Animals

A total of 24 male Wistar albino rats (weighing 200–250 g, 12 weeks of age) were supplied by the Experimental Research Center of Ondokuz Mayis University. All of the animals were housed under controlled environmental conditions (21°C ± 1°C, 40%–70% humidity, 12/12 dark–light cycle, standard rat diet and water). The experimental protocol was approved by the Ethics Committee for Experimental Animal Studies of Ondokuz Mayis University.

Experimental groups

All surgical procedures were conducted under anesthesia after intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). Dexketoprofen was supplied as pure powder by Menarini Ricerchere S.P.A. Sede e Laboratory, Pomezia, Rome. Powder dexketoprofen trometamol was prepared in 25 mg/mL aliquots with physiologic serum (SF). Before surgery, the ventral abdomen was shaved and cleaned with 10% povidone iodine. The 24 rats were divided into 4 groups to undergo 1-hour ischemia and different subsequent reperfusion intervals, as described below.

Dexketoprofen was administered (25 mg/kg, intraperitoneally) 15 minutes before induction of ischemia. One-hour reperfusion (group DIR1, n = 6) and 6-hour reperfusion (group DIR6, n = 6) were produced by removing the vascular clamps after 1 hour of partial hepatic ischemia.

Physiologic serum (1 mL/kg) was administered to the control groups 15 minutes before induction of ischemia. One-hour reperfusion (group IR1, n = 6) and 6-hour reperfusion (group IR6, n = 6) were produced by removing the vascular clamps after 1 hour of partial hepatic ischemia.

After SF or dexketoprofen administration, a midline laparotomy was performed, and a rat model of 70% hepatic ischemia was achieved by occluding the portal circulation with a traumatic vascular clamp to the median and left lateral lobs of the liver, as described by Lin et al.²³ Reperfusion was produced by removing the vascular clamps after 1 hour of partial hepatic ischemia. The rats were humanely killed after the reperfusion period.

Measurement of hepatic tissue and serum MDA

The hepatic tissue homogenate was prepared as described by Celik and Suzek,²⁴ with minor modifications. The tissues were homogenized for 5 minutes in 50 mM ice-cold KH₂PO₄ solution (1:5 wt/vol) using a Dounce homogenizer (Sigma-Aldrich Co, LLC, St Louis, Missouri) and then centrifuged at 2142g for 20 minutes at 4°C.

A 0.5-mL aliquot of supernatant was added to 2.5 mL 20% trichloroacetic acid and 1 mL 67% thiobarbituric acid in a 10-mL centrifuge tube. The mixture was then incubated at 90°C for 30 minutes in a water bath, after which the tubes were rapidly cooled to stop the reaction. Following the addition of 4 mL n-butanol, the mixture was vortexed and centrifuged. The supernatants (20 μL) were injected into a high-performance liquid chromatography - fluorescence detection (HPLC-FLD) (Ex: 515 nm; Em: 553 nm) system. Serum MDA levels were measured according to the method described by Yoshioka et al²⁵ and Agarwal and Chase.²⁶

Measurement of serum aspartate aminotransferase and alanine aminotransferase

The blood samples were centrifuged at 3000g at 4°C for 10 minutes. The serum of the centrifuged blood samples was transferred into microcentrifuge tubes and stored at −80°C until enzyme analyses were performed. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) measurements were performed by the endpoint method using an autoanalyzer (Autolab, AMS Srl, Selective Access, Saba, The Netherlands) and commercial kits (Audit Diagnostics, Carrigtwohill, Ireland) according to the manufacturers' instructions, as previously described.²⁷

Measurement of serum leptin

The rat serum leptin levels were detected using a commercial enzyme-linked immunosorbent assay kit.²⁸

Histopathologic examination

Fresh portions of the left and median lobes of the liver of each rat were excised rapidly, fixed in 10% buffered formalin, and embedded in paraffin. Sections were cut to 5-μm thickness and stained with hematoxylin and eosin for routine microscopic examination. The stained sections were examined for the presence of hydropic degeneration, necrosis, and polymorphonuclear cell infiltration. The histopathologic changes in the liver specimens were analyzed at 10 different high-power fields on a scale of 0 to 3 (none = 0, mild = 1, moderate = 2, severe = 3), as described by Demirel et al.²⁹

Statistical analysis

Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) for Windows (Version 15.0, SPSS Inc, Chicago, Illinois). All results were expressed as mean ± SD. Differences between 2 groups (e.g., IR1 versus IR6, DIR1 versus DIR6) were analyzed using an independent sample t test and a Mann-Whitney U test. A value of P ≤ 0.05 was considered statistically significant.

Serum leptin levels after IR injury

Our results demonstrated significant alterations in serum leptin levels between the 1-hour and 6-hour reperfusion groups after 1-hour partial hepatic ischemia. Serum leptin levels were significantly higher in the IR6 group than in the IR1 group (P = 0.044) without medical intervention (Fig. 1A). There were no significant differences in serum leptin levels between the 1-hour (DIR1) and 6-hour (DIR6) reperfusion groups when the rats were pretreated with dexketoprofen. There were no significant differences in serum leptin levels between the dexketoprofen-treated and the nontreated groups (IR1 versus DIR1, IR6 versus DIR6).

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Serum MDA levels and liver tissue concentration

Tissue MDA levels were significantly higher (P = 0.004) in the IR6 group than in the IR1 group; however, there was no significant difference in tissue MDA concentration between the dexketoprofen-treated groups (DIR1 versus DIR6) (Fig. 1B). In addition, there were no significant differences in serum MDA levels among the 4 reperfusion groups (IR1 versus DIR1, P = 0.873; IR6 versus DIR6, P = 0.539) (Fig. 1C).

Serum activities of AST and ALT after injury

Our results demonstrated that serum AST and ALT activities in the IR6 group were not significantly higher than in the IR1 group, and there was no significant difference between the treated groups (DIR1, DIR6). In addition, serum AST and ALT activities were significantly higher in the IR1and IR6 groups than in the DIR1and DIR6 groups (Fig. 1D and 1E).

Histopathologic observations

Liver histopathology was evaluated on the basis of neutrophil infiltration, hydropic degeneration, and necrosis.

In the IR1 group, there was no appreciable neutrophil infiltration into the liver tissue; however, we observed mild or moderate necrosis and moderate hydropic degeneration in this group. In the dexketoprofen-treated (DIR1) group, we observed only mild hydropic degeneration, and there was no appreciable necrosis or neutrophil infiltration in the liver tissue.

After 6 hours of reperfusion (IR6), neutrophil infiltration, moderate necrosis, and moderate–severe hydropic degeneration were observed in the liver tissues. Similarly, mild or moderate neutrophil infiltration, hydropic degeneration, and necrosis were observed in the dexketoprofen-treated (DIR6) group (Fig. 2).

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Discussion

IR injury is a multifactorial process that includes ischemic organ damage and inflammation-related reperfusion injury. Kupffer cells, lymphocytes, polymorphonuclear neutrophils, endothelial cells, and ROS play significant roles in the pathogenesis of liver IR injury.³⁰

Neutrophil-induced parenchymal cell injury, as described by Jaeschke, includes 3 steps: sequestration of neutrophils, transendothelial migration, and adherence-dependent cytotoxicity.³¹ Previous studies have investigated the association between hepatic IR injury and NSAIDs, such as ibuprofen³² and diclofenac.³³

In our study, hepatic parenchymal damage was assessed by measuring serum AST and ALT activities and by histopathologic evaluation. Although serum AST and ALT activities were similar in the IR1 and IR6 groups, the values obtained were higher than those of sham groups in previous studies.^34–37 Serum AST and ALT activities were decreased by the administration of dexketoprofen, indicating that it might reduce liver damage in IR injury.

The present study shows that leptin levels were not altered by the administration of dexketoprofen. To our knowledge, no previous study has reported the effect of NSAIDs on endogenous leptin levels during IR injury. Leptin, a circulating hormone produced by adipose tissue, plays a significant role in energy homeostasis, angiogenesis, inflammation,³⁸ blood pressure homeostasis,³⁹ and innate and adaptive immunity.¹⁶ Leptin is a 167 aa peptide that is structurally similar to cytokines such as IL-6, IL-11, and IL-12. It acts via the Ob-R receptor, which is structurally similar to other class I cytokine receptors, and it is expressed by immune cells: neutrophils, monocytes, and macrophages.⁴⁰ Recent studies have shown that leptin attenuates IR injury in various tissues, such as the brain,^41,42 liver,⁴³ and intestine.⁴⁴ Increased leptin levels during infection and inflammation might indicate that leptin plays a protective role in host response to inflammation.⁴⁵ Lin et al established a rat model of 70% liver IR injury²³ and found that serum leptin levels were significantly higher in a 6-hour reperfusion group than in a 4-hour reperfusion group. Similarly, our results suggest that endogenous leptin levels were elevated after 6 hours of reperfusion, but there were no significant differences in serum leptin levels between the dexketoprofen-treated and nontreated groups.

IR injury is a multifactorial process that includes complex molecular mechanisms. Activation of the inflammatory response, calcium overload, and the production of free oxygen radicals play important roles in lipid peroxidation and ultimately result in liver damage.⁴⁶ MDA is the most frequently used indicator of lipid peroxidation and indicates oxidative tissue damage.⁴⁷ In the present study, tissue MDA concentration was significantly higher during IR in the IR6 group than in the IR1 group, as demonstrated in previous studies.^41,34,48 In addition, our results showed that tissue MDA concentration was suppressed by the administration of dexketoprofen. This effect was also related to dexketoprofen-reduced neutrophil infiltration, as well as lipid peroxidation.

After dexketoprofen pretreatment, no necrosis was observed in the 1-hour reperfusion group, and intraperitoneal dexketoprofen treatment resulted in reduced hydropic degeneration, neutrophil recruitment, and necrosis in the 6-hour reperfusion group. Our results demonstrate that dexketoprofen pretreatment can reduce the histopathologic changes associated with IR injury.

In conclusion, this study focused on the effect of dexketoprofen on endogenous leptin levels and lipid peroxidation during different time points of liver IR injury. Our results suggest that dexketoprofen administration can protect the liver from IR injury by decreasing inflammation and lipid peroxidation, but there is no association between serum leptin levels and dexketoprofen. As there are insufficient studies on this subject, we suggest that further studies are required to determine leptin fluctuation during IR injury.