Protective effects of carbon monoxide-releasing molecule-2 on the barrier function of intestinal epithelial cells

Abstract

Objective: To investigate the protective effects and mechanisms of carbon monoxide-releasing molecule-2 (CORM-2) on barrier function of intestinal epithelial cells.

Materials and methods: After pre-incubation with CORM-2 for 1 hour, cultured intestinal epithelial IEC-6 cells were stimulated with 50 µg/ml lipopolysaccharides (LPS). Cytokines levels in culture medium were detected using ELISA kits. Trans-epithelial electrical resistance (TER) of IEC-6 cell monolayers in Transwells were measured with a Millipore electric resistance system (ERS-2; Millipore) and calculated as Ω/cm2 at different time points after LPS treatment. The permeability changes were also measured using FITC-dextran. The levels of tight junction (TJ) proteins (occludin and ZO-1) and myosin light chain (MLC) phosphorylation were detected using Western blotting with specific antibodies. The subsequent structural changes of TJ were visualized using transmission electron microscopy (TEM).

Results: CORM-2 significantly reduced LPS-induced secretion of TNF-α and IL-1β. The LPS-induced decrease of TER and increase of permeability to FITC-dextran were inhibited by CORM-2 in a concentration dependent manner (P<0.05). LPS-induced reduction of tight junction proteins and increase of MLC phosphorylation were also attenuated. In LPS-treated cells, TEM showed diminished electron-dense material and interruption of TJ and desmosomes between the apical lateral margins of adjoining cells, which were prevented by CORM-2 treatment.

Conclusions: The present study demonstrates that CORM-2, as a novel CO-releasing molecule, has ability to protect the barrier function of LPS-stimulated intestinal epithelial cells. Inhibition of inflammatory cytokines release, restoration of TJ proteins and suppression of MLC phosphorylation are among the protective effects of CORM-2.

Figure 1. Effect of CORM-2 on cell viability.

IEC-6 cells were treated with CORM-2 in the absence or presence of LPS (/ml) for 24 h, and cell viability was assessed using the MTT assay. Results are the mean h, and cell viability was assessed using the MTT assay. Results are the mean±SD of 3 independent experiments. ANOVA test P>0.05.

Figure 2. Effect of CORM-2 on the permeability of LPS-treated IEC-6 cell monolayer.

When cells reached confluence in the transwell system, CORM-2 was added and incubated for 1 h. The cells were washed and treated with 50 h. The cells were washed and treated with 50 µg/ml LPS for 24 h. (a) Mean±SD of TER values from 3 independent experiments are shown. (b) Permeability of FITC-dextran across the cell monolayer indicated CORM-2 could decrease the LPS-induced increase. Results are the mean±SD of 3 independent experiments. *ANOVA test, p<0.05 as compared to control group, #p<0.05 as compared to LPS group.

Figure 3. Effect of CORM-2 on the release of proinflammatory cytokines in LPS-treated IEC-6 cells.

IEC-6 cells were pretreated with CORM-2 for 1 h and cells were washed and stimulated with 50 h and cells were washed and stimulated with 50 µg/ml LPS for 24 h. TNF h. TNF-α (a) and IL-1β (b) in the culture medium were measure using ELISA kits. Mean±SD from 3 independent experiments are shown. *ANOVA test, P<0.05 when compared to control group (no treatment), #ANOVA test, P<0.05 when compared to LPS alone group.

Figure 4. Effect of CORM-2 on TJ protein expression in LPS-treated IEC-6 cells.

IEC-6 cells were pretreated with CORM-2 for 1 h and the cells were washed and stimulated with 50 h and the cells were washed and stimulated with 50 µg/ml LPS for 24 h. The cells were washed with PBS and harvested with RIPA buffer. The protein concentration was determined and proteins subjected to Western blotting (Methods). A typical image is shown (Upper panel). The relative ratios (Mean±SD) of ZO-1/β-actin (Middle panel) and occludin/β-actin (Lower panel) are calculated based on the densities of bands on Western blots from 3 independent experiments. *ANOVA test, P<0.05 when compared to control group (no treatment), #ANOVA test, P<0.05 when compared to LPS alone group.

Figure 5. Effect of CORM-2 on the levels of MLC phosphorylation.

(A) IEC-6 cells were treated with 50 µg/ml LPS and harvested at different time points. MLC phosphorylation (p-MLC) was detected using phosphorylation-specific antibody. Sample loading was normalized using anti-MLC antibody. A typical blot is shown (Upper panel). (B) IEC-6 cells were pretreated with CORM-2 for 1 h followed by LPS (50 µg/ml) treatment for 1 h. A typical Western blot shows that CORM h. A typical Western blot shows that CORM-2 reduced LPS-enhanced MLC phosphorylation (Upper panel). The relative ratios (Mean±SD) of p-MLC/MLC are calculated based on the densities of bands on Western blots from 3 independent experiments (Lower panels of (A) and (B)). *ANOVA test, P<0.05 when compared to control group (no treatment), #ANOVA test, P<0.05 when compared to LPS alone group.

Figure 6. Changes of TJ observed by Transmission Electron Microscopy.

Ultrastructure of TJs in a monolayer of cultured IEC-6 cells was observed by a transmission electron microscope. Panel (A) Normal control. (B) LPS–treated cells. The electron-dense materials were diminished in TJ and desmosomes. The space between adjoining cells was widened. (C) CORM-2 attenuated the LPS disruption of TJ and desmosomes. Arrows indicate the location of the TJs (Scale bar = 0.2 µm).