Carbon monoxide-releasing molecule-2 attenuates LPS induced barrier injure of Caco-2 cells

Objective To investigate the effects and mechanisms of carbon monoxide-releasing molecule-2 (CORM-2) on LPS induced barrier injure of Caco-2 cells. Methods The model of Caco-2 monolayer cells damage induced by LPS was established by using 50 μg/ml LPS for 24 hours. After preconditioning with different concentrations (10 μmol/L, 50 μmol/L, and 100 μmol/L) of CORM-2 for 1 hour, the cultured well-grown Caco-2 monolayer cells were stimulated with 50μg/ml lipopolysaccharides (LPS) for 24 hour. The 100 μmol/L CORM-2 was put into 37℃ and 5% CO2 incubator for 18 hours until the CO has been fully released, and it became an inactive CORM - 2(iCORM-2). The cultured Caco-2 monolayer cells were divided into six groups: the control group, the LPS group, the LC1(10 μmol /L CORM-2 preconditioning) group, the LC2(50 μmol/L CORM-2 preconditioning) group, the LC3(100 μmol/L CORM-2 preconditioning) group, and the LC4 (iCORM-2 preconditioning) group. The apoptosis rates of different groups of Caco-2 monolayer cells were detected using flow cytometry. Cytokines (TNF-α,IL-1β and HMGB-1) levels of different groups were detected using ELISA kits. The levels of tight junction proteins (occludin ZO-1, claudin-1 and claudin-4) of every group were detected using Western blotting with specific antibodies. The structural changes of tight junction proteins were visualized by immunofluorescence technique. Results Compared with control group, cell apoptosis rate and release of inflammatory cytokines such as TNF-α, IL-1β and HMGB-1 in LPS group were significantly higher, and the levels of tight junction proteins were apparently decreased (P<0.05) in LPS group. Compared with LPS group, cell apoptosis rate and release of inflammatory cytokines such as TNF-α, IL-1β and HMGB-1 decreased, and the levels of tight junction proteins were attenuated obviously, P<0.05, in CORM-2 preconditioning groups. And the higher the concentration of CORM-2, the more obvious the protective effects. Conclusions This study demonstrates that CORM-2, as one of exogenous CO-releasing molecules, has the capacity to protect the barrier damage of LPS-stimulated Caco-2 monolayer cells in a concentration dependent manner. The higher the concentration of CORM-2 was, the stronger the protective effects were. The protective effects of CORM-2 include reducing Caco-2 monolayer cells apoptosis rate, inhibiting inflammatory cytokines production and release, and restoring distribution and levels of tight junction proteins. Key words: Exogenous carbon monoxide-releasing molecule-2; Lipopolysaccharides; Caco-2 monolayer cell; Tight junction proteins