minutes before laparotomy, the IRþOct group rabbits received an injection of octreotide (Jilin Province A-Think Pharmaceutical Co., Ltd., Jilin, China) 20 ug/kg intraperitoneally and 30 ug/kg subcutaneously. Control and IR group rabbits received the same volume isotonic saline, and all rabbits underwent a laparotomy. HIRI models were set up in the IR and IRþOct groups by using Pringle’s method [19], which consisted of ischemia for 30 minutes followed by reperfusion for 120 minutes. All animals were killed, and the left liver lobe of each rabbit was removed and fixed in liquid nitrogen and then stored at 80 C for protein profiles analysis.
3283
Two-Dimensional Electrophoresis Eighty milligrams of rabbit liver left lobe tissues of each group were lysed in 400 uL of lysis buffer (urea 7 mol/L, thiourea 2 mol/L, DTT 100 mmol/L, 4% CHAPS, Tris 40 mmol/L, 2% Pharmalyte, 1 mg/mL DNase I) at 37 C for 1 hour and then centrifuged at 15,000 rpm for 30 minutes at 4 C. The supernatant was transferred and the concentration of the total proteins was determined by using a 2-dimensional quantification kit (Amersham Biosciences, Sweden). Two-dimensional electrophoresis (2-DE) was performed to separate 800-mg protein samples, as described by Feng et al [19]. After sodium dodecyl sulfateepolyacrylamide gel electrophoresis, the protein spots were visualized by staining with colloidal Coomassie (Bio-Rad Laboratories, Minneapolis, Minn, United States). Triplicate gels were made for each cell line.
Image Analysis To minimize the contribution of experimental variations, 3 separate 2-dimensional gels were prepared for each tissue. The stained 2-dimensional gels were scanned with the MagicScan software on ImageScanner (Amersham Biosciences, Uppsala, Sweden) and were analyzed by using the PDQuest system (Bio-Rad Laboratories, California, United States). Spot intensities were quantified by calculating the spot volume after normalization of the image using the total spot volume normalization method multiplied by the total area of all the spots. An average 2-DE map for each group was constructed with the PDQuest image analysis software after spot matching, and the expression level of a protein spot was determined by its average intensity volume of 3 gels. The average 2-DE maps were used to perform the various expression analysis. The differences of the matched protein spots between the tissues was calculated by the ratio of their average expression levels, and proteins were classified as being differentially expressed between the tissues when average spot intensity was showed a difference 2-fold variation. Significant differences in protein expression levels were determined by the Student t test with a set value of P < .05.
Mass Spectrometry Analysis All the differential protein spots exhibiting consistent differences between the tissues in triplicate experiments were excised from stained gels using punch, in-gel trypsin digested. They were analyzed with a Voyager System DE-STR 4307 matrix-assisted laser desorption/ionizationetime-of-flight (MALDI-TOF) mass spectrometry (MS) to produce a peptide mass fingerprint as previously described by Feng et al [19]. The protein spots identified according to MALDI-TOF MS were also subjected to analysis of electrospray ionization quadrupole time-of-flight MS (Micromass, Waters Corporation, Milford, Mass, United States). Briefly, the samples were loaded onto a precolumn (320 mm 50 mm, 5-mm C18 silica beads; Waters Corporation) at 30 mL/min flow rates for concentrations and fast desalting through a Waters CapLC autosampler and then eluted to the reversed-phase column (75 mm 150 mm, 5 mm, 100Å; LC Packing) at a flow rate of 200 nL/min after flow splitting for separation. MS/MS spectra were performed in data-depended mode in which up to 4 precursor ions above an intensity threshold of 7 counts per second were selected for MS/MS analysis from each survey “scan.”
Western Blot Analysis Western blot analysis was performed to detect the expression of heat-shock protein (HSP) 70 and HSP27 in the frozen liver tissues, as previously described by Feng et al [19]. Briefly, 40 mg of lysates
3284 were separated by 10% sodium dodecyl sulfateepolyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Blots were blocked with 5% nonfat dry milk for 2 hours at room temperature and then incubated with 1:1000 dilution of monoclonal mouse antieHSP70 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif, United States), 1:3000 dilution of monoclonal mouse anti-HSP27 antibody (Santa Cruz Biotechnology, Santa Cruz, California, United States) for 2 hours at room temperature, and then by incubation with 1:3000 dilution of horseradish peroxidaseeconjugated secondary antibody for 1 hour at room temperature. The signal was visualized with an enhanced chemiluminescence detection reagent. b-actin was detected simultaneously by using 1:3000 dilution of monoclonal mouse antieb-actin antibody (Santa Cruz Biotechnology, Santa Cruz, California, United States), as a loading control. Three independent experiments were conducted.
Database Search In peptide mass fingerprint map database searching, Mascot Distiller was used to obtain the monoisotopic peak list from the raw mass spectrometry files. Peptide matching and protein searches against the Swiss-Prot database were performed by using the Mascot search engine (http://www.matrixscience.com/) with a mass tolerance of 50 ppm. Protein scores of >64 (threshold) indicate identity or extensive homology (P < .05) and were considered significant. In the tandem mass spectrometry data database query, the MS/MS data (PKL format file) was imported into the Mascot search engine with a MS/MS tolerance of 0.3 Da to search the Swiss-Prot database. For the protein matching of 2 unique peptides, individual ion scores of >28 (threshold) indicate identity or extensive homology (P < .05) and were considered significant. For the protein matching only 1 unique peptide, individual ion scores of >35 (threshold) indicate identity or extensive homology (P < .05) and were considered significant.
RESULTS Screening for HIRI-Octreotide Associated Proteins by 2-DE
A comparative proteomic study of the IR, IRþOct, and control rabbits was conducted to identify proteins involved in the octreotide-associated HIRI process. Two representative, high-resolution 2-dimensional maps (pH 3e10 gradient) from liver tissues of 3 groups were demonstrated in Fig 1. In total, 1012 29, 1007 34, and 1019 26 protein spots were separated and visualized respectively. The match rates were 92.8%, 94.2%, and 91.6%. We analyzed and compared quantitatively 2-DE using PDQuest image analysis
Fig 1. Representative 2-dimensional electrophoresis (2-DE) maps (pH 3e10 gradient) in 3 groups: (A) sham operative group (control); (B) ischemia/reperfusion group (IR); and (C) the ischemia/ reperfusion þ octreotide group (IRþOct).
YANG, SUN, GUAN ET AL
and found 31 differentially expressed proteins, including the expression level of 16 proteins such as HSP70. Phosphatidylethanolamine-binding proteins (PEBPs) were up-regulated and 11 proteins were down-regulated in the IRþOct group compared with the control group. Six proteins were elevated and 4 proteins were decreased in the IRþOct group compared with the IR group. Representative protein spots identified by MALDI-TOF-MS are shown in Fig 2. Spots 5, 6, 15, 16, 20 were up-regulatedin the ischemia/ reperfusion+octreotide group than in the ischemia/reperfusion group and the control group. Proteins Identified by Using MALDI-TOF-MS
After comparing the average 2-DE maps, differential protein spots (2-fold) were detected and subjected to the analysis of MALDI-TOF MS. Then, peptide matching and protein searches against the Swiss-Prot database were performed by using the Mascot search engine. 16 differential proteins were successfully identified. The MS/MS results of spot 5 are shown in Fig 3. The database search results showed that the access number of spot 5 in Swiss-Prot database is BAB18615 and its molecular weight is 53,484. Thus, the protein spot 5 was identified as HSP70. Table 1 summarizes the additional information for all the proteins identified. These proteins were metabolism enzymes involving mitochondrial, molecular chaperone, antiapoptotic proteins and other proteins, or initiation factors involving cellular structures, signal transduction and transcript translation. Validation of HSP27 and HSP70 by Western Blot Analysis
To verify the expression of differential proteins screened by proteomic analysis, we selected 2 proteins, HSP70 (protein spot 5) and HSP27 (protein spot 30), whose expression exhibited significant differences between the IRþOct group and the control group and detected their expression level according to Western blot analysis. As demonstrated in Fig 4, the expression levels of HSP70 were significantly elevated (4.5-fold higher in the IR group and 9.3-fold higher in the IRþOct group than that in the control group). Meanwhile, the expression of HSP27 was up-regulated 7.2-fold higher in the IR group and 10.9-fold higher in the IRþOct group than in the control group. More importantly, the expression levels of HSP27 and HSP70 were promoted in the IRþOct group by
HEPATOCELLULAR PROTEIN PROFILES
3285
Fig 2. Representative discrepancy protein spots identified by matrix-assisted laser desorption/ionizationetime-of-flight (MALDI-TOF) mass spectrometer (MS). Proteins spots were zoomed and showed that the expression level of (A) spots 5, 15, 16, and (B) spots 6 and 20 were up-regulated more in the ischemia/ reperfusion þ octreotide group (IRþOct) than in the ischemia/reperfusion (IR) group and the control group.
more than 2.0- and 1.5-fold, respectively, than that in the IR group, which is consistent with the results from proteomic analysis. These findings imply that HSP27 and HSP70 may play an important role in the protection of octreotide against IR. DISCUSSION
HIRI is a complicated pathophysiological process. It could result in excessive intracellular calcium [20], increasing free oxygen radicals in activated neutrophils and Kupffer cells and apoptosis in liver parenchymal cells [21,22]. Using proteomics technology, Hirsch et al [23] identified 1559
Fig 3. Database search results of spot 5 heat-shock protein 70. (A) Peptide matching and protein searches against the Swiss-Prot database were performed by using the Mascot search engine, producing results of its peptide mass fingerprint. (B) The top score of spot 5 was 142, serial number was BAB18615; it was identified as HSP70.
proteins in an HIRI model and found that IR caused certain inflammation-related proteins, such as calgranulin, C3, myeloperoxidase, copper/zinc superoxide dismutase, and hydrogen peroxide, to be up-regulated. We also confirmed that HIRI causes hepatocyte damage, and that octreotide, a synthetic somatostatin octapeptide, may be a promising pharmacologic agent for preconditioning against HIRI [7]. In the present study, we investigated the mechanism of HIRI and the protection mechanism of octreotide by using a proteomics model. We obtained high-resolution, reproducible 2-DE maps (pH 3e10 gradient) (Fig 1) of liver tissues from the 3 groups (control, IR, and IRþOct
3286
YANG, SUN, GUAN ET AL Table 1. Differential Expression Proteins Identified by Mass Spectrometry Between the IR, IRDOct, and Control Groups
No.
Swiss-Prot Accession No.
2 3 5 6 8 9 10 13 20 21 22 23 25 29
AAH22368 Q5VWY1 BAB18615 Q8MK67_RA-BIT A29821 AAA67526 AAB58347 Q96E67 gij999892 gij21730859 gij31543380 FIHUA Q6GMY0 Q6IBM4
30 31
HHHU27 A33370
Expression Ratio
Protein Name
Molecular Weight (Da)
Scores
Function
IR:Control
IRþOct:Control
NADH-ubiquinone oxidoreductase Mitochondrial short-chain enoyl-coenzyme A Heat-shock protein 70 Phosphatidylethanolamine-binding protein HSPA5 precursor Heat-shock protein 9B (mortalin-2) Envelope glycoprotein ACTB protein Triosephosphate isomerase Superoxide dismutase RNA-binding protein Translation initiation factor eIF-5A Keratin 8 HNRPH1 protein
79,465 31,367 70 kD 21,097 72,187 70,000 70,895 40,194 26,807 22,295 21,142 16,821 53,717 49,099
161 80 128 104 139 98 130 134 106 72 79 64 97 85
Y0.4 [2.6 [2.1 [3.3 [[4.2 [2.0 Y0.4 [[4.1 [2.5 [2.2 Y0.48 Y0.43 Y0.36 Y0.45
[3.5 Y0.3 [[5.3 [[7.4 [3.6 [[5.0 Y0.3 [2.3 [[4.6 [2.8 1.6 YY0.22 1.3 YY0.22
Heat shock protein 27 Hþ-transporting 2-sector ATPase b-chain precursor
22,768 56,525
98 99
Metabolism Fatty acid b-oxidation Chaperone Anti-apoptosis Chaperone Chaperone Signal transduction Metabolism Metabolism Signal transduction Signal transduction Transcript Anti-apoptotic Ubiquinone-binding protein Molecular chaperone Hydrogen ion transporting ATP synthase
[2.1 YY0.24
[[7.2 Y0.42
Abbreviations: [, protein spots that are up-regulated >2-fold compared with the control group; [[, protein spots that are up-regulated >4-fold compared with the control group; ATP, adenosine triphosphate; Control, sham operative group; IR, ischemia/reperfusion group; IRþOct, ischemia/reperfusion þ octreotide group; RNA, ribonucleic acid.
groups) and compared 2-DE data by using PDQuest image analysis. We found 31 differentially expressed proteins, 20 up-regulated proteins in the IR group, 17 up-regulated proteins in the IRþOct group, 7 down-regulated proteins
in the IR group, and 11 down-regulated proteins in the IRþOct group. These proteins were then identified according to MALDI-TOF-MS, and a total of 16 differential expression proteins such as HSP27 and HSP70 were
Fig 4. Up-regulation of heat-shock protein (HSP) 70 and HSP27 were confirmed by Western blot analysis in the ischemia/ reperfusion þ octreotide group (IRþOct) group. (A) Western blot analysis was performed to confirm the up-regulation of HSP70 and HSP27 in response to octreotide treatment. Representative results are shown from 1 of 3 independent experiments. (B) Summary of mean band intensity of HSP70 and HSP27 from Western blot images was plotted on the right. Error bars indicate SE. Upper *P ¼ .0134, lower *P ¼ .0117 upper **P ¼ .0037, lower **P ¼ .0019, *P < .05, **P < .05. Abbreviations: Ctrl, sham operative group; IR, ischemia/reperfusion group.
HEPATOCELLULAR PROTEIN PROFILES
successfully identified. These proteins were metabolism enzymes involving mitochondria, antiapoptotic proteins, molecular chaperone, and other proteins or initiation factors involving cellular structures, signal transduction, and transcript translation (Table 1). In this study, the verified protein spots 2, 3, 13, 20, and 31 were related to material and energy metabolism. Among them, spot 2 was identified as NADH-ubiquinone oxidoreductase, which is also referred to mitochondrial precursor or NADH2 dehydrogenase (ubiquinone). It is one of the main components of the mitochondrial inner membrane respiratory chain. NADH-ubiquinone oxidoreductase is also involved in ATP synthesis by participating oxidative phosphorylation of cells as a hydrogen carrier. In addition, protein spot 20 was triosephosphate isomerase, which could catalyze glucose to 1,6 diphosphate in glycolysis. Our results showed that the expression of NADH2 dehydrogenase was significantly down-regulated in the IR group; furthermore, the extent of the down-regulation was bigger in the IRþOct group compared with the IR group. The expression of triosephosphate isomerase in the IR group was significantly upregulated; interestingly, the up-regulated magnitude was bigger in the IRþOct group relative to the IR group. This may be because, after IR injury, the ATP demand increased, and NADH2 dehydrogenase consumption also increased when liver cells made up ATP. In addition, the energy supply of liver cells was mainly produced from aerobic oxidation of tricarboxylic acid cycle in the IRþOct group, which compensated with more ATP compared with the IR group according to glycolysis. The voltage-dependent anion channels located in the outer mitochondrial membrane may control the transport of some ions and metabolites. They regulate mitochondrial-mediated cell death in 2 main ways: one, as the main components of the mitochondrial permeability transition pore, and the other through the interaction with the Bcl-2 family of proteins [24e26]. Our previous study found that serious mitochondria damage occurred in the hepatic ischemia-reperfusion process, and it was associated with expression of apoptosisrelated proteins such as Bcl-2/Bax [15]. In this study, our results showed that PEBP from spot 6 was up-regulated in the IR and IRþOct groups, and that expression of PEBP in the IRþOct group was much higher by 2.2-fold than that in the IR group (Table 1). It has been reported by Zhang et al [27] that PEBP4 could promote mouse cells transposition by inhibiting the activities of ERK1/2 and JNK and by upregulating the express of cyclooxygenase-2; thus, it could decrease cell apoptosis induced by epirubicin. Based on the function of PEBP against apoptosis, our findings suggest that the apoptosis of liver cells resulting from HIRI might be related to PEBP. More importantly, the results imply that octreotide might exert a protective effect against liver cells apoptosis from HIRI by up-regulating PEBP. We also showed that the expression levels of HSP70 and HSP27 were up-regulated in the IR group and the IRþOct group and were especially higher in the IRþOct group compared with the control group; this was according to
3287
proteomics analysis and was also verified by Western blot analysis (Fig 4). HSP is a nonsecreted protein, which is called a “molecular chaperone” involved in important physiological activities of the living cell (eg, protein translocation, folding and assembly, maintaining cell homeostasis). Consistent with our findings, HSP70 has undergone extensive research as an anti-cell damage marker in HIRI [28,29] and pharmacologic preconditioning models [30,31]. HSP 27 has also been considered as one of the anti-cell damage markers in cardiac IRI [32,33] and the kidney IRI model [34]. The elevation of HSP70 and HSP27 suggest that both might be critical modulates in response to octreotide preconditioning against HIRI for anti-cell damage. Further investigation regarding their detailed mechanisms will help us elucidate the role of octreotide against HIRI. In conclusion, the mechanism of HIRI and the protection mechanism of octreotide on HIRI are complicated, involving the influence of multiple factors. Our study found that octreotide may play an important role in the protection of HIRI through up-regulation of the expression of the antiinjury substances such as HSP70 and HSP27, significantly raising the PEBP which alleviates IR-related apoptosis, and down-regulating mitochondrial metabolic enzymes such as NADH2 dehydrogenase and triosephosphate isomerase. These findings strongly support the promise of octreotide preconditioning for liver and liver transplantation surgery and also shed some light on the protective mechanism of octreotide against HIRI. REFERENCES [1] Jaeschke H. Molecular mechanisms of hepatic ischemiareperfusion injury and preconditioning. Am J Physiol Gastrointest Liver Physiol 2003;284:G15e26. [2] Fernandez L, Heredia N, Peralta C, Xaus C, RoselloCatafau J, Rimola A, et al. Role of ischemic preconditioning and the portosystemic shunt in the prevention of liver and lung damage after rat liver transplantation. Transplantation 2003;76: 282e9. [3] Montalvo-Jave EE, Pina E, Montalvo-Arenas C, Urrutia R, Benavente-Chenhalls L, Pena-Sanchez J, et al. Role of ischemic preconditioning in liver surgery and hepatic transplantation. J Gastrointest Surg 2009;13:2074e83. [4] Niemann CU, Hirose R, Liu T, Behrends M, Brown JL, Kominsky DF, et al. Ischemic preconditioning improves energy state and transplantation survival in obese Zucker rat livers. Anesth Analg 2005;101:1577e83. [5] Fernandez L, Carrasco-Chaumel E, Serafin A, Xaus C, Grande L, Rimola A, et al. Is ischemic preconditioning a useful strategy in steatotic liver transplantation? Am J Transplant 2004;4: 888e99. [6] Oberg K, Norheim I, Theodorsson E, Ahlman H, Lundqvist G, Wide L. The effects of octreotide on basal and stimulated hormone levels in patients with carcinoid syndrome. J Clin Endocrinol Metab 1989;68:796e800. [7] McCrirrick A, Hickman J. Octreotide for carcinoid syndrome. Can J Anaesth 1991;38:539e40. [8] Chieffo C, Cook D, Xiang Q, Frohman LA. Efficacy and safety of an octreotide implant in the treatment of patients with acromegaly. J Clin Endocrinol Metab 2013;98:4047e54. [9] Glatstein M, Scolnik D, Bentur Y. Octreotide for the treatment of sulfonylurea poisoning. Clin Toxicol (Phila) 2012;50: 795e804.
3288 [10] Li J, Wang R, Tang C. Somatostatin and octreotide on the treatment of acute pancreatitisdbasic and clinical studies for three decades. Curr Pharm Des 2011;17:1594e601. [11] Sharkey AJ, Rao JN. The successful use of octreotide in the treatment of traumatic chylothorax. Tex Heart Inst J 2012;39:428e30. [12] Turkcapar N, Bayar S, Koyuncu A, Ceyhan K. Octreotide inhibits hepatic fibrosis, bile duct proliferation and bacterial translocation in obstructive jaundice. Hepatogastroenterology 2003;50: 680e3. [13] Bloechle C, Kusterer K, Konrad T, Hosch SB, Izbicki JR, Knoefel WT, et al. Rat liver injury induced by hypoxic ischemia and reperfusion: protective action by somatostatins is independent from changes in glucose metabolism. Horm Metab Res 1994;26:270e5. [14] Li JQ, Qi HZ, He ZJ, Hu W, Si ZZ, Li YN. Protective effect of octreotide on liver warm ischemia reperfusion injury. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2006;31:792e6 [in Chinese]. [15] Yang J, Sun H, Takacs P, Zhang Y, Liu J, Chang Y, et al. The effect of octreotide on hepatic ischemia-reperfusion injury in a rabbit model. Transplant Proc 2013;45:2433e8. [16] Uto H, Kanmura S, Takami Y, Tsubouchi H. Clinical proteomics for liver disease: a promising approach for discovery of novel biomarkers. Proteome Sci 2010;8:70. [17] Josic D, Hixson DC. Liver proteomics. Methods and protocols. Methods Mol Biol 2012;909:vevi. [18] Beretta L. Liver proteomics applied to translational research in liver disease and cancer. Proteomics Clin Appl 2012;4:359e61. [19] Feng XP, Yi H, Li MY, Li XH, Yi B, Zhang PF, et al. Identification of biomarkers for predicting nasopharyngeal carcinoma response to radiotherapy by proteomics. Cancer Res 2010;70: 3450e62. [20] Takemoto Y, Uchida M, Nagasue N, Ohiwa K, Kimoto T, Dhar DK, et al. Changes in calcium content of the liver during hepatic ischemia-reperfusion in dogs. J Hepatol 1994;21:743e7. [21] Zhou W, Zhang Y, Hosch MS, Lang A, Zwacka RM, Engelhardt JF. Subcellular site of superoxide dismutase expression differentially controls AP-1 activity and injury in mouse liver following ischemia/reperfusion. Hepatology 2001;33:902e14. [22] Oikawa K, Ohkohchi N, Sato M, Masamume A, Satomi S. Kupffer cells play an important role in the cytokine production and activation of nuclear factors of liver grafts from non-heart-beating donors. Transpl Int 2002;15:397e405.
YANG, SUN, GUAN ET AL [23] Hirsch J, Hansen KC, Choi S, Noh J, Hirose R, Roberts JP, et al. Warm ischemia-induced alterations in oxidative and inflammatory proteins in hepatic Kupffer cells in rats. Mol Cell Proteomics 2006;5:979e86. [24] Walz W. Chloride/anion channels in glial cell membranes. Glia 2002;40:1e10. [25] Lang F, Foller M, Lang KS, Lang PA, Ritter M, Gulbins E, et al. Ion channels in cell proliferation and apoptotic cell death. J Membr Biol 2005;205:147e57. [26] Murphy E, Imahashi K, Steenbergen C. Bcl-2 regulation of mitochondrial energetics. Trends Cardiovasc Med 2005;15:283e90. [27] Zhang Y, Wang X, Xiang Z, Li H, Qiu J, Sun Q, et al. Promotion of cellular migration and apoptosis resistance by a mouse eye-specific phosphatidylethanolamine-binding protein. Int J Mol Med 2007;19:55e63. [28] Xu X, Ye Q, Xu N, He X, Luo G, Zhang X, et al. Effects of ischemia-reperfusion injury on apolipoprotein M expression in the liver. Transplant Proc 2006;38:2769e73. [29] Dai CL, Kume M, Yamamoto Y, Yamagami K, Yamamoto H, Nakayama H, et al. Heat shock protein 72 production in liver tissue after experimental total hepatic inflow occlusion. Br J Surg 1998;85:1061e5. [30] Matsuo K, Togo S, Sekido H, Morita T, Kamiyama M, Morioka D, et al. Pharmacologic preconditioning effects: prostaglandin E1 induces heat-shock proteins immediately after ischemia/ reperfusion of the mouse liver. J Gastrointest Surg 2005;9:758e68. [31] Bedirli A, Sakrak O, Muhtaroglu S, Soyuer I, Guler I, Riza Erdogan A, et al. Ergothioneine pretreatment protects the liver from ischemia-reperfusion injury caused by increasing hepatic heat shock protein 70. J Surg Res 2004;122:96e102. [32] Chen H, Wu XJ, Lu XY, Zhu L, Wang LP, Yang HT, et al. Phosphorylated heat shock protein 27 is involved in enhanced heart tolerance to ischemia in short-term type 1 diabetic rats. Acta Pharmacol Sin 2005;26:806e12. [33] Efthymiou CA, Mocanu MM, de Belleroche J, Wells DJ, Latchmann DS, Yellon DM. Heat shock protein 27 protects the heart against myocardial infarction. Basic Res Cardiol 2004;99: 392e4. [34] Park KM, Cho HJ, Bonventre JV. Orchiectomy reduces susceptibility to renal ischemic injury: a role for heat shock proteins. Biochem Biophys Res Commun 2005;328:312e7.