An Experimental Study Of The Effects Of Anti-inflammatory Natural Product Chitooligosaccharides On Inflammatory Bowel Disease

PART I Effect of Chitooligosaccharides on Lipopolysaccharide-induced damage of intestinal epithelial cellsObjective To investigate the effect of chitooligosaccharides on the damage of intestinal epithelial cells induced by Lipopolysaccharides (LPS).Methods Intestinal epithelial cells Caco-2 were divided into five groups:normal group, model group, and high-, middle-and low-dose chitooligosaccharides groups. Lipopolysaccharides (LPS)-stimulated Caco-2 intestinal epithelial cells were used as an in vitro model of inflammatory bowel diseases. Caco-2 cells were pre-incubated with various concentrations of chitooligosaccharides (0.25,0.5 and 1.0 mg/ml) for 2h prior to being co-stimulated or not with LPS at a concentration of 1μg/mL for 48h. Cell viability was measured using MTT assay. The levels of proinflammatory mediators tumor necrosis factor(TNF)-α, interleukin-8 and prostaglandin (PG)E2 were analyzed using Enzyme-linked Immunosorbent Assay. Proportion of cells undergoing apoptosis was determined by flow cytometric analysis of cells stained with Annexin V and propidium iodide(PI). The ROS levels of Caco-2 cells were determined using DCFH-DA staining and flow cytometric analysis. The activities of SOD、GSH-Px and MDA levels in culture supernatants and cell lysates were measured to investigate the effect of chitooligosaccharides on the redox homeostasis in intestinal epithelial cells. And the expression of toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), cyclooxygenase (COX)-2, apoptosis-related protein caspase-3 and bcl-2 was examined by western blot.Results Compared with only LPS-treated group, chitooligosaccharides at the concentration of 0.25,0.5, 1.0mg/ml significantly and concentration-dependently reduced the release of proinflammatory mediators TNF-α, PGE2 and NO(P< 0.05; P< 0.01),0.5 and 1.0mg/ml chitooligosaccharides downregulated the COX-2 expression, iNOS activity and ROS level, upregulated the activities of SOD、GSH-Px, and decreased MDA levels in Caco-2 cells (P<0.05; P<0.01), suggesting chitooligosaccharides inhibited the LPS-induced inflammatory response and corresponding oxidant damage of intestinal epithelial cells. The cell viability was not affected by treatment with chitooligosaccharides and/or LPS. In addition, chitooligosaccharides decreased the apoptotic (Annexin V+/PI-) cell populations, accompanied by reduced levels of the pro-apoptotic protein caspase-3 and elevated levels of the anti-apoptotic bcl-2, suggesting chitooligosaccharides inhibited the LPS-induced intestinal epithelial cell apoptosis. In presence of chitooligosaccharides, the protein expressions of TLR4 and NF-κB in LPS-stimulated Caco-2 cells were reduced than those of the only LPS-treated group (P<0.05; P<0.01), these effects of chitooligosaccharides being presented in a concentration-dependent manner.Conclusions The present study suggests the potential medical use of chitooligosaccharides in the control of inflammatory bowel diseases, which may be due to the downregulation of TLR4/NF-κB pathway.PART Ⅱ Ang Ⅱ-TLR4-NF-κB-COX2 signaling pathway is involved in the protective effect of chitooligosaccharides on DSS-induced mouse colitisObjective To determine the effect of chitooligosaccharides, a kind of marine natural product, on the development of inflammatory bowel disease (IBD) using the dextran sodium sulfate (DSS) model of mouse colitis, and explore its possible mechanism.Methods C57BL/6J mice were randomly divided into 6 groups(10 mice in each group):normal control group, DSS model group, low, medium and high-dose chitooligosaccharides -treated groups, and sulfasalazine(SASP)-treated (positive control) group. Mice in the normal group were given distilled water. Experimental colitis was induced in mice from other groups by dissolving 3% DSS in their drinking water for 7 days. Mice in low, medium and high-dose chitooligosaccharides -treated groups were orally administered with 125,250 and 500mg/kg chitooligosaccharides every day for a week, respectively. Mice in SASP-treated group were given 50 mg/kg SASP as positive control. Animals in normal control and DSS model groups were orally administered with normal saline. The body weight changes and the occurrence of bloody diarrhea were observed at the same time of each day. After the last administration, colon were collected, colon length were determined and colon tissue pathology were scored. Serum angiotensin converting enzyme(ACE) activity was detected by ACE detection kit. Level of Prostaglandin(PG)E2 in colon mucosa tissue was detected by ELISA Kit. In addition, the mRNA and protein expressions of ACE, Toll-like receptor(TLR)4, Nuclear transcription factor-κB (NF-κB) and cyclooxygenase (COX)2 in colon were measured by Real-time PCR and immunofluorescence, respectively.Results Compared with DSS model group, chitooligosaccharides treatment (125-500mg/kg) significantly decreased DSS-induced reduction of body weight, relieved bloody diarrhea event, improved the degree of colon shortening of model mice, reduced the score of colon tissue pathology and decreased the level of PGE2 in colon mucosa tissue. In addition, the mRNA and protein expressions of ACE,TLR4,NF-κB and COX2 were significantly decreased in colon of DSS mice with chitooligosaccharides treatment.Conclusions chitooligosaccharides have a protective effect on the development of experimental colitis, the mechanism of which appears be related to their capacity to down-regulate the angiotensin-TLR4-NF-KB-COX2 signalling pathway.