Homeostasis alteration within small intestinal mucosa after acute enteral refeeding in total parenteral nutrition mouse model

Abstract

Feeding strategies to care for patients who transition from enteral nutrient deprivation while on total parenteral nutrition (TPN) to enteral feedings generally proceed to full enteral nutrition once the gastrointestinal tract recovers; however, an increasing body of literature suggests that a subgroup of patients may actually develop an increased incidence of adverse events, including death. To examine this further, we studied the effects of acute refeeding in a mouse model of TPN. Interestingly, refeeding led to some beneficial effects, including prevention in the decline in intestinal epithelial cell (IEC) proliferation. However, refeeding led to a significant increase in mucosal expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), as well as an upregulation in Toll-like receptor 4 (TLR-4). Refeeding also failed to prevent TPN-associated increases in IEC apoptosis, loss of epithelial barrier function, and failure of the leucine-rich repeat-containing G protein-coupled receptor 5-positive stem cell expression. Transitioning from TPN to enteral feedings led to a partial restoration of the small bowel microbial population. In conclusion, while acute refeeding led to some restoration of normal gastrointestinal physiology, enteral refeeding led to a significant increase in mucosal inflammatory markers and may suggest alternative strategies to enteral refeeding should be considered.

Keywords: enteral nutrition; intestinal epithelial cell proliferation; tumor necrosis factor-α.

Fig. 1.

Acute refeeding reversed loss of intestinal epithelial cell (IEC) proliferation post-total parenteral nutrition (TPN). A: proliferating cell nuclear antigen (PCNA) immunofluorescence staining was performed to measure IEC proliferation. Proliferation index was calculated as the ratio of PCNA-positive cell number to total cell number in each crypt; 15 U-shaped crypts of each slide were counted. B: proliferation index for each group. C: bromodeoxyuridine (BrdU, red) and green fluorescent protein (GFP, green) immunofluorescence staining were performed to detect IEC proliferation and stem cell maker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) expression (arrows) in EGFP-LGR5 transgenic mice. Proliferation index was calculated as the ratio of BrdU-positive cell number to total cell number in each crypt; 15 U-shaped crypts were counted for each slide. GFP-positive crypt numbers were counted in 100 crypts for each mouse. E: Lgr5 mRNA expression was measured in jejunum mucosal, corrected for β-actin expression. TGF-β1, transforming growth factor-β1. Results are means ± SD. For each experiment, n = 5–6 mice for each group, *P < 0.05 and ***P < 0.001. ns, Not significant.

Fig. 2.

Proliferative signaling pathways are partially restored with refeeding. A: phospho (p)-AKT expression was measured with Western blot methods (adjusted for β-actin), and p-AKT protein expression was calculated as the ratio of density of p-AKT to total AKT. B: Wnt-β-catenin signaling was measured with Western blot and real-time PCR. C: epidermal growth factor receptor (EGFR) and its ligands were measured with real-time PCR, and corrected for β-actin expression. Results are expressed as means ± SD. EGF, epidermal growth factor. For each experiment, n = 5–6 for each group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3.

IEC apoptosis persists with refeeding after TPN. A: TdT-dUTP nick end-labeling (TUNEL) staining was performed to detect IEC apoptosis. B: apoptosis index was calculated as the ratio of TUNEL-positive cell number to total cell number in each villi; 15 villi were selected for each section. Results are means ± SD. For each experiment, n = 5–6 for each group. ***P < 0.001.

Fig. 4.

Mucosal cytokine and Toll-like receptor (TLR) expression increases its proinflammatory response with acute refeeding. A: mucosal proinflammatory cytokine expression was measured with real-time PCR. B: regulatory cytokines IL-10 and TGF-β expression were measured with real-time PCR. C: mucosal Toll-like receptor expression. MCP1, monocyte chemoattractant protein 1. Results are means ± SD. For each experiment, n = 5–6 for each group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5.

Mucosal microbiota and defensin expression with acute refeeding. A: phylum level analysis after RDP classification of pyrosequenced ileal mucosa-associated bacteria samples. B: α-defensin (cryptins) expression was measured with quantitative PCR (qPCR), corrected for β-actin expression. C: matrix metalloproteinase 7 (MMP7) expression (red) with DAPI (blue nuclear counterstain) was measured with immunofluorescence staining. D: lysosome expression (red) with DAPI (blue nuclear counterstain) and PCNA (green) was measured with immunofluorescence staining. Results are means ± SD. For each experiment, n = 5–6 for each group. **P < 0.01.

Fig. 6.

Epithelial barrier function failed to improve with acute refeeding. A: transepithelial resistance (TER) was measured using Ussing chambers. B: permeability was measured with FITC-dextran-40 (FD40) concentration. C: zonula occludens (ZO)-1 and occludin expression was measured with real-time PCR and immunofluorescence staining. D: claudin-2 expression was measured with real-time PCR and immunofluorescence staining. Results are means ± SD. For each experiment, n = 5–6 for each group. *P < 0.05, **P < 0.01, and ***P < 0.001.