| Carrageenans were sulfated polysaccharides widely used in the fields of food and medicine due totheir good bioactivity and physical chemistry properties. There are security problems to using carrageenans.One side there was several proofs showed that the animals had inflammation blowel diseases as coloniculcers who eated carrageenan at long time. The other side some researchs indicate it was safe to eatcarrageenan. So it was very important to determine the inflammation mechanism of carrageenan. Thispaper was investigate the immune adjustment effect and mechanism of carrageenan on human colonepithelial cells(Caco-2and HT-29cells). An in vitro model system using coculture was constructed toanalyze the effect of carrageenans on intestinal epithelial cells, and human intestinal epithelial-like Caco-2monolayers and activated macrophage-like THP-1cells were used in this study.λ-，κ-andι-carrageenans(CGN) were the types of carrageenans(CGN).λ-CGN，κ-CGN and ι-CGNwere the undegraded carrageenans; λ10-40kd，κ10-40kd and ι10-40kd were the degraded carrageenanswhich molecular weight were rang10KD and40KD; λ<5000，κ<5000and ι<5000were the degradedcarrageenans which molecular weight under5000KD.Cell viabilitiy of the human colon epithelial cells wasmeasured by MTT colormetric survival assay, while the effect of the cytokine secrection of the humamcolon epithelial cells induced by carrageenans was analyzed using the CBA assay kit and ELISA assaykit.The signal mechanism in cells immune adjustment was analyzed by Western blotting andEMSA(electrophoretic mobility shift assay).The binding size of κ-CGN(κ-carrageenan) and the cells wasdetected by fluorescence microscope. Carrageenans had little effect on the human colon epithelial cellsgrowth, when carrageenans treated the cells at the low concentration(≦10μg/mL),but the carrageenans ofλ-10-40kd and κ<5000markedly inhibited Caco-2cells proliferation at very high concentration (1mg/mL).The κ-CGN had the strongest effect of up-regulation human colon cells secreted IL-8in a dose-dependentmanner. It could up-regulation IL-8as control was2.5times in Caco-2cells and6times in HT-29cellswhen the dose at40μg/mL. κ-CGN was up-regulation TLR4(Toll like receptor4) in a dose-dependentmanner, it leaded to activation NF-κB and up-regulation transduction NF-κB into the nucleus in bothCaco-2cells and HT-29cells. The inhibiton of TLR4and NF-κB could totally remove the effect ofup-regulation IL-8in Caco-2induced by κ-CGN but HT-29cells. Flu-CGN was binding with TLR4on thecells membrane and the fluorescence intensity on HT-29cells membrane was stronger than it on Caco-2cell. We found that κ-CGN could up-regulation p-ERK, p-JNK and p-p38which were the importantproteins of MAPKs signal pathway in the HT-29cells. The inhibiton of MAPKs could reduce IL-8inHT-29cells. Also we had detected AP-1found that κ-CGN up-regulation transduction AP-1into thenucleus in HT-29cells. κ-CGN had ability to enhance the effect of LPS.When κ-CGN treated the coculturemodel at≦10μg/mL, there were many cytokins be determined in the cell supernatant. Furthermore,κ-CGN induced significantly apootosis evidenced by Annexin V-FITC and PI double staining. The antibodyTNF-R1could increase the effect of κ-CGN treated the intestinal model. So we could knew that the role ofTNF-α was the messenger between Caco-2and THP-1cells.In conclusion, λ-10-40kd and κ<5000could be development as antitumor drugs.Because of thedifferent stuctures and molecular weight the carrageenans had the different the effect of the human coloncells secreted cytokins which was induced by carrageenans. The κ-CGN had the strongest effect ofup-regulation human colon cells secreted IL-8, through TLR4-NF-κB-IL-8signal pathways. But there wereother signal pathways in HT-29cells, as MAPKs signal pathway. κ-CGN could induce inflammation at twoways, one was κ-CGN up-regulation TLR4in bowel, then it indirect induced inflammation by LPS. The... |
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