Identification of dihalogenated proteins in rat intestinal mucosa injured by indomethacin

Abstract

Previous studies have shown that activated neutrophils and their myeloperoxidase (MPO)-derived products play a crucial role in the pathogenesis of non-steroidal anti-inflammatory drug (NSAID)-related small intestinal injury. The aim of the present study is to identify dihalogenated proteins in the small intestine on indomethacin administration. Intestinal damage was induced by subcutaneous administration of indomethacin (10 mg/kg) in male Wistar rats, and the severity of the injury was evaluated by measuring the area of visible ulcerative lesions. Tissue-associated MPO activity was measured in the intestinal mucosa as an index of neutrophil infiltration. The dihalogenated proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using novel monoclonal antibodies against dibromotyrosine (DiBrY), and they were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) peptide mass fingerprinting and a Mascot database search. Single administration of indomethacin elicited increased ulcerative area and MPO activity in the small intestine. 2D-PAGE showed an increased level of DiBrY-modified proteins in the indomethacin-induced injured intestinal mucosa and 6 modified proteins were found. Enolase-1 and albumin were found to be DiBrY modified. These proteins may be responsible for the development of neutrophil-associated intestinal injury induced by indomethacin.

Hemopexin is upregulated in rat intestinal mucosa injured by indomethacin

“…These findings are consistent with the results from previous reports of indomethacin-induced intestinal injury in rodents. 13,14 Furthermore, we performed 2D-PAGE to identify the upregulated proteins in the mucosa injured by indomethacin and confirmed that the expression of HPX was altered (Table 1). Thus, the proteomic approach offers many opportunities and challenges in the identification of new markers and therapeutic targets, as well as in the understanding of disease pathogenesis.…”

“…13 As shown in Figure 2a,b, the images of several spots were increased in intestinal mucosa after indomethacin treatment compared to normal intestinal mucosa. Among them, consecutive five spots located at the same molecular weight (about 70 kDa), but at different isoelectric points were found (Fig.…”

“…11 Furthermore, we have also identified the posttranslational oxidative modified protein involved in the pathogenesis of gastric ulcer and intestinal inflammation using a proteomic approach. 12,13 The aim of the present study was to identified the proteins associated with indomethacin-induced intestinal injuries in rats by the 2D-DIGE/MALDI-TOF analysis.…”

The Role of Methylglyoxal-Modified Proteins in Gastric Ulcer Healing

“…To date, the most consistently successful proteomic methodology is the combination of 2D-PAGE followed by MS protein identification using peptide mass fingerprints and tandem MS peptide sequencing. We have also identified several novel disease markers and therapeutic targets in esophagitis [71], H. pylori-infected gastric mucosa [72], indomethacin-induced intestinal injury [73], inflammatory bowel disease [74][75][76], and gastrointestinal cancer [60].…”

Nonsteroidal anti-inflammatory drug-induced visible and invisible small intestinal injury

“…Recruitment of neutrophils and their myeloperoxidase activity ultimately induce inflammation and ulceration. (6,7) …”