Reinforced Epithelial Barrier Integrity via Matriptase Induction with Sphingosine-1-Phosphate Did Not Result in Disturbances in Physiological Redox Status

Abstract

Objectives. The relationship among matriptase function, cellular redox status, and maintenance of intestinal barrier integrity has not been established yet. The aim of this study is to reveal if the crosstalk between matriptase activators and intestinal epithelial monolayers can lead to perturbations in physiological redox regulation in vitro. Methods. The effects of suramin and sphingosine-1-phosphate (S1P) were tested on viability of intestinal porcine epithelial IPEC-J2 cells using MTS assay. Measurements of transepithelial electrical resistance (TER) were performed to determine changes in barrier integrity of cell monolayers. Amplex Red assay was used to monitor extracellular hydrogen peroxide production. Occludin distribution pattern was detected prior to and after matriptase activation using immunofluorescent staining technique. Results. TER reduction was observed in suramin-treated IPEC-J2 cell monolayers, which could be attributed to cell cytotoxic properties of 48 hr 50 μM suramin administration. In contrast, S1P treatment increased TER significantly and elevated occludin accumulation in tight junctions. It was also found that extracellular hydrogen peroxide levels were maintained in IPEC-J2 cells exposed to matriptase activators. Discussion. S1P administration not accompanied by redox imbalance might be one of the key strategies in the improvement of barrier function and consequently in the therapy of intestinal inflammations.

Figure 1

(a) 48 hr incubation of IPEC-J2 cells with S1P at different concentrations at 50, 100, and 200 ng/mL. Values represent average absorbance values of produced MTS formazan in metabolically active cells ± SEMs. No significant differences were found between control and cell monolayers exposed to S1P at concentration up to 200 ng/mL. (b) 48 hr incubation of IPEC-J2 cells with suramin at different concentrations at 50, 100, and 200 μM. Values represent average absorbance values of produced MTS formazan in metabolically active cells ± SEMs. Significant differences were found between controls and cell monolayers treated with higher concentrations (100 μM, ^∗ p = 0.0222 and 200 μM, ^∗ p = 0.01025) of suramin.

Figure 2

(a) Transepithelial electrical resistance values of IPEC-J2 cell monolayers after 0.5, 2, 24, and 48 hr treatment with S1P (200 ng/mL) and suramin (50 μM) were measured and compared to controls in nine parallel experiments. At the start of the treatment IPEC-J2 cells were not completely differentiated. The values are expressed in average relative TERs ± SEMs. Asterisk indicates significant differences between treated and mock groups (^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001). (b) Transepithelial electrical resistance values of differentiated IPEC-J2 cell monolayers after 0.5, 2 hr treatment with S1P (200 ng/mL) and suramin (50 μM) were measured and compared to controls in nine parallel experiments. The values are expressed in average relative TERs ± SEMs. Asterisk indicates significant differences between treated and mock groups (^∗ p < 0.05 and ^∗∗∗ p < 0.001).

Figure 3

Fluorescence intensities of supernatants of control and S1P (200 ng/mL)- and suramin (50 μM)-treated groups determined with Amplex Red assay. At the start of the treatment IPEC-J2 cells were not completely differentiated. The measured values are indicated as average fluorescence intensities ± SEMs (n = 5). There were no significant differences between mock and matriptase activator-treated groups (p > 0.05) up to 48 hr.

Figure 4

Immunofluorescence staining of occludin (in red) in control and in S1P (200 ng/mL) and suramin (50 μM)-treated IPEC-J2 cells after 48 hr treatment. Occludin can be seen in red and cell nuclei were stained with DAPI in blue. S1P-induced more intensive occludin staining in cell membranes, which was indicated by white arrows. In case of suramin administration, occludin shows more diffuse distribution pattern compared to that in control panel. 600x magnification.