In vitro model to estimate gut inflammation using co-cultured Caco-2 and RAW264.7 cells

Abstract

A system for assessing the anti-inflammatory effect of food factors was developed by establishing a co-culture system with intestinal epithelial Caco-2 cells (apical side) and macrophage RAW264.7 cells (basolateral side). In this system, the stimulation of RAW264.7 cells with lipopolysaccharide was followed by a decrease in transepithelial electrical resistance, which is a marker of the integrity of the Caco-2 monolayer and an increase in TNF-alpha production from RAW264.7 cells and IL-8 mRNA expression in Caco-2 cells. Treatment with anti-TNF-alpha antibodies or budesonide suppressed in increase in TNF-alpha production and IL-8 mRNA expression. These results indicated that this co-culture model could imitate the gut inflammation in vivo. In addition, fucoidan, sulphated polysaccharides from brown algae, was employed as a candidate of evolution and added to the apical side of this model. Fucoidan suppressed IL-8 gene expression through a reduction in TNF-alpha production from RAW264.7 cells stimulated with lipopolysaccharide.

Different Suppressive Effects of Fucoidan and Lentinan on IL-8 mRNA Expression inin VitroGut Inflammation

“…The TNF-produced led Caco-2 cells to secrete interleukin (IL)-8, one of inflammation markers. 16) Moreover, this indicated that this gut inflammation model is applicable in the search for anti-inflammatory factors against intestinal inflammation. In present study, we focused on the suppressive effects of lentinan and fucoidan on interleukin (IL)-8 mRNA expression using this model, and found that in Caco-2 cells, lentinan and fucoidan suppressed IL-8 mRNA expression in different ways.…”

“…A co-culture experiment was performed as described by Tanoue et al 16) TNFcontents were quantified by cytotoxicity assay with L929 cells (actinomycin D-treated murine fibroblast cell line) using murine rTNF-as the standard, as described by Kerékgyártó et al 10) Semi-quantitative RT-PCR was performed as described previously. 16) To semi-quantify the induction of IL-8 mRNA expression in Caco-2 cells, IL-8 mRNA expression levels were normalized relative to the expression of GAPDH mRNA, and IL-8/GAPDH ratios were calculated by densitometric analysis using Image J software. The contents of lentinan and fucoidan in the culture supernatant were quantified by a method described by Mizuno et al 17,18) Budesonide, an oral medicine for Crohn's disease patients, was added in the apical compartment in this system as a positive control.…”

“…16) To study the anti-inflammatory activity of polysaccharides as a food factor, lentinan and fucoidan, from Lentinula edodes and Laminaria japonica Areschoug respectively, were applied in this model. LPS treatment from the basolateral compartment enhanced TNF-production about 10-fold as compared with the control (Tables 1 and 2).…”

Lipoxin A4Reduces Lipopolysaccharide-Induced Inflammation in Macrophages and Intestinal Epithelial Cells through Inhibition of Nuclear Factor-κB Activation

“…The coculture system was constructed according to the method of a previous report (Tanoue et al, 2008). In brief, Caco-2 cells were seeded at 3.75 ϫ 10 5 cells/well in Transwell insert plates (4.67 cm 2 , 0.4-m pore size; Corning Costar, Cambridge, MA).…”

Screening of budesonide nanoformulations for treatment of inflammatory bowel disease in an inflamed 3D cell-culture model

“…Several cell culture models incorporating immunocompetent cells, such as macrophages or B-cells, to simulate intestinal tissue have been developed (tanoue et al, 2008;des Rieux et al, 2007;Spottl et al, 2006). In our model, the inflammatory response is promoted by addition of a pro-inflammatory cytokine to the triple culture of epithelial and immunocompetent cells to mimic the inflammation in IBD.…”