Original ContributionNeutrophils, Lipid Peroxidation, and Nitric Oxide in Gastric Reperfusion Injury in Rats
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.
Section snippets
Reagents
NG-nitro-l-arginine (l-NNA) , NG-nitro-d-arginine (d-NNA), l-arginine, sodium nitroprusside (SNP) were purchased from Sigma Chemical (St. Louis, MO). Thiobarbituric acid (TBA) was obtained from Wako Pure Chemical (Osaka, Japan). 1,1,3,3-Tetramethoxy propane was obtained from Tokyo Kasei (Tokyo, Japan). Platelet activating factor (PAF) antagonist CV-3988, rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate, was provided by Takeda Chemical Industry (Osaka, Japan), and
Effects of l-NNA Treatment on Serum NO3− Levels Before and After Ischemia-Reperfusion
Fig. 1 compares the serum concentration of nitrate in samples obtained from the celiac artery before and after ischemia-reperfusion. The concentration of nitrate was 16.2 ± 1.6 μM in rats treated with d-NNA for 3 days, which decreased to 5.0 ± 0.9 μM after ischemia-reperfusion. The nitrate concentration in l-NNA-treated rats (5.36 ± 1.04 μM) significantly decreased before ischemia compared to rats treated with d-NNA, and further decreased to 2.02 ± 0.52 μM after ischemia-reperfusion.
Effects of SNA and l-NNA on Ischemia/Reperfusion-Induced Gastric Mucosal Injury
Neither d
Discussion
Extensive studies have implicated reactive oxygen species and lipid peroxidation in the model of ischemia-reperfusion used in this study.7, 8 Even though endogenous NO may serve a vital protective role during reperfusion, details remain unknown. The present study showed that treatment with SNP, an NO donor, during ischemia-reperfusion significantly reduced the reperfusion-induced gastric mucosal injury, and significantly inhibited the increase in TBA-reactive substances of the gastric mucosa,
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