Anti-Oxidative Effect of Simvastatin in Liver and Lung Tissue after Hepatic Ischemia/Reperfusion in Rat

MATERIALS AND METHODS

Animals: Male wistar rats were purchased from Tabriz Medical University Animal Care Center and had free access to laboratory chow and water and housed in plastic cages and they were kept according to university guidelines for care and use of laboratory animals. This project was conducted from 2008 to 2009. Animals were randomly assigned into three groups of fifteen animals in each group. Group 1, the sham group, underwent laparotomy but did not experience I/R; group 2, the I/R group, underwent ischaemia for 30 min and were reperfused for 24 h; Group 3 underwent ischaemia and reperfusion like group 2 but also received 10 mg kg^-1 simvastatin dissolved in water by oral gavage for three days before the operation.

Surgery procedure: Total hepatic ischemia model performed for induction of ischemia. Rats anesthetized with ketamin (50 mg kg^-1) and xylazine (10 mg kg^-1), abdomen wall shaved and prepared with betadaine, midline incision performed and portal triad clamped for 30 min, after 30 min ischemia clamps removed and liver reperfused for 24 h, after removing the clamps the abdomen wall closed with sutures (Dibazar et al., 2008). After surgery process animals had free access to food and water. Twenty four hours after surgery animals anesthetized and liver and lung tissue excised and preserved in -20C until biochemical analysis for measuring tissue GPx, SOD and MDA level.

Assay of antioxidant enzymes: In order to measure anti-oxidant enzyme activity, the tissue samples were homogenized in 1.15% KCL solution. SOD activity in tissue was determined by using xanthine and xanthine oxidase to generate superoxide radicals which then react with 2-(4-iodophenyl)-3-(4-nitrojenol)-5-phenyltetrazolium chloride to form a red formazan dye. The SOD activity was then measured by the degree of inhibition of this reaction (Ransod, Randox Laboratories Ltd. United Kingdom). Results obtained SOD Unit/mg protein (Paoletti et al., 1986).

Gpx activity in tissue was measured using the method described by Paglia and Valentine (1967). GPx catalyses the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione reductase and NADPH, the oxidised glutathione is immediately is converted to reduced form with a concomitant oxidation of NADPH to NADP⁺. The decrease in absorbance at 340 nm is measured. (Ransod, Randox Laboratories Ltd. United Kingdom).Results obtained GPx Unit/mg protein.

Tissue MDA level: MDA levels were measured using the thiobarbituric acid reactive substances (TBARS) method (Kaya et al., 2004).

Statistical analysis: Comparison among groups was performed using one-way Analysis of Variance (ANOVA) followed by Tukey’s test. Significance was set at p<0.05; data are reported as Mean±SD.

RESULTS

Gpx and SOD: Hepatic I/R decreased GPx and SOD levels both in liver and lung tissue significantly (p<0.05, Table 1 and 2). Results showed that GPx and SOD levels were higher in simvastatin + I/R group comparing to I/R group significantly in liver tissue, 24 h after I/R induction (p<0.05, Table 1).

The results also showed that GPx and SOD levels were higher in simvastatin + I/R group comparing to I/R group significantly in lung tissue 24 h after I/R induction (p<0.05, Table 2).The levels of GPx and SOD were lower in liver tissue of rats in I/R+simvastatin group comparing to sham group(P<0.05, Table 1). The levels of GPx and SOD were lower in lung tissue of rats in I/R+simvastatin group comparing to sham group (p<0.05, Table 2).

MDA level: Hepatic I/R increased MDA level both in liver and lung significantly (p<0.05, Table 1, 2).

Table 1:	Level of GPx, SOD and MDA in rat liver tissue after 24 h hepatic I/R
The values are shown as a Mean±SD for rats in each group and difference of (p<0.05) considered significant. GPx: Glutathione peroxidase, SOD: Superoxide dismutase, MDA: Malondialdehyde

Table 2:	Level of GPx, SOD and MDA in rat lung tissue after 24 h hepatic I/R
The values are shown as a Mean±SD for rats in each group and difference of (p<0.05) considered significant. GPx: Glutathione peroxidase, SOD: Superoxide dismutase, MDA: Malondialdehyde

Measurement of MDA level indicated that its level in liver tissue was higher in I/R group than simvastatin+I/R group significantly (p<0.05, Table 1). Results also showed that MDA level in lung tissue was higher in I/R group comparing to I/R+simvastatin group significantly (p<0.05). MDA levels were higher in liver and lung tissue of rats in I/R+simvastatin group comparing to sham group significantly (p<0.05) (Table 1, 2).

DISCUSSION

The objective of this study was to evaluate the anti-oxidant effect of simvastatin administration in preventing the damage associated with liver I/R in liver and lung tissue. It was previously understood that increasing production of oxygen radicals and post ischemic damage of the liver have a two phase time course: an initial phase of injury (roughly within the first 2 h after reperfusion) and a late phase, from approximately 3 to 24 h of blood reflow (Colletti et al., 1990; De Tata et al., 2005). In reperfusion phase there is an overproduction of oxygen radicals in activated Kupffer cells (De Tata et al., 2005) and in the mitochondria of hepatocytes and endothelial cells, where oxygen reflow encounters highly reduced respiratory chains (Jaeschke et al., 1990).

Oxidative stress play an important role in inducing tissue injury after I/R. Oxidative stress is generally defined as an imbalance that favors the production of ROS over their inactivation by antioxidant defense systems; however, the precise mechanisms by which ROS produce cellular injury remain elusive. The majority of ROS are products of mitochondrial respiration. The one-electron reduction of molecular oxygen produces a relatively stable intermediate, the superoxide anion radical, which can be regarded as the precursor of most ROS in biological systems. Despite their content of various antioxidants and detoxifying enzymes, the mitochondria appear to be the most powerful intracellular source of ROS. The dismutation by superoxide dismutase results in hydrogen peroxide production. Subsequent interaction of and H₂O₂ in a Haber-Weiss reaction, or iron- or cupper-driven cleavage of H₂O₂ in a Fenton reaction, can generate the highly reactive hydroxyl radical (Orrenius et al., 2007). For attenuating devastating effects of ROS, organisms have developed a variety of antioxidant defense systems, especially the endogenous antioxidant enzymes system including SOD and GPx (Yuan et al., 2005).

MDA is a sensitive index to show lipid peroxidation. Lipid peroxidation induces both structural and functional injury to the cell organelle membranes. Reactive oxygen species-induced lipid peroxidation plays important role in the extent of tissue injury.

The overall process of lipid peroxidation consists of three stages: initiation, propagation and termination. Once formed, peroxyl adicals (ROO·) can be rearranged via a cyclisation reaction to endoperoxides (precursors of malondialdehyde) with the final product of the peroxidation process being malondialdehyde (MDA). MDA is mutagenic in bacterial and mammalian cells and arcinogenic in rats. Present results showed that simvastatin treatment decreased liver and lung tissue MDA contents significantly (Valko et al., 2006). One of the most effective intracellular enzymatic antioxidants is SOD. SOD is an important antioxidant enzyme in cells and catalyzes the conversion of superoxide ions into oxygen and hydrogen peroxide. SOD destroys O₂^•G with remarkably high reaction rates, by successive oxidation and reduction of the transition metal ion at the active site in a Ping-Pong type mechanism (Valko et al., 2006). GPx is glutathione related enzyme, which can catalyze the synthesis of GSH to ease the burden of lipid peroxidation. GPx acts in conjunction with the tripeptide glutathione (GSH), which is present in cells in high (micromolar) concentrations. The substrate for the catalytic reaction of GPx is H₂O₂, or an organic peroxide ROOH (Valko et al., 2006). Present study showed that liver and lung tissue GPx and SOD level 24 h after hepatic I/R were higher than I/R group and simvastatin maintained cellular antioxidant enzymes levels significantly.

CONCLUSION

Present results showed that simvastatin attenuated hepatic I/R induced liver and lung tissue anti-oxidant enzymes (SOD and GPx) suppression and decreased tissue MDA level as an index of oxidative injury and final product of lipid per-oxidation. As simvastatin is a safe drug and result of our previous research and also this research show the efficacy of simvastatin pretreatment in decreasing liver and lung injury after hepatic I/R, so it may be useful in attenuating hepatic I/R injury and it may be effective for performing safer hepatic surgeries.