J Gastrointest Surg (2011) 15:1679–1688 DOI 10.1007/s11605-011-1648-x
2011 SSAT POSTER PRESENTATION
Ischemic Preconditioning-Like Effect of Polyunsaturated Fatty Acid-Rich Diet on Hepatic Ischemia/Reperfusion Injury Ana Maria Mendonça Coelho & Marcel Cerqueira Cesar Machado & Hilton Kenji Takahashi & Sandra N Sampietre & José Tadeu Stefano & Andre Zonetti A. Leite & Rui Curi & Luiz A. Carneiro D’Albuquerque
Received: 25 March 2011 / Accepted: 26 July 2011 / Published online: 9 August 2011 # 2011 The Society for Surgery of the Alimentary Tract
Abstract Aim The aim of this study was to investigate a possible preconditioning effect of oral diet enriched with polyunsaturated fatty acids (PUFAs) on liver ischemia/reperfusion (I/R) injuries. Methods Wistar male rats were fed a standard diet or polyunsaturated fatty acid-rich diet (PRD) enriched with (GII) or without (GIII) ω-3 PUFA. Rats were submitted to partial liver ischemia during 1 h and evaluated in pre- and post-I/R conditions. In pre-I/R condition, livers were collected for determination of fatty acid composition, liver mitochondrial function, malondialdehyde (MDA) content, and histological analysis. Four hours after liver reperfusion serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), serum levels of tumor necrosis factor-alpha, interleukin-6, interleukin-10, and prostaglandin-E2, liver mitochondrial function, MDA content, and histology were evaluated. Results In the pre-I/R condition, GII and GIII groups had an increase on PUFA content and exhibited slight increased macrosteatosis and microsteatosis in the liver. After 4 h of reperfusion, PRD-fed rats showed a marked decrease on steatosis, diminished necrosis, an increase in MDA formation, and mitochondrial uncoupling. We also observed a marked decrease in plasma levels of cytokines and ALT and AST activities in post-I/R condition in PRD groups. Conclusion In this experimental model in the rat, PRD has a preconditioning effect protecting the liver from I/R injury and should be object of future clinical studies. Keywords Liver ischemic/reperfusion lesion . Preconditioning . Polyunsaturated fatty acid-rich diet . Hepatic mitochondrial dysfunction
This work was presented at the 52nd Annual Meeting of the Society for Surgery of the Alimentary Tract on May 2011, in Chicago, IL. A. M. M. Coelho : M. C. C. Machado (*) : S. N. Sampietre : J. T. Stefano : A. Z. A. Leite : L. A. C. D’Albuquerque Department of Gastroenterology (LIM/37-LIM/07), Medical School, University of Sao Paulo, R. Peixoto Gomide, 515 13 andar, 01409001 Sao Paulo, Sao Paulo, Brazil e-mail:
[email protected] H. K. Takahashi : R. Curi Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil
Introduction Ischemia is a condition caused by partial or absolute blockage of blood flow through an organ, which results in relative deficiency of oxygen supply. Reperfusion and restoration of oxygen supply, paradoxically, aggravate this condition, causing ischemia/reperfusion (I/R) injury.1–3 Liver I/R injury occurs in several clinical situations such as hemorrhagic shock, hepatic resection, liver transplantation, and in multiple organ failure. Injury in liver arises as a result of multiple pathophysiological processes.4,5 Oxygen deprivation in liver leads to metabolic imbalance with mitochondrial dysfunction and energy deficiency. Hepatocyte and endothelial cells swelling after reperfusion contribute to narrowing sinusoidal blood vessels, leukocyte entrapment, and platelet aggregation, resulting in occlusion and failure of hepatic microcirculation. The subsequent inflammatory response with activation of macrophages,
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neutrophils, and Kupffer cells, which produce several mediators such as reactive oxygen species (ROS), proteases, and cytokines, causes further cell damage.6 Thus, mitochondrial dysfunction and energy deficiency early in the ischemic phase trigger a chain of deleterious pathophysiological responses, ultimately causing hepatocyte death and liver dysfunction.7 Some studies have suggested the ischemic preconditioning may ameliorate hepatic I/R.8–11 The concept of preconditioning was introduced in 1961 to describe a pathological stimulus that occurs below the threshold of definitive lesion that leads to a protective effect.12 Previous studies have demonstrated that in heart and in brain, preconditioning is associated with a moderate uncoupling of mitochondria respiratory chain.13,14 Previous study reported that a lipid emulsion (Intralipid) raises hepatocyte uncoupling protein 2 productions that may have a protective effect on hepatocyte by inhibiting mitochondrial production of ROS.15 In spite of the information above, the effect of polyunsaturated fatty acid (PUFA)-rich diets on liver I/R injury has not been extensively investigated. The aim of this study was to investigate the preconditioning effect of a PUFA-rich diet on liver injury of rats submitted to ischemia/ reperfusion. Mitochondrial function was evaluated by mitochondrial oxidation and phosphorylation activities in rat liver under ischemia/reperfusion injury. The degree of the liver lesion was estimated by measuring serum activities of aspartate aminotransferase and alanine aminotransferase and by tissue histological analysis. The inflammation state of the rats was evaluated by measuring the serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and prostaglandin-E2 (PGE2).
Material and Methods Treatment of Animals Male Wistar rats weighing 180–200 g were housed in cages with a controlled 12 h-light/dark cycle with free access to a standard chow and water for 1 week. Rats were then randomly divided into three groups: GI (n =20) that received standard diet (SD) containing 4% soybean oil, 23% protein, and 62% carbohydrate (Nuvital, Brazil); GII (n=20) rats were fed a polyunsaturated fatty acid-rich diet (PRD) containing 27% soybean oil enriched with 1% codfish liver oil, 23% protein, and 38% carbohydrate; and GIII (n=20) rats were fed a PRD without codfish liver oil for 4 weeks. Animal weights were not different among the groups after 4 weeks, ranging from 250 to 280 g. This study was designed in accordance with the Guidelines for the Care
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and Use of Laboratory Animals.16 The experimental protocol was approved by the Ethics Committee of the University of São Paulo. Surgical Procedure and Sample Collection The rats were submitted to the following experimental protocols: Pre-ischemia/reperfusion condition (pre-I/R): ten rats from each group were killed without being submitted to surgery. Rats were anesthetized for blood sampling through cardiac puncture and killed by exsanguination. Liver tissue was collected for biochemical and histological examination. Ischemia/reperfusion condition (post-I/R): ten rats from each group were submitted to partial liver ischemia. Animals underwent 1 h of warm liver ischemia followed by reperfusion (I/R). The animals were anesthetized with intraperitoneal ketamine (30 mg/kg) and xylazine (30 mg/kg) and submitted to orotracheal intubation, and ventilated with a tidal volume of 0.08 ml/g body weight, at a respiratory rate of 60/min, and FiO2 of 0.21 (Small Animal Ventilator model 683, Harvard Apparatus, Holliston, MA, USA). During the surgical procedure, body temperature was monitored using a rectal digital thermometer (YSI Precision 4000A Thermometer, USA), being maintained at 37°C. Median laparotomy was performed and the hepatic pedicle of median and left anterolateral segments were dissected, exposed, and clamped with a nontraumatic microvascular bulldog clamp during 1 h that induces ischemia to 70% of the total liver volume. In this model, intestinal congestion is avoided allowing the possibility to study the effects of isolated liver ischemia. The incision was closed, and after a 60-min ischemic period, the abdomen was reopened allowing clamp removal and liver reperfusion.17,18 At 4 h after liver reperfusion, rats were re-anesthetized for blood sampling through cardiac puncture and killed by exsanguination. The liver tissue for post-ischemic analysis was obtained from median and left anterolateral segments previously submitted to I/R injury and collected for biochemical and histological examination. No mortality is observed in this model of partial liver ischemia. Determination of Liver Fatty Acid Composition Total lipids from the liver of pre-ischemia/reperfused rats were extracted as previously described by Folch et al.19 The lipids were saponified using 2 ml of an alkaline methanol solution (1 mol per ml NaOH solution in 90% methanol), at 37°C, for 2 h, in a shaking water bath. Afterwards, the solution was acidified to pH 3 with HCl (1 mol/ml). Fatty
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acids were then extracted three times with 2 ml hexane. After extraction procedure and saponification, the fatty acids were derivatized with 4-bromomethyl-7 coumarin,20 and the analysis carried out in a liquid chromatographer (Shimadzu model LC-10A, Shimadzu, Kyoto, Japan). The samples were eluted using a C8 column (25 cm×4.6 i.d., 5 μm of particles) with C8 pre-column (2.5 cm×4.6 i.d., 5 μm of particles), 1 ml per minute of acetonitrile/water (77:23 v/v) flow and fluorescence detector (325 nm excitation and 395 emission). Liver Mitochondrial Oxidation and Phosphorylation Activities Liver mitochondria were prepared as previously described.21 Briefly, rat livers were rapidly excised and placed in medium containing 250 mM sucrose, 10 mM Tris–HCl, and 1 mM EGTA, pH 7.3, at 4°C. The tissue was scissor-minced and homogenized in ice using a Teflon Potter homogenizer. The homogenate was centrifuged at 600×g for 10 min. The supernatant was centrifuged for 10 min at 10,000×g to obtain the mitochondrial pellet. Mitochondrial suspension containing 30–40 mg/ml of mitochondrial protein was prepared and stored on ice before the assay of mitochondrial respiration. The mitochondrial oxygen consumption was polarographically22 measured using a Gilson 5/6H Oxygraph (Gilson Medical Electronics, Inc., Middleton, WI) in a closed reaction vessel fitted with a Clark oxygen electrode (Yellow Springs Instruments Co., Yellow Springs, OH) at 28°C. The incubation medium consisted of 120 mM KCl, 2 mM sodium phosphate, 10 μM rotenone, and 1 mM EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′tetraacetic acid) and was buffered at pH 7.3 with 5 mM Tris–HCl. Mitochondria were energized with potassium succinate as substrate at a final concentration of 10 mM. After a brief equilibration period, state 3 (activated state, S3) respiration was induced by the addition of 280 nmol adenosine diphosphate (ADP). The added ADP was phosphorylated to adenosine triphosphate (ATP), and the state 4 (basal state, S4) respiration was then measured. The oxygen consumption ratio in the presence of ADP to that in absence (respiratory control rate, RCR) and the ADP/O ratio were calculated as indices of mitochondrial oxidation and phosphorylation activities.23 RCR ADP/O
oxygen consumption in the S3/oxygen consumption in the S4 moles of ATP formed from ADP per atom of oxygen consumed
S3 and S4 were measured and reported as nmol oxygen per milligram mitochondrial protein per minute.
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Mitochondria protein content was determined by the method of Lowry et al.24 Serum Activities of Aspartate Aminotransferase and Alanine Aminotransferase The extension of hepatocellular injury was assessed by measuring serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The enzyme activities were assayed by using the optimized ultraviolet method (COBAS MIRA) from Roche (Roche Diagnostics, Rotkrenz, Switzerland). Results are expressed as units per liter (U/l). Lipid Peroxidation Analysis Malondialdehyde (MDA) formation was used as indicative of the occurrence of lipid peroxidation in the tissues and was estimated as thiobarbituric acid-reactive substances (TBARS). Liver tissues (100 mg/ml) were homogenized in 1.15% KCl buffer and centrifuged at 14,000×g for 20 min. An aliquot of the supernatant was then added to a reaction mixture consisting of 1.5 ml 0.8% thiobarbituric acid, 200 μl 8.1% (v/v) sodium dodecyl sulfate, 1.5 ml 20% acetic acid (pH 3.5), and 600 μl distilled water. The mixture was then heated at 90°C for 45 min. After cooling to room temperature, the samples were cleared by centrifugation (10,000×g for 10 min), and the absorbance was measured at 532 nm using malondialdehyde bis (dimethyl acetal) as external standard. The content of lipid peroxides was expressed as nmol MDA per mg of protein.25 Determination of Inflammatory Mediators Serum levels of TNF-α, IL-6, and IL-10 from both pre- and post-I/R conditions were determined by ELISA using commercial kits (Biosource International Cytoscreen, Camarillo, CA, USA). PGE2 was also determined by ELISA using Cayman Chemical kit (MI, USA). Histological Analysis of the Liver from Pre- and Post-I/R Conditions Liver samples were fixed in 10% buffered formalin for standard hematoxylin and eosin staining. Histological evaluation of the liver sections was performed by the same pathologist in a blinded manner. The severity of histological injury was analyzed according to the scoring system proposed by Quireze et al.26 The following features were considered: hepatocellular steatosis (micro- and macrosteatosis), cellular necrosis, and tumefaction. Each feature was assigned with score from 0 to 3 based on its absence (0) or presence to a mild (1), moderate (2), or severe (3)
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degrees. The intensity of steatosis was semiquantitatively evaluated: absence (0); low steatosis, 5% to 15% (1); mild steatosis, 16% to 30% (2); moderate steatosis, 31% to 60% (3); and severe steatosis, > 60% (4).27
analysis was performed by the Mann–Whitney test, and results were presented as median and range. The level of p < 0.05 was considered statistically significant. The GraphPad Prism Software (GraphPad Software, San Diego, CA) was used for statistical analysis.
Determination of Glucose-6-Phosphate Content Liver samples were homogenized in 0.6 N perchloric acid and the glucose-6-phosphate content was analyzed as described by Lang and Michal.28
Results Effect of the PRD Diet on Liver Fatty Acid Composition in Pre-I/R Condition
Determination of Glycogen Content For determination of glycogen content, liver samples were digested for 1 h in 1M-KOH at 70°C. The digest was acidified with glacial acetic acid to pH 4.8. Amyloglucosidase (in acetate buffer, pH 4.8) was added, and the samples were incubated at 40°C for 2 h to allow complete degradation of glycogen to glucose. The glucose concentration was measured enzymatically using a Bioclin’s Glucose Reagent Kit (Bioclin, São Paulo, Brazil)
Effect of PRD Diet on Mitochondrial Uncoupling in Pre- and Post-I/R Conditions
Statistical Analysis Continuous variables (fatty acid composition, liver mitochondrial function, MDA content, activities of AST and ALT, TNFα, IL-6, IL-10, and PGE2, glucose-6-phosphate, and glycogen content) were compared using Student’s t test, and results were presented as mean values±SEM. Histological
Table 1 Fatty acid composition (percentage) in liver from rats fed the standard and HFD (PUFA-rich) diets
GI refers to the standard diet, GII refers to PRD enriched with ω-6 and ω-3 PUFA, and GIII refers to PRD enriched with ω-6 PUFA. Data are expressed as mean±SEM of seven animals per group *p
