The Synergistic Effects of Hypoxia/Reoxygenation or Tissue Acidosis and Bacteria on Intestinal Epithelial Cell Apoptosis

BACKGROUND Clinical data indicate that gut perfusion deficits must be rectified within 24 hours after traumatic injury to decrease organ failure and death. Ischemia/reperfusion injury to the gut causes enterocyte apoptosis (Apo), which may contribute to intestinal barrier failure. The temporal response of enterocyte Apo to acidosis and hypoxia/reoxygenation (H/R) in vitro is unknown. The purpose of this study was to examine the effect of various time points of acidosis or H/R on enterocyte apoptosis and monolayer integrity in an in vitro model. METHODS Caco-2 cell monolayers were made acidic (Dulbecco’s modified Eagle’s medium, pH 6.9) by hydrochloric acid or exposed to 95% nitrogen/5% carbon dioxide (hypoxia) and then 21% oxygen (reoxygenation). Escherichia coli C-25 were added to the apical media in subsets. Apo and necrosis were quantified by flow cytometry. Permeability was determined by fluorescein isothiocyanate-dextran. Transepithelial electrical resistance (TEER) indexed monolayer. RESULTS Extracellular acidosis and C-25 significantly increased apoptosis of Caco-2 cells at 18 hours (extracellular acidosis [EC] + C-25, 14.5 ± 3.0; control, 3.8 ± 0.8; p < 0.001 by analysis of variance). Similarly, the H/R + C-25 group showed a significant increase in apoptosis at 12 hours (H/R + C-25 vs. control, 22.86 ± 2.12 vs. 3.74 ± 0.7; p < 0.001 by analysis of variance). The permeability difference was not significant for EC + C-25 versus control at 18 hours (0.68 ± 0.25 vs. 0.43 ± 0.0.0.36, respectively; p > 0.05). The H/R + C-25 group had a profound increase in permeability over control at 12 hours (10.8 ± 0.5 vs. 2.1 ± 0.3, respectively; p < 0.001). The TEER was significantly lowered for EC versus control at 18 hours (458 ± 1.5 vs. 468 ± 8.2) and at 0, 6, and 18 hours for EC + C-25 (409 ± 28.1, 443 ± 16.8, and 438 ± 8.9 vs. 455 ± 6.5, 467 ± 6.5, and 469 ± 8.2, respectively). There was no significant change in the H/R and H/R + C-25 groups. CONCLUSION Synergism of H/R or tissue acidosis and bacteria caused increased Apo, TEER, and permeability in vitro.