Indomethacin and several other nonsteroidal anti-inflammatory drugs (NSAID) are known to produce gastric mucosal injury.1,2) The pathogenesis of NSAID-induced gastric mucosal injury is reported for inhibition of prostaglandin (PG) biosynthesis 3) and depletion of gastric mucosal blood flow. 4,5) A number of studies have highlighted the importance of alterations in mucosal blood flow after NSAID administration. By this depletion of gastric mucosal blood flow, polymorphonuclear leukocytes (PMN) were infiltlated to gastric mucosal tissue. 6) We clarified these phenomena that PMN infiltlates, generates oxygen radicals and induces direct gastric mucosal injury, and then increasing lipid peroxidation further aggravate damage in previous study. 7) We demonstrated that neutrophil-derived free radicals are also important factors in the gastric mucosal injury induced ischemia-reperfusion in pylorus-ligated rats.8) But the generation system of oxygen free radicals is not clear in the indomethacin induced-gastric mucosal injury. We examined this and report here the mechanism of pathogenesis in this type of injury.Superoxide radicals have several generation systems, with activation of xanthine-xanthine oxidase (XOD) enzyme in vein cellular tissue and activation of NADPH oxidase enzyme in PMN being the main ones. The direct effect of indomethacin on XOD activity in vitro and NADPH oxidase in ex vivo was therefore examined.Cytokines, IL-1 and TNF a are well known to play important roles in inflammation.9) The first step of PMN infiltration into an inflammation site is believed to take place in many cytokines and the growth binding factors IL-1 and TNF a from mast cells and macrophages. We measured cytokines to learn whether indomethacin acts to directly activate the oxygen radical generation system or is a second any effect. MATERIALS AND METHODS AnimalsMale Donryu rats (SPF) 8 weeks old were obtained from Charles River Co., Tokyo, and ICR mice (SPF) 8 weeks old were obtained from SLC Japan Co., Tokyo.Materials Indomethacin, ferricytochrome C (horse heart type III), NADPH Na (type III), NBT and BCIP were obtained from Sigma, and xanthine oxidase solution from Boehringer Mannheim Yamanouchi, Tokyo. Diphenylene iodonium chloride was from ALEXIS Co., U.S.A.; Bio-Rad protein assay was from BIO RAD; anti p47 phox was from Trunsduction Laboratories and mouse IL-1 and the TNF a assay system were obtained from Genzyme. Other chemicals were of reagent grade and were used without purification.Assay of Xanthine Oxidase (XOD) Activity XOD reduced cytochrome c was monitored spectrophotometrically at 550 nm. The reaction mixture consisted of 100 mM potassium phosphate buffer (pH 7.4) (1.2 ml), 0.1 mM cytochrome c (0.2 ml), 5 mM xanthine (0.2 ml) and 0.1 U/ml xanthine oxidase solution (0.2 ml). This mixture was preincubated for 5 min at 37°C and indomethacin was added at a fianl concentration of 0.1, 0.5, 1, 5, or 10 mM. Indomethacin was disorbed by 100% ethanol solution and finally diluted with distilled water at Ͻ1% ethanol concentration.G...