Purinergic P2X7 receptor antagonist ameliorates intestinal inflammation in postoperative ileus

Abstract

Postoperative ileus (POI) is a postsurgical gastrointestinal motility dysfunction caused by mechanical stress to the intestine during abdominal surgery. POI leads to nausea and vomiting reduced patient quality of life, as well as high medical costs and extended hospitalization. Intestinal inflammation caused by macrophages and neutrophils is thought to be important in the mechanism of POI. Surgery-associated tissue injury and inflammation induce the release of adenosine triphosphate (ATP) from injured cells. Released ATP binds the purinergic P2X7 receptor (P2X7R) expressed on inflammatory cells, inducing the secretion of inflammatory mediators. P2X7R antagonists are thought to be important mediators of the first step in the inflammation process, and studies in chemically induced colitis models confirmed that P2X7R antagonists exhibit anti-inflammatory effects. Therefore, we hypothesized that P2X7R plays an important role in POI. POI models were generated from C57BL/6J mice. Mice were treated with P2X7R antagonist A438079 (34 mg/kg) 30 min before and 2 hr after intestinal manipulation (IM). Inflammatory cell infiltration and gastrointestinal transit were measured. A438079 ameliorated macrophage and neutrophil infiltration in the POI model. Impaired intestinal transit improved following A438079 treatment. P2X7R was expressed on both infiltrating and resident macrophages in the inflamed ileal muscle layer. The P2X7R antagonist A438079 exhibits anti-inflammatory effects via P2X7R expressed on macrophages and therefore could be a target in the treatment of POI.

Keywords: P2X7 receptor; anti-inflammation; macrophages; postoperative ileus.

Fig. 1.

P2X7 receptor (P2X7R) blockade inhibited leukocyte infiltration in a postoperative ileus model in control mice. Effect of purinergic P2X7R blockade on leukocyte infiltration induced by intestinal manipulation (IM) in a postoperative ileus model in control mice. Myenteric nerve plexus inhibition via P2X7R blockade was performed by administration of A438079 (34 mg/kg, s.c.), as described in the Materials and Methods. A: Immunohistochemical or histochemical staining of CD68-positive macrophages or myeloperoxidase (MPO)-stained neutrophils in the intestine of control mice. Representative images from four independent experiments are shown. Black bar indicates 50 μm and white bar indicates 100 μm. Red and green signals indicate PGP 9.5–positive myenteric neurons and CD68-positive macrophages, respectively. B and C: Number of infiltrating macrophages (B) and neutrophils (C) in images shown in (A). * or ***, significantly different from control at P<0.05 or P<0.001; # or ##, significantly different from IM at P<0.05 or P<0.01, respectively (n=4 each). Each column shows the mean ± SEM.

Fig. 2.

P2X7 receptor (P2X7R) inhibition ameliorates diminished gastrointestinal transit in a mouse model of postoperative ileus (POI). A: Data shown are mean ± SEM of the ratio (%) of fluorescein isothiocyanate (FITC) content. B: Geometric center calculated from A. ***P<0.001; significantly different from control. #P<0.05; significantly different from POI. Data shown are mean ± SEM from four independent experiments.

Fig. 3.

Expression of P2X7 receptor (P2X7R) on cells of the ileal muscle layer of control and IM-treated mice. Double-immunostaining of CD68 (red) and purinergic P2X7R (green) in the ileal muscle layer of control and intestinal manipulation (IM)-treated mice. Scale bar, 100 μm. Typical results are shown for three independent experiments.