Fig 1.
H2O2 and DEM induce intracellular oxidative stress in IPEC-J2 cells.
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.
Fig 2.
Effect of H2O2 and DEM on the intracellular oxidation state of glutathione.
IPEC-J2 cells were pre-treated with or without the antioxidants Trolox or ascorbic acid and incubated with A: 0.5 mM H2O2, B: 4 mM DEM for 1 h. Glutathione (GSH) and glutathione disulphide (GSSG) concentrations were analysed using HPLC. Results are represented 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.
Fig 3.
Viability of H2O2 and DEM-treated IPEC-J2 cells pre-treated with antioxidants.
IPEC-J2 cells were pre-treated with or without the antioxidants Trolox or ascorbic acid and incubated with A: 1 mM H2O2, B: 4 mM DEM for 1 h. Results are represented as means ± SE (n = 4), normalized to the control treatment. Significant differences between treatments are represented by different letters. T: 2 mM Trolox pre-treatment, AA: 1 mM ascorbic acid pre-treatment.
Fig 4.
Membrane integrity of H2O2 and DEM-stressed IPEC-J2 cells with or without antioxidants pre-treatment.
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 (p<0.001). B: TEER is significantly reduced when incubated with 2 mM DEM or more (p<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.
Fig 5.
Membrane FD-4 permeability of H2O2 and DEM-stressed IPEC-J2 cells with or without antioxidant pre-treatment.
Membrane permeability was quantitated one day after oxidative stressing by measuring the amount of FD-4 that permeated through the IPEC-J2 monolayer. A: FD-4 permeation is significantly increased when incubated with 1 mM H2O2 or more (p<0.001). B: FD-4 permeation is significantly increased when incubated with 2 mM DEM or more (p<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. 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.
Fig 6.
H2O2 and DEM affect the tight junction distribution in IPEC-J2 cells.
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.
Fig 7.
Wound healing capacity of H2O2 and DEM-stressed IPEC-J2 cells pre-treated with antioxidants.
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. Results are represented as means ± SE, n = 4. Significant differences between treatments are represented by different letters. T: 2 mM Trolox pre-treatment, AA: 1 mM ascorbic acid pre-treatment.
Fig 8.
Microscopic representation of the wound healing capacity of H2O2 and DEM-stressed IPEC-J2 cells pre-treated with antioxidants.
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.