κ-Carrageenan Enhances Lipopolysaccharide-Induced Interleukin-8 Secretion by Stimulating the Bcl10-NF- κ B Pathway in HT-29 Cells and Aggravates C. freundii-Induced Inflammation in Mice

Abstract

Background. The dietary usage of carrageenan as common food additive has increased observably over the last 50 years. But there is substantial controversy about its safety. Methods. We investigated whether the κ-carrageenan could enhance lipopolysaccharide-induced IL-8 expression by studying its actions on the TLR4-NF-κB pathway. The aggravating effect of κ-carrageenan on Citrobacter freundii DBS100-induced intestinal inflammation was also investigated in a mouse model. Results. Our data show that κ-carrageenan pretreatment promoted LPS-induced IL-8 expression in HT-29 cells. Although CD14, MD-2, and TLR4 were upregulated, the binding of LPS was not enhanced. However, the pathway of Bcl10-NF-κB was triggered. Interestingly, κ-carrageenan competitively blocked the binding of FITC-LPS. Furthermore, pretreatment with κ-carrageenan for one week previous to gavage with C. freundii DBS100 markedly aggravated weight loss, mortality, and colonic damage. The secretion of cytokines was unbalanced and the ratio of Tregs was decreased significantly. In addition, κ-carrageenan, together with C. freundii DBS100, enhanced the transcription and secretion of TLR4 and NF-κB. Conclusions. κ-Carrageenan can synergistically activate LPS-induced inflammatory through the Bcl10-NF-κB pathway, as indicated by its aggravation of C. freundii DBS100-induced colitis in mice. General Significance. Our results suggest that κ-carrageenan serves as a potential inflammatory agent that magnifies existing intestinal inflammation.

Figure 1

κ-Carrageenan enhances LPS-induced IL-8 secretion in HT-29 cells. (a) The effect of LPS on time-course and dose-response in HT-29 cells. (b) κ-Carrageenan increased LPS-induced expression of IL-8. IL-8 expression was assessed by ELISA in HT-29 cells treated with or without different concentrations of LPS (0.01, 0.1, and 1 μg/mL) for 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h or treated with κ-carrageenan at the indicated doses in the absence or presence of 1 μg/mL LPS. ^∗∗ P < 0.01, compared with the blank group. ^## P < 0.01, compared with the LPS treated alone.

Figure 2

κ-Carrageenan enhances LPS-induced TLR4 expression in HT-29 cells. (a) The effect of κ-carrageenan on the expression of TLR4 in HT-29 cells. (b) κ-Carrageenan increased LPS-induced expression of TLR4. Cells were treated with κ-carrageenan for 24 h or were pretreated with κ-carrageenan for 1 h and then with LPS (1 μg/mL) for 24 h. (c) TLR4 expression in HT-29 cells is affected by κ-carrageenan. Cells were treated with κ-carrageenan for 24 h and then were incubated with 1 μg PE-TLR4 for 25 min. ^∗ P < 0.05, ^∗∗ P < 0.01, compared with the blank group. ^# P < 0.05, ^## P < 0.01 compared with those in the LPS alone treated group.compared with the LPS treated alone. (d) y-axis shows the mean fluorescent intensity of PE-TLR4.

Figure 3

The impact of κ-carrageenan on mRNA expression of CD14 and MD-2 in LPS-induced HT-29 cells and binding of LPS. (a) The enhanced effect of κ-carrageenan on mRNA expression of CD14 and MD-2 in LPS-induced HT-29 cells. HT-29 cells were cultured with κ-carrageenan for 24 h or were pretreated with κ-carrageenan for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. (b) Effect of κ-carrageenan on FITC-LPS combined with cells. HT-29 cells were treated with the indicated concentrations of κ-carrageenan for 24 h and after cells were washed with PBS, and then 1 μg/mL FITC-LPS was added for 25 min. The intensity of fluorescently labeled cells is shown as MFI of FITC-LPS. (c) κ-Carrageenan competitively blocks the binding of LPS. HT-29 cells were treated with 1 μg/mL LPS or the indicated concentrations of κ-carrageenan for 1 h, and then 1 μg/mL FITC-LPS was added for 25 min. ^∗∗ P < 0.01, compared with the blank group. ^## P < 0.01 (n = 3), compared with the LPS treated alone.

Figure 4

κ-Carrageenan potentiates the LPS-stimulated activation of NF-κB. (a) The effect of κ-carrageenan on the expression of Bcl10, NF-κB, and p-IκBα in HT-29 cells. (b) κ-Carrageenan enhances the LPS-stimulated secretion of Bcl10, NF-κB, and p-IκBα. HT-29 cells were treated with κ-carrageenan for 1 h. (c) κ-Carrageenan induces transcriptional activation of NF-κB. ^∗ P < 0.05, ^∗∗ P < 0.01, compared with the blank group. ^# P < 0.05, ^## P < 0.01, compared with the LPS treated alone.

Figure 5

The impact of κ-carrageenan on weight loss and the mortality with Citrobacter freundii DBS100-stimulated inflammation. (a) Weight changes in percentage. (b) Survival ratios. κ-Carrageenan was orally administered for 1 week prior to bacterial inoculation. Infection was performed by oral gavage with 10⁹ CFU/mouse of C. freundii DBS100. After ten days, the weight and the survival rate of the animals were recorded. Repeated measures ANOVA was used to measure differences between the groups for A; differences over time for B and differences between the interaction of experiment and time for AB shown. ANOVA and LSD post hoc test were used to examine pairwise comparisons between the groups at individual time points. ^∗∗ P < 0.01, compared with the blank group. ^## P < 0.01, compared with the C. freundii DBS100-treated group.

Figure 6

The impact of κ-carrageenan on colon damage following Citrobacter freundii DBS100. (a) Blank group; (b) DBS100 treated alone; (c) LOW treated alone; (d) LOW + DBS100 cooperative accessing; (e) MED treated alone; (f) MED + DBS100 cooperative accessing; (g) HIG treated alone; (h) HIG + DBS100 cooperative accessing; (i) colon damage scores at 10 days after DBS100 administration. ^∗∗ P < 0.01, compared with the blank group. ^## P < 0.01, compared with the C. freundii DBS100-treated group. CG, congestion; EM, edema.

Figure 7

The impact of κ-carrageenan on histological changes and the HAI scores of Citrobacter freundii DBS100-stimulated mice. (a) Blank group; (b) DBS100 treated alone; (c) LOW treated alone; (d) LOW + DBS100 cooperative accessing; (e) MED treated alone; (f) MED + DBS100 cooperative accessing; (g) HIG treated alone; (h) HIG + DBS100 cooperative accessing. H&E: ×200. (i) The HAI scores of colonic in C. freundii DBS100-stimulated mice. ^∗∗ P < 0.01, compared with the blank group; ^## P < 0.01, compared with the DBS100-treated group. M, mucosa; SM, submucosa; ML, muscle layer.

Figure 8

SEM of morphology changes. (a) Blank group; (b) DBS100 treated alone; (c) LOW treated alone; (d) LOW + DBS100 cooperative accessing; (e) MED treated alone; (f) MED + DBS100 cooperative accessing; (g) HIG treated alone; (h) HIG + DBS100 cooperative accessing, ×200-fold. CP, crypts; IM, intact microvilli; DM, disintegrating microvilli; MS, mucus secretion at crypt opening; UE, ulcers and empty.

Figure 9

Ratio of CD4+CD25+CD127dim/CD4+ (Tregs) in mice. ^∗∗ P < 0.01, compared with the blank group. ^## P < 0.01, compared with the Citrobacter freundii DBS100 treated alone group.

Figure 10

The impact of κ-carrageenan on the secretion of TLR4 and NF-κB in the colonic mucosa of experimental mice. (a) The effect of κ-carrageenan on the secretion of TLR4 and NF-κB in mice, ×200 fold. (b) The impact of κ-carrageenan on the expression of TLR4 and NF-κB at the mRNA level. ^∗ P < 0.05, ^∗∗ P < 0.01, compared with the blank group. ^## P < 0.01, compared with the Citrobacter freundii DBS100-treated group.