The protective effects of carnosine and melatonin in ischemia-reperfusion injury in the rat liver

Summary

The reperfusion following liver ischemia results in hepatocyte damage and apoptosis. The aim of this study was to investigate the effects of two antioxidant agents, carnosine and melatonin, in rat liver ischemia-reperfusion injury. Five study groups were formed; I. sham, II. ischemia-reperfusion, III. ischemia-reperfusion+melatonin, IV. ischemia-reperfusion+carnosine, V. ischemia-reperfusion+melatonin+carnosine. Then 250 mg/kg carnosine and 10 mg/kg melatonin were administered intraperitoneally 30 min before ischemia and immediately after the reperfusion. Sinusoidal dilatation, congestion and neutrophil infiltration were observed in the ischemia-reperfusion group while these symptoms were less pronounced in the treatment groups. Alanine aminotransferase, aspartate aminotransferase and myeloperoxidase levels were increased in the ischemia-reperfusion group while they were lowered in the treatment groups. Glutathione level was low in the ischemia-reperfusion group while it tended to increase in the ischemia-reperfusion+carnosine administered and ischemia-reperfusion+carnosine+melatonin administered groups. There was an increase in the number of apoptotic cells in the ischemia-reperfusion group while this number was lowered in the treatment groups. Carnosine was more effective than melatonin in the reversal of structural and biochemical alterations that resulted from ischemia-reperfusion injury. The administration of melatonin and carnosine together yielded better outcomes compared to the sole administration of each agent.

Introduction

Ischemia starts a series of biochemical reactions that cause cell dysfunction and may progress to cell death. The restoration of blood flow to the ischemic tissue is requisite to remove toxic metabolites produced during the ischemic period. However, severe metabolic disorders may appear due to the infusion of toxic metabolites and various inflammatory mediators that are formed during ischemia into the systemic circulation and reperfusion may cause further tissue damage (Carden and Granger, 2000). Ischemia-reperfusion damage (IRD) is the cellular damage that appears due to the restoration of oxygen delivery to the hypoxic organ. IRD in the liver was first observed in the liver transplant that was experimentally performed by Toledo-Pereyra et al. (1975). Congestion, progressive thrombosis and graft necrosis leading to organ failure developed in the transplanted liver (Bilzer and Gerbes, 2000). The pathophysiology of liver IRD consists of many mechanisms that cause liver damage. Various cellular and molecular interactions such as Kupffer cell activation, formation of reactive oxygen species (ROS), cytokine and chemokine secretion, vasoconstriction, impairment of nitric oxide and endothelin balance, accumulation of neutrophil leukocytes, alteration in mitochondrial permeability, unbalanced influx of calcium (Ca²⁺) into the cell and pH paradox may be involved in the process. These complex mechanisms cause cell death, organ dysfunction and eventually organ loss (Kaplowitz, 2000; Jaeschke, 2003; Teoh and Farrell, 2003).

Carnosine (CAR) (β-alanyl-l-histidine), first described in the 1900s, was the first discovered of all neuropeptides (Gulewitsch and Amiradzibi, 1900). Muscles and nerves are diffusely distributed in tissues. Water soluble CAR functions in the cytosol where the oxidation mediators (metals and oxygen radicals) are abundantly located. Due to its biological function of scavenging active oxygen radicals, it has an antioxidant property. It acts as a scavenger of hydroxyl and superoxide radicals and, most effectively, of the singlet oxygen molecule. CAR has been observed to have a protective effect, based on its scavenging effect on hydroxyl and superoxide radicals, in brain, kidney, liver and skeletal muscle (Boldyrev et al., 1999; Fouad et al., 2007; Fujii et al., 2003, Fujii et al., 2005; Stvolinsky et al., 1999, Stvolinsky et al., 2000; Zalesova and Kuleva, 1998).

Pineal gland is the main source of melatonin (MEL) (N-acetyl-5-metoksitriptamin) in the circulation. It is also produced in small amounts in the retina, gastrointestinal system and by leukocytes (Reiter et al., 2007). MEL is tiny (Reiter et al., 2001a, Reiter et al., 2001b; Reiter and Tan, 2004) and highly lipophilic, and for this reason, it is found abundantly in all parts of the cell. MEL protects the DNA, lipids and proteins against oxidative damage (Reiter et al., 2000, Reiter et al., 2001a, Reiter et al., 2001b; Tan et al., 1993). The free radical scavenging and antioxidant effects of MEL have been shown in many studies (Cuzzocrea et al., 2000; Reiter and Maestroni, 1999; Cabeza et al., 2001; Pei et al., 2002).

In the present study, we aimed to compare the protective effects of CAR with MEL against liver IRD through histological (hematoxylin and eosin), immunohistochemical (apostain), electron microscopical and biochemical (ALT, alanine aminotransferase; AST, aspartate aminotransferase; GSH, glutathione; MPO, myeloperoxidase) parameters.