Fucoidan derived from Cladosiphon okamuranus Tokida ameliorates murine chronic colitis through the down-regulation of interleukin-6 production on colonic epithelial cells

Abstract

Our previous study indicated that the interleukin (IL)-6/STAT-3 signal was up-regulated in inflammatory bowel disease (IBD) in both humans and animal models. We also discovered phosphorylated STAT-3 in the nucleus of the colonic epithelial cells in IBD mice. Intestinal epithelial cells (IEC) have been shown to secrete IL-6. Therefore, the secretion of IL-6 from IEC may be one of the mechanisms of STAT-3 phosphorylation in IEC during the pathogenesis of IBD, and inhibition of IL-6 production by IEC may be beneficial in preventing IBD. We examined the preventative effect of various types of fucoidans on IL-6 production in a lipopolysaccharide (LPS)-stimulated murine colonic epithelial cells line, CMT-93, in vitro. We also determined in vivo the effect of fucoidans on murine chronic colitis induced with dextran sodium sulphate. Among fucoidans, those from Cladosiphon okamuranus Tokida and Kjellmaniella crassifolia inhibited IL-6 production in CMT-93 cells with the down-regulation of NF-kappaB nuclear translocation. Analysis of the effect of fucoidan on murine colitis in vivo showed that the disease activity index and myeloperoxidase activity decreased in mice fed Cladosiphon fucoidan, but not Fucus fucoidan. Cytokine profiles in colonic lamina propria indicated that the synthesis of interferon (IFN)-gamma and IL-6 decreased and that of IL-10 and transforming growth factor (TGF)-beta increased in mice fed Cladosiphon fucoidan, compared with mice fed a standard diet or Fucus fucoidan. The levels of IL-6 mRNA in colonic epithelial cells was lower in colitis-induced Balb/c mice fed Cladosiphon fucoidan than those fed a standard diet. Fucoidan improves murine chronic colitis by down-regulating the synthesis of IL-6 in the colonic epithelial cells. Fucoidan derived from C. o. Tokida may be useful as a dietary substance for the patients with inflammatory bowel disease.

Fig. 1

Effects of various doses of LPS on IL-6 synthesis in the murine colonic epithelial cell line CMT-93. Cells were cultured in the absence or presence of various doses of LPS. After 72 h, the amount of IL-6 in the culture supernatant was determined by sandwich ELISA. Similar results were obtained from five independent experiments. Data are mean ±s.d.

Fig. 2

Effect of various fucoidans on IL-6 synthesis in LPS-stimulated CMT-93 cells (a). Various fucoidans were isolated from marine seaweed as described in Materials and methods. Cells were cultured in the presence of both purified fucoidans (2·5 µg/ml) and LPS (10 µg/ml) for 72 h. Dose-dependent inhibition of IL-6 release by fucoidans in an LPS-stimulated colonic epithelial cell line CMT-93 (b). CMT-93 was cultured with various doses of the fucoidan derived from C. o. Tokida or from K. crassifolia in the presence of LPS for 72 h. The amount of IL-6 in the culture supernatant was measured by an IL-6-specific sandwich ELISA. Three independent experiments gave similar results. Data are mean ±s.d.

Fig. 3

The effects of fucoidan in NF-κB activities in LPS-stimulated CMT-93 cells. The amounts of nuclear-localized p65 NF-κB in LPS-stimulated CMT-93 were determined by transfactor ELISA system. The amounts of nuclear-localized NF-κB in LPS-stimulated CMT-93 cells were down-regulated by treatment of Cladosiphon fucoidan. Two independent experiments gave similar results. Data are mean of duplicate assays.

Fig. 4

Disease activity index (a) and colon length (b) in colitis-induced Balb/c mice fed normal diet or fed diets containing fucoidans (n = 10, each group). Disease activity index and colon length were determined in colitis-induced Balb/c mice fed normal diet, or fed Cladosiphon fucoidan or Fucus fucoidan as described in Materials and methods. Similar results were obtained from three independent experiments. Data are mean ± s.d.

Fig. 5

MPO activity of the colonic tissue in colitis-induced Balb/c mice fed normal diet or fed fucoidan-containing diets (n = 10, each group). Colonic tissues were homogenated in HTMB buffer with a polytron-homogenizer. After centrifugation, MPO activity in the supernatants was measured as described in Materials and methods. Down-regulation of MPO activity in the colonic tissue in colitis-induced Balb/c mice fed Cladosiphon fucoidan observed in separate experiments is plotted as dots. Bars represent mean values.

Fig. 6

Cytokine profiles in the culture supernatants of TCR-β/CD28-stimulated LI–LPLs in colitis-induced Balb/c mice fed standard diet or fucoidan-containing diets (n = 10, each group). LI–LPLs were stimulated with or without MoAbs TCR-β/CD28. After 48 h culture, amounts of each cytokine were analysed by sandwich ELISA. Similar results were obtained from two independent experiments. Data are mean ± s.d.

Fig. 7

Immunoglobulin contents of colonic tissue in colitis-induced Balb/c mice fed normal diet, Cladosiphon fucoidan or Fucus fucoidan (n = 10, each group). The IgG, IgG1 and IgG2a contents in the colonic tissue were examined by sandwich ELISA using colonic tissue homogenates isolated from the three groups of mice. The total IgG contents of the mice fed Cladosiphon fucoidan was lower than that of the others and the differences were significant. The IgG2a content was significantly lower in the mice fed Cladosiphon fucoidan than Fucus fucoidan. Each plot shows individual data and bars represent mean values.

Fig. 8

RT-PCR analysis of cytokines or TLR-4 mRNA in colonic epithelial cells from colitis-uninduced Balb/c mice, or from colitis-induced Balb/c mice fed normal diet or Cladosiphon fucoidan. Total RNA was obtained from the colonic epithelial cell fraction of three experimental groups of mice. RT-PCR was performed with each specific primer for G3PDH, IL-6, TNF-α and TLR-4. After electrophoresis, the gel was stained with ethidium bromide and PCR products were visualized by trans-illuminator.