Pharmacology Journal

Regardless of the cause, intestinal ischemia is a serious and growing clinical problem with an unacceptable mortality rate over 60%. When blood flow to the intestine is decreased, mucosal lesions develop in the upper part of the villi and during reperfusion, development of these lesions is rapid.

Formation of toxic oxygen metabolites has been suggested to play an important role in the development of damage during ischemia/reperfusion (I/R) injury. Oxygen radical formation results in damage to an array of biomolecules found in tissues, including nucleic acids, membrane lipids, enzymes and receptors. Membrane-associated polyunsaturated fatty acids are readily attached by reactive oxygen species in a process that results in the peroxidation of lipids. Peroxidation of membrane lipids disrupts membrane fluidity and cell compartmentation, which can result in cell lysis. Thus, oxygen radical-initiated lipid peroxidation and protein damage may contribute to the impaired cellular function and necrosis associated with reperfusion.

Another potential source of reactive oxygen species in postischaemic tissue is the polymorphonuclear leukocyte (PMN, neutrophil). Activated neutrophils adhere to microvascular endothelium and subsequently emigrate and secrete the enzyme myeloperoxidase (MPO), which catalyses the formation of reactive oxygen species.

Pentoxifylline (PTX), a methylxanthine derivative known for many years for its haemorheological properties, has proven to be a potent inhibitor of tumour necrosis factor (TNF) production. Recent reports suggest that PTX can enhance the chemotactic response of neutrophils, but may inhibit the phagocytosis and superoxide production by neutrophils and monocytes. It has also been shown that it has beneficial effects in patients with lung ischemia-reperfusion injury.

In the present study, we investigated the effect of PTX on I/R induced intestinal injury, assessing the following biomarkers: malondialdehyde (MDA), an end product of lipid peroxidation (LP); glutathione (GSH), a key antioxidant; and myeloperoxidase (MPO) activity, an index of PMN stimulation.

This study was carried out on Wistar albino rats weighing 200–250 g. After 12 h fasting periods, the rats were anaesthetized with an intraperitoneal injection of 10 mg/kg ketamin hydrochloride. After shaving the abdomen and preparation with betadine, a midline laparotomy was performed. The small bowel was exteriorized and the ligament of treitz was cut to expose the superior mesenteric artery (SMA). The SMA was dissected with microforceps and a microclamp was applied to the SMA right after its origin for occlusion. After 1 h of ischemia, the microclamp on the SMA was removed for 2 h of reperfusion. At the end of 2 h, the animals were sacrificed by cervical dislocation and ileal tissue samples were obtained.

Experimental design

The animals were divided into four groups of 10 rats.

Group 1, Sham-operated group (S): these animals underwent laparotomy and SMA dissection without I/R injury.

Group 2, Sham-operated and PTX treatment group (S + PTX): these animals received 50 mg/kg PTX via the superior vena cava and underwent sham operation.

Group 3, ischemia/reperfusion group (I/R): these animals were treated with the above mentioned procedure for I/R injury.

Group 4, I/R and PTX treatment group (I/R + PTX): these animals received 50 mg/kg PTX via the superior vena cava before I/R procedure.

Biochemical determination

The ileum samples were homogenized with ice-cold 150 mM KCl for MDA and GSH determination. The MDA content of homogenates was determined spectrophotometrically by measuring the presence of thiobarbituric acid reactive substances. Results were expressed as nmol MDA/g tissue weight. Glutathione was determined by the spectrophotometric method, which was based on the use of Ellman’s reagent. Results were expressed as μmol GSH/g tissue weight.

To determine MPO activity, the ileal tissue samples were homogenized (10% w/v) in 0.5% hexadecyltrimethylammonium bromide (HTAB) pH=6. The homogenate was centrifuged at 12000 r.p.m. for 10 min at 4 °C. The pellet was rehomogenized with 0.5% HTAB. Myeloperoxidase activity was assessed by measuring the H₂O₂-dependent oxidation of o-dianisidine. One unit of enzyme activity was defined as the amount of the MPO present that caused a change in absorbance in 1.0 min at 460 nm at 37 °C. Results were expressed in U/min.

Statistics

All data were expressed as mean ± SEM. Groups of data were compared with an analysis of variance ( ANOVA) followed by Tukey’s multiple comparison tests. Values of P < 0.05 were regarded as significant.

Filed under: Gastrointestinal