Background and Purpose
Dietary fibre comprises a complex group of polysaccharides that are indigestible but are fermented by gut microbiota, promoting beneficial effects to the intestinal mucosa indirectly through the production of short chain fatty acids. We found that a polysaccharide, rhamnogalacturonan (RGal), from the plant Acmella oleracea, has direct effects on intestinal epithelial barrier function. Our objective was to determine the mechanism whereby RGal enhances epithelial barrier function.
Experimental Approach
Monolayers of colonic epithelial cell lines (Caco‐2, T84) and of human primary cells from organoids were mounted in Ussing chambers to assess barrier function. The cellular mechanism of RGal effects on barrier function was determined using inhibitors of TLR‐4 and PKC isoforms.
Key Results
Apically applied RGal (1000 μg ml−1) significantly enhanced barrier function as shown by increased transepithelial electrical resistance (TER) and reduced fluorescein isothiocyanate (FITC)‐dextran flux in Caco‐2, T84 and human primary cell monolayers, and accelerated tight junction reassembly in Caco‐2 cells in a calcium switch assay. RGal also reversed the barrier‐damaging effects of inflammatory cytokines on FITC‐dextran flux and preserved the tight junction distribution of occludin. RGal activated TLR4 in TLR4‐expressing HEK reporter cells, an effect that was inhibited by the TLR4 inhibitor, C34. The effect of RGal was also dependent on PKC, specifically the isoforms PKCδ and PKCζ.
Conclusion and Implications
RGal enhances intestinal epithelial barrier function through activation of TLR4 and PKC signalling pathways. Elucidation of RGal mechanisms of action could lead to new, dietary approaches to enhance mucosal healing in inflammatory bowel diseases.
Aims: The anticoagulant effect and cytotoxicity of a high molecular weight polysaccharide fraction (1000RS) obtained from the tunic of the ascidia Microcosmus exasperatus were evaluated. Methods: Anticoagulant properties of 1000RS was evaluated by activated Partial Thromboplastin Time (aPTT), Thrombin Time (TT), Prothrombin Time (PT), anti-factor Xa and lupic anticoagulant (dRVVT) assays. Cytotoxicity was tested on murine hematopoietic cells using MTT assay. Results: This galactose rich fraction showed to be a potential anticoagulant due to its inhibitory effect on the intrinsic coagulation pathway. At the same time, anticoagulant doses of this fraction have no effect on cellular viability, which means that it can be used as a therapeutic agent. Conclusion: In vitro anticoagulant effect of 1000RS occurs at innocuous doses, however, it still need to be tested using in vivo models and its cytotoxicity evaluated in normal human cell lines.
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