Trolox and ascorbic acid reduce direct and indirect oxidative stress in the IPEC-J2 cells, an in vitro model for the porcine gastrointestinal tract

Oxidative stress in the small intestinal epithelium is a major cause of barrier malfunction and failure to regenerate. This study presents a functional in vitro model using the porcine small intestinal epithelial cell line IPEC-J2 to examine the effects of oxidative stress and to estimate the antioxidant and regenerative potential of Trolox, ascorbic acid and glutathione monoethyl ester. Hydrogen peroxide and diethyl maleate affected the tight junction (zona occludens-1) distribution, significantly increased intracellular oxidative stress (CM-H(2)DCFDA) and decreased the monolayer integrity (transepithelial electrical resistance and FD-4 permeability), viability (neutral red) and wound healing capacity (scratch assay). Trolox (2 mM) and 1 mM ascorbic acid pre-treatment significantly reduced intracellular oxidative stress, increased wound healing capacity and reduced FD-4 permeability in oxidatively stressed IPEC-J2 cell monolayers. All antioxidant pre-treatments increased transepithelial electrical resistance and viability only in diethyl maleate-treated cells. Glutathione monoethyl ester (10 mM) pretreatment significantly decreased intracellular oxidative stress and monolayer permeability only in diethyl maleate-treated cells. These data demonstrate that the IPEC-J2 oxidative stress model is a valuable tool to screen antioxidants before validation in piglets

Microscopic representation of the wound healing capacity of H₂O₂ and DEM-stressed IPEC-J2 cells pre-treated with antioxidants.

<p>IPEC-J2 cells were pre-treated with or without the antioxidants Trolox or ascorbic and incubated with A: 0.5 mM H₂O₂, B: 4 mM DEM for 1 h. The microscopic illustrations show the start of the wound healing assay (T = 0) and the final measuring point after 6 h (T = 6). AO: antioxidant treatment, ROS: oxidative stress inducer, T: 2 mM Trolox pre-treatment, AA: 1 mM ascorbic acid pre-treatment.</p

Membrane integrity of H₂O₂ and DEM-stressed IPEC-J2 cells with or without antioxidants pre-treatment.

<p>Membrane integrity, assessed by transepithelial electrical resistance (TEER) was determined after an overnight incubation with plain growth medium in order to stabilise the TEER values. A: TEER is significantly reduced when incubated with 1 mM H₂O₂ or more (<i>p</i><0.001). B: TEER is significantly reduced when incubated with 2 mM DEM or more (<i>p</i><0.001). Results are represented as means ± SE, n = 3. IPEC-J2 cells were pre-treated with or without the antioxidants Trolox, ascorbic acid or GSH-MEE and incubated with C: 1 mM H₂O₂, D: 4 mM DEM for 1 h. Results are represented as means ± SE, n = 4. The dotted black line marks the confluency threshold of 1 kΩcm². Significant differences between treatments are represented by different letters. T: 2 mM Trolox pre-treatment, AA: 1 mM ascorbic acid pre-treatment, G: 10 mM GSH-MEE pre-treatment.</p

H₂O₂ and DEM induce intracellular oxidative stress in IPEC-J2 cells.

<p>IPEC-J2 cells were pre-treated with or without the antioxidants Trolox, ascorbic acid or GSH-MEE and loaded with CM-H₂DCFDA probe prior to oxidative stressing. A: 0.5 mM H₂O₂, B: 4 mM DEM for 1 h. Results are presented as means ± SE, n = 3. Significant differences between treatments are represented by different letters. T: 2 mM Trolox pre-treatment, AA: 1 mM ascorbic acid pre-treatment, G: 10 mM GSH-MEE pre-treatment.</p

H₂O₂ and DEM affect the tight junction distribution in IPEC-J2 cells.

<p>Immunocytochemical staining with ZO-1 of IPEC-J2 cells was performed to investigate the effect of A: no treatment, B: 1 mM H₂O₂ or C: 4 mM DEM for 1 h on the tight junction distribution. The arrowheads point towards the tight junction-associated protein ZO-1 at the cell-cell contact complex. The dotted circle marks the tight junction distribution in an IPEC-J2 cell. The dotted square focusses on the increased presence of vesicles, especially seen in dividing or H₂O₂-stressed cells. Scale bar: 30 μm.</p