The protective effect of hydroxyxanthone on the leakiness of intestinal epithelia by the proinflammatory cytokine related to bacterial endotoxin

Abstract

Leaky gut characterized as decreased transepithelial electrical resistance and increased paracellular permeability to macromolecules is associated with several fatal diseases including sepsis. Pro-inflammatory cytokine IL-1β is a critical mediator of underlying mechanism through activation of myosin light chain kinase pathway. Blocking the inducible phosphorylated myosin light chain (p-MLC) indicates the successful treatment. Xanthones predominantly found in mangosteen (Garcinia mangostana) has been indicated as a potent anti-inflammatory action. Hydroxyxanthones (HDX), the major natural xanthones, have been recently synthesized in various forms.

This study aimed to examine and screen the action of various forms of synthetic HDXs, in preventing the increased paracellular permeability during challenging with IL-1β by inhibiting the p-MLC expression in an in vitro model of intestinal Caco-2 cell culture. Caco-2 cells were cultivated in standard media with 10% fetal bovine serum for 7 or 21 days representing colonic-like or differentiated jejunal-like intestinal epithelial cells, respectively. Screening of non-cytotoxic forms of HDXs over 24 and 48 h were determined primarily by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The transepithelial resistance (TER) measured by volt-ohm meter and paracellular permeability of fluorescein isothiocyanate-dextran (FD-4; MW=4,400 Da) were accomplished for evaluating of paracellular permeability. Semi-quantitative Western blot analysis was performed to monitoring of p-MLC protein expression in response to drug treatments.

In the MTT assay, all HDXs 1-monoHDX, 1,3-diHDX, 1,3,6-triHDX or 1,3,6,8-tetraHDX at 10 or 100 µM had no cytotoxicity. In an exception, 1,3,6,8-tetraHDX 100 µM decreased the viability of 7-day cell at 48 h. In normal condition, 1-monoHDX or 1,3-diHDX (10 µM) treatment promoted the increased TER in 7-day cells at 12 h, but 1,3-diHDX and 1,3,6-triHDX decreased TER in 21-day cells during 12-48 h. In 7-day cells challenged with IL-1β to decrease TER and increase paracellular permeability of FD-4, 1-monoHDX or 1,3-diHDX at 10 µM restored all the effect of IL-1β on the paracellular permeability at 12 and 24 h (n=5 experiments; p<0.05). In 21-day cells which were not responding to IL-1β, however 1-monoHDX also enhanced the TER from the initial values (p<0.05) at 12 and 24 h. In an accordance, Western blot analysis showed that p-MLC protein expression was increased in IL-1β treatments at 24 h. HDXs decreased the p-MLC stimulated by IL-1β at 24 h. These results suggest that HDXs can protect the increased paracellular permeability induced by IL-1β by inhibiting the increased p-MLC expression in colonic-like cells. In conclusion, 1-monoHDX and 1,3-diHDX at a concentration of 10 µM may be beneficial and be a candidate for treatment of the leaky gut in the future.