Ischemia-Reperfusion Iloprost , a prostacyclin ( PGI 2 ) analogue , reduces liver injury in hepatic ischemia – reperfusion in rats 1 Iloprost , um análogo da prostaciclina ( PGI 2 ) , reduz danos da isquemia / reperfusão hepática em ratos

Purpose: To evaluate the effects of iloprost a prostacyclin analogue on the hepatic IR injury in rats. Methods: Forty male SpragueDawley rats (250-300 g) were divided into four groups each containing 10 rats;—(1) controls: data from unmanipulated animals; (2) sham group: rats subjected to the surgical procedure, except for liver I/R, and given saline; (3) I/R group: rats that underwent liver ischemia for 45 min followed by reperfusion for 45 min; (4) I–R/ Iloprost group: rats pretreated with iloprost (10 μg kg-1, i.v). Liver tissues were taken to determine SOD, CAT, GSH, and MDA levels and for biochemical and histological evaluation. Results: The plasma ALT and AST levels were increased in group 3 than in group 4. MDA values and the liver injury score decreased, while the SOD, CAT, and GSH values increased in group 4 compared to group 3. In group 3, hepatocytes were swollen with marked vacuolization. In group 4, there were regular sinusoidal structures with normal morphology without any signs of congestion. Conclusion: We demonstrated hepatoprotective effects of iloprost against severe ischemia and reperfusion injury in rat liver.


Introduction
Liver injuries, liver tumor resection, hemorrhagic shock with fluid resuscitation, and liver transplantation are responsible for liver injury caused by ischemia/reperfusion (I/R).Various mechanisms have been proposed to explain the mechanisms of ischemia-reperfusion (IR) injury.The implicated factors include free oxygen radicals, leukocyte migration and activation, microcirculatory abnormalities, sinusoidal endothelial cell damage, activation of the coagulation cascade, Kupffer cell activation due to the release of inflammatory cytokines, and proteolytic enzymes [1][2][3][4][5] .Data obtained by several researchers indicate that the generation of oxygen-derived free radicals is probably the most important factor involved [6][7][8] .Upon reperfusion endothelial cells produce reactive oxygen species (ROS) such as superoxide anions, hydroxyl radicals, and hydrogen peroxide in larger amounts while nitric oxide (NO) synthesis decreases significantly 9 .During the process of I/R injury, then, inflammatory reactions are activated, resulting in the formation of inflammatory cytokines, such as tumor necrosis factor-, interleukin-1,-8, platelet-activating factor and arachidonic acid metabolites 10 .
Several enzymes and drugs have been used to prevent such injury in humans and animals.However, the role of iloprost in hepatic I/R injury is unclear.Iloprost is the long-acting stable analogue of prostaglandin I 2 PGI 2 11 .PGI 2 is one of the major cyclooxygenase products of endothelial cells.It inhibits platelet aggregation, leukocyte activation, chemotaxis, and superoxide anion production; is known to be a potent vasodilator 12 and has proven to be effective in attenuating the changes in microvascular permeability, which is the final result of I/R 13 .According to these properties, this study examined the effect of iloprost during liver ischemia/ reperfusion-induced oxidative stress in rats.

Methods
Forty male Sprague-Dawley rats weighing 250-300 g were used in the study.All of the experimental protocols were performed according to the guidelines for the ethical treatment of experimental animals.

Animals and experimental protocol
The rats were housed individually in cages, and allowed free access to standard rat chow and water before and after the experiments.The animal rooms were windowless and under controlled temperature (22 ± 2ºC) and lighting conditions.The animals were fasted overnight before the experiments, but were given free access to water.They were anesthetized using 100 mg kg -1 ketamine and 20 mg kg -1 xylazine body weight, i.p.The right femoral vein was cannulated to administer drugs and saline.
The animals were randomized into four groups (n=10, each)-(1) controls: unmanipulated animals, rats not subjected to any surgical procedure or liver manipulation; (2) sham group: rats subjected to the surgical procedures described below, except for liver I/R, and administered saline vehicle and maintained under anesthesia for an equivalent duration (i.e., 45 min and 45 min); (3) I/R group: rats subjected to the surgical procedures described below that underwent liver ischemia for 45 min followed by reperfusion for 45 min (n=10); (4) I-R/Iloprost group: rats that received iloprost (10 µg kg -1 , i.v.; Ilomedin ® , Schering, Berlin, Germany) in 1ml of 0.9% NaCl solution over a period of 3 min from the tail vein 10 min before the removal of vascular microclamp.

Liver ischemia/reperfusion
As described previously 14 , the ligament attachments connecting the liver, diaphragm, abdominal wall, and neighboring organs were divided.After the organ was isolated carefully, the liver hilus was exposed to find the common hepatic artery and portal vein.A vascular microclamp was used to interrupt the blood supply to three-quarters of the liver for 45 min, and this was followed by 45 min of reperfusion.Other rats were subjected to a sham operation (sham-operated), which was identical to the surgical procedure used for the I/R group rats without clamping; the rats were kept under anesthesia for the same length of time.At the end of the experiments, the rats were killed with an overdose of sodium pentobarbital.

Measuring serum liver enzymes
The abdominal aorta was punctured and 5 ml of blood was taken and put into heparinized tubes.Plasma was separated by centrifugation (3000 rpm for 10 min at room temperature) for biochemical studies.The activities of alanine aminotransferase (ALT, a specific marker for hepatic parenchymal injury), and aspartate aminotransferase (AST, a nonspecific marker for hepatic injury) in plasma were determined in units per liter using standard auto-analyzer methods on an Abbott Aeroset (Abbott Laboratories, Abbott Park, IL, USA).Just before the rats were sacrificed, the livers were removed for histopathological evaluation.

Histopathological study
The livers were divided into two pieces.One was placed in 10% formalin solution immediately, left overnight, and then embedded in paraffin blocks.The blocks were cut in 4-µm sections and stained with hematoxylin-eosin, using standard protocols.The severity of hepatic injury in the sections was evaluated using a point-counting method on an ordinal scale as follows: grade 0, minimal or no evidence of injury; grade 1, mild injury consisting of cytoplasmic vacuolation and focal nuclear pyknosis; grade 2, moderate to severe injury with extensive nuclear pyknosis, cytoplasmic hypereosinophilia, and loss of intercellular borders; and grade 3, severe necrosis with disintegration of hepatic cords, hemorrhage, and neutrophil infiltration 15 .

Biochemical analyses
The other piece was washed in ice-cold 0.9% saline solution, weighed, and stored at -70°C.Tissue homogenates were prepared as 1.0 g 10 ml -1 in 250 mM sucrose, 1 mM EDTA, 1 mM DL-dithiothreitol, and 15 mM Tris HCl (pH 7.4), using an all-glass Potter Elvehjem homogenizer (Selecta, Barcelona, Spain).Each homogenate was centrifuged for 20 min at 800 x g.The resulting supernatant fraction was used to determine enzyme activities.The protein concentrations in the supernatant were determined using the Bradford method 16 .

Malondialdehyde determination
Liver MDA levels were determined using the method of Wasowicz et al. 17 based on the reaction of MDA with thiobarbituric acid at 95 to 100ºC.Fluorescence intensity was measured in the upper n-butanol phase using fluorescence spectrophotometry (F-4010; Hitachi, Tokyo, Japan) adjusted for excitation at 525 nm and emission at 547 nm.The arbitrary values obtained were compared with a series of standard solutions (1,1,3,3tetramethoxypropane).The results are given in nanomoles per milligram of wet tissue (nmol mg wet tissue 1 ).

Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH) determination
SOD activity was measured using the xanthine-oxidasecytochrome c method, as described by McCord and Fridovich 18 .The final concentrations in the cuvettes were 50 mM potassium phosphate (pH 7.8), 0.1 mM EDTA, 10 mM cytochrome c, 50 mM xanthine, 50 or 2 mM cyanide, 1 U catalase, and 0.05-0.1 mg of tissue.The reaction was initiated by adding 1 U xanthine-oxidase.The inhibition of xanthine-oxidase was followed spectrophotometrically at 550 nm.One unit of SOD activity was defined as the amount of enzyme that produced 50% inhibition of the control rate of cytochrome c reduction.
CAT activity was assayed according to the method of Beers and Sizer 19 .The final concentrations in the cuvettes were 500 mM potassium phosphate (pH 7), 100 mM H2O2, and 0.05-0.1 mg of tissue.The decrease in the absorbance at 240 nm after adding the substrate was followed spectrophotometrically.

Protein assays
The protein content of the homogenates was determined using the procedure of Lowry et al. 21.

Statistical analysis
Data were entered and analyzed on an IBM-compatible personal computer using SPSS version 9.0.All values were expressed as the mean ± SE.The significance of the data obtained was evaluated using analysis of variance (ANOVA).Differences between means were analyzed using the post-ANOVA test (Tukey's b); p-values less than 0.05 were considered significant.

Results
The ALT and AST levels were increased significantly in groups 3 and 4 in comparison with groups 1 and 2 (p=0.05 in all cases).However, the ALT and AST levels were decreased significantly in group 4 compared to group 3 (p<0.05)(Figure 1).The MDA, SOD, CAT, and GSH-Px values for the different groups are shown in Figure 2. In group 3, MDA significantly increased compared to groups 1, 2, and 4 (p<0.05 in all cases).In The histopathological score was 0.1 ± 0.2, 0.1 ± 0.3, 3.8 ± 0.1, and 1,3 ± 0.3 in groups 1 to 4, respectively.The histopathological score was higher in groups 3 and 4 than in groups 1 and 2 (p<0.05 in all cases).Moreover, the histopathological score was significantly lower in group 4 than in group 3 (p<0.05).
In histologic examination of the liver tissues with hema-

Discussion
The present study demonstrates that iloprost, while improving liver functions, significantly decreased the I/R induced elevations of lipid and protein oxidation, and they also maintained GSH levels.Furthermore, histologic findings also support the the protective role of iloprost.
It is widely accepted that the formation of ROS in the early phase of reperfusion plays a major role in initiating and propagating oxidative stress after reperfusion in different organs, including the liver [22][23][24] .During ischemia, cells can not keep their membrane integrity; and this causes release of calcium and phospho-lipid A 2 as well as formation of polyunsaturated fatty acids and fatty acid radicals.If oxygenation is reestablished at that stage of ischemia, fatty acid radicals react with oxygen and perform the lipid peroxidation reaction.This reaction increases membrane permeability and stimulates chemotaxis of leukocytes, which can release oxygen-derived free radicals and proteolytic enzymes when activated [25][26] .Despite their important initiative function ROS are not responsible for the whole pathophysiological process by their own namely, other inflammatory mediators originating frompostischemic tissues, such as eicosanoids, can also, contribute significantly to the pathophysiology of I/R injury 27 .Especially PGI 2 and PGE1 induce vasodilatation, inhibit platelet and leukocyte aggre-toxylin and eosin staining, we demonstrated that no morphological damage was observed in any rat in groups 1 or 2 (Figures 3A, B).In group 3, the hepatocytes were swollen with marked vacuolization and congestion was noted in the enlarged sinusoids (Figure 3C).In group 4, regular sinusoidal structures were noted with normal morphology and no signs of congestion (Figure 3D).gation, exhibit anti-inflammatory activity such as suppression of tumor necrosing factor-production and probably have direct cyto-protective effects 28 .Prostacyclin is a member of the prostaglandin family of lipid mediators and is the dominant cyclooxygenase metabolite of arachidonic acid in vascular endothelium 29 .
Iloprost, a PGI 2 analogue, mimics the pharmacodynamic properties of this compound like potent inhibition of platelet activation and aggregation, vasodilation and yet ill-defined direct cytoprotection 30 .Kawashima et al. 31 , in an isolated lung perfusion model, showed that iloprost ameliorates postischemic lung reperfusion injury.In a hind limb I/R induced experimental lung injury model, Koksela et al. 9 demonstrated that iloprost attenuates ischaemia induced remote organ reperfusion injury.Antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, remove oxygen free radicals 32 .MDA is the end product of lipid peroxidation and is a well-known parameter for determining the increased free radical formation in tissue 33,34 .In this study, we found that reperfusion injury produces oxidative stress in the liver, as shown by the increased MDA content and decreased SOD, CAT, and GSH activities.However, the mean SOD, CAT, and GSH levels increased in the I-R/ Iloprost group compared to the I/R group.This was probably related to the stimulation of PGI 2 expression by a PGI 2 analogue Iloprost, the blockage of ROS production, and a decrease in the consumption of the antioxidant enzyme.In addition, the MDA levels significantly decreased in the I-R/Iloprost group when compared to the I/R group.Ischemia/ reperfusion is also associated with the release of enzymes, e.g., ALT, and AST, which are markers of cytolysis.In this study, the biochemical parameters were better in the I-R/ Iloprost group than in the I/R group.Histological examination of the liver revealed regular sinusoidal structures in the I-R/ Iloprost group, versus swollen, markedly vacuolized cells in the I/R group.

Conclusion
The administration of iloprost prevented hepatic malfunction, inhibited the generation of free radicals, and improved hepatic microcirculatory impairment after hepatic I/R injury.These results may have important implications for the therapeutic potential of a PGI 2 analogue iloprost in treating hepatic ischemia.

FIGURE 1 -
FIGURE 1 -Effects of liver ischemia/reperfusion and iloprost on liver function.The AST (a) and ALT (b) values in the iloprost group were significantly lower than in group 3. *p<0.05compared with group 1 and 2; p<0.05 compared with group 3.The values are the mean ± SE.Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase

FIGURE 3 -
FIGURE 3 -(A, B) In groups 1 and 2, there were normal liver parenchyma with regular morphology.H&E X 200.(C) In group 3, the hepatocytes are swollen with marked vacuolization and congestion in the sinusoids H&E X 200.(D) In group 4, the hepatocytes and sinusoids show normal morphology, reflecting a well preserved liver parenchyma.H&E X 200.