Neutrophils, Lipid Peroxidation, and Nitric Oxide in Gastric Reperfusion Injury in Rats

Abstract

Nitric oxide (NO) modulation of ischemia-reperfusion injury was investigated by measuring lipid peroxide and neutrophil accumulation in rat stomachs treated with N^G-nitro-l-arginine (l-NNA), a specific NO synthase inhibitor. Ischemia-reperfusion injury was induced in the rat stomach. Treatment with l-NNA for 3 days at a dose of 3 mg/kg/day significantly enhanced this injury. This enhancement was reversed by the simultaneous administration of l-arginine at a dose of 30 mg/kg/day. Both thiobarbituric acid (TBA)-reactive substances, an index of lipid peroxidation, and myeloperoxidase (MPO) activity, an index of tissue-associated neutrophil accumulation, were increased in the gastric mucosa after ischemia-reperfusion. l-NNA treatment enhanced these increases in TBA-reactive substances and MPO activity. The increase in the area of gastric erosions correlated closely with accumulation of TBA-reactive substances as well as the increase in MPO activity. Enhancement of ischemia-reperfusion injury by l-NNA treatment was inhibited by injection with anti-neutrophil antibody, anti-platelet activating factor (PAF) antagonist, and anti-leukotriene B₄ (LTB₄) receptor antagonist. In addition, the increase in TBA-reactive substances and MPO activity was decreased by these antibodies or antagonists. Enhancement of reperfusion-induced gastric mucosal injury associated with inhibition of NO synthesis may involve neutrophil infiltration and lipid peroxide accumulation in the gastric mucosa, mediated by PAF and LTB₄.

Introduction

Recent studies have documented that reactive oxygen species and lipid peroxidation are involved in the pathogenesis of gastric mucosal injuries induced by stress,1, 2 nonsteroidal anti-inflammatory drugs,3, 4 and Helicobacter pylori infection.5, 6 In 1989, we described a novel gastric mucosal injury induced by ischemia-reperfusion in rats to investigate the role of reactive oxygen species in gastric injury using a experimental model,[7] in which gastric ischemia is induced by applying and removing a vascular clamp at the celiac artery. Using this model, we documented that lipid peroxides accumulate in the gastric mucosa in parallel with the increase in the area of gastric erosions during reperfusion.[7] This observation has led to the recognition that oxygen-derived free radicals including superoxide and hydroxyl radicals are involved in the pathogenesis of this injury by initiating and promoting lipid peroxidation.8, 9 Polyunsaturated fatty acids of the cellular membranes are degraded by lipid peroxidation with subsequent disruption of membrane integrity, suggesting that lipid peroxidation mediated by oxygen radicals is an important cause of the damage and destruction of cell membranes.10, 11 As a source of oxygen radicals in this injury, xanthine oxidase in endothelial cells and NADPH oxidase present at the surface of neutrophils are considered to be important enzymes. Recent reports have demonstrated that neutrophil infiltration in the gastrointestinal mucosa via neutrophil-endothelial cell interactions play a significant role in the pathogenesis of ischemia-reperfusion-induced gastrointestinal injuries because activated neutrophils generate tissue damaging products such as reactive oxygen species, protease, collagenase, and a ferrous iron-ferritin complex.12, 13, 14

Previous reports have demonstrated that several factors, including leukotrienes,[15] platelet-activating factor (PAF),[16] nitric oxide (NO),[17] and adhesion molecules, have been implicated in lipid peroxidation or neutrophil accumulation associated with reperfusion-induced gastric mucosal injury. NO, synthesized by an NO synthase from l-arginine, is a highly reactive species that maintains the microcirculation in a state of active vasodilation,[18] prevents platelet and leukocyte adherence,19, 20 and preserves physiologic vascular impermeability.[21] Although it has been shown that exogenous NO has protective actions in the stomach against HCl-induced mucosal injury22, 23 and ischemia-reperfusion injury,[24] there are few reports investigating the involvement of endogenous NO in the development of gastric mucosal injury induced by ischemia-reperfusion, and it remains to be elucidated. Impairment of release and production of NO in endothelial cells has been shown to occur to a significant degree in postreperfusion injury.[25] In addition, superoxide anions produced by endothelial cells and neutrophils have been shown to inactivate NO; hyperoxia due to reperfusion aggravates the inactivation of NO.26, 27 Depletion of NO has been implicated as a key event in the acute inflammatory response observed in tissues exposed to ischemia-reperfusion.20, 28 This contention is supported by several studies that have shown that augmentation of NO levels during reperfusion reduces neutrophil accumulation and decreases markers of tissue injury elicited by ischemia-reperfusion,24, 28, 29 and that NO inhibition exacerbates acute injuries elicited by ischemia-reperfusion.30, 31, 32 However, there is other evidence that supports an injurious role for NO if it is present in substantial excess. Several studies have documented that pretreatment with the NO synthase inhibitor reduces ischemia-reperfusion injury in rabbit skeletal muscle,[33] isolated rabbit heart,[34] and rat cerebrum.[35]

We recently have found that prior inhibition of NO synthesis enhances gastric mucosal injury after celiac artery occlusion and reperfusion in the rat.[36] However, we did not determine which mechanisms were involved in this enhancement of reperfusion injury. The present study was designed to assess the role of lipid peroxidation and neutrophil accumulation mediated by inflammatory mediators in the enhancement of gastric mucosal injury elicited by NO inhibition during ischemia-reperfusion in rats.