Chitin-glucan improves important pathophysiological features of irritable bowel syndrome

Abstract

Background: Irritable bowel syndrome (IBS) is one of the most frequent and debilitating conditions leading to gastroenterological referrals. However, recommended treatments remain limited, yielding only limited therapeutic gains. Chitin-glucan (CG) is a novel dietary prebiotic classically used in humans at a dosage of 1.5-3.0 g/d and is considered a safe food ingredient by the European Food Safety Authority. To provide an alternative approach to managing patients with IBS, we performed preclinical molecular, cellular, and animal studies to evaluate the role of chitin-glucan in the main pathophysiological mechanisms involved in IBS.

Aim: To evaluate the roles of CG in visceral analgesia, intestinal inflammation, barrier function, and to develop computational molecular models.

Methods: Visceral pain was recorded through colorectal distension (CRD) in a model of long-lasting colon hypersensitivity induced by an intra-rectal administration of TNBS [15 milligrams (mg)/kilogram (kg)] in 33 Sprague-Dawley rats. Intracolonic pressure was regularly assessed during the 9 wk-experiment (weeks 0, 3, 5, and 7) in animals receiving CG (n = 14) at a human equivalent dose (HED) of 1.5 g/d or 3.0 g/d and compared to negative control (tap water, n = 11) and positive control (phloroglucinol at 1.5 g/d HED, n = 8) groups. The anti-inflammatory effect of CG was evaluated using clinical and histological scores in 30 C57bl6 male mice with colitis induced by dextran sodium sulfate (DSS) administered in their drinking water during 14 d. HT-29 cells under basal conditions and after stimulation with lipopolysaccharide (LPS) were treated with CG to evaluate changes in pathways related to analgesia (µ-opioid receptor (MOR), cannabinoid receptor 2 (CB2), peroxisome proliferator-activated receptor alpha, inflammation [interleukin (IL)-10, IL-1b, and IL-8] and barrier function [mucin 2-5AC, claudin-2, zonula occludens (ZO)-1, ZO-2] using the real-time PCR method. Molecular modelling of CG, LPS, lipoteichoic acid (LTA), and phospholipomannan (PLM) was developed, and the ability of CG to chelate microbial pathogenic lipids was evaluated by docking and molecular dynamics simulations. Data were expressed as the mean ± SEM.

Results: Daily CG orally-administered to rats or mice was well tolerated without including diarrhea, visceral hypersensitivity, or inflammation, as evaluated at histological and molecular levels. In a model of CRD, CG at a dosage of 3 g/d HED significantly decreased visceral pain perception by 14% after 2 wk of administration (P < 0.01) and reduced inflammation intensity by 50%, resulting in complete regeneration of the colonic mucosa in mice with DSS-induced colitis. To better reproduce the characteristics of visceral pain in patients with IBS, we then measured the therapeutic impact of CG in rats with TNBS-induced inflammation to long-lasting visceral hypersensitivity. CG at a dosage of 1.5 g/d HED decreased visceral pain perception by 20% five weeks after colitis induction (P < 0.01). When the CG dosage was increased to 3.0 g/d HED, this analgesic effect surpassed that of the spasmolytic agent phloroglucinol, manifesting more rapidly within 3 wk and leading to a 50% inhibition of pain perception (P < 0.0001). The underlying molecular mechanisms contributing to these analgesic and anti-inflammatory effects of CG involved, at least in part, a significant induction of MOR, CB2 receptor, and IL-10, as well as a significant decrease in pro-inflammatory cytokines IL-1b and IL-8. CG also significantly upregulated barrier-related genes including muc5AC, claudin-2, and ZO-2. Molecular modelling of CG revealed a new property of the molecule as a chelator of microbial pathogenic lipids, sequestering gram-negative LPS and gram-positive LTA bacterial toxins, as well as PLM in fungi at the lowesr energy conformations.

Conclusion: CG decreased visceral perception and intestinal inflammation through master gene regulation and direct binding of microbial products, suggesting that CG may constitute a new therapeutic strategy for patients with IBS or IBS-like symptoms.

Keywords: Abdominal pain; Chitin-glucan; Inflammation; Intestinal barrier; Irritable bowel syndrome; Microbial cell walls chelation; Molecular modelling.

Figure 1

Long-lasting visceral hypersensitivity in rats. Chitin-glucan analgesic effect evaluated by pain thresholds at week (W)-2-0-3-5-7.

Figure 2

Dextran sodium sulfate-induced colitis in C57BL/6 mice. DSS: Dextran sodium sulfate; DAI: Disease activity index.

Figure 3

Time- and dose-related analgesic effects of chitin-glucan in rats with long-lasting TNBS-induced hypersensitivity. Inhibition of pain perception in % at week (W) 0-3-5-7 compared to W-2 in untreated animals receiving tap water (Control in blue), untreated animals sensitized by TNBS (TNBS in black), TNBS sensitized rats treated with phloroglucinol (Phloro in grey), TNBS sensitized rats treated with chitin-glucan at 1.5 g/d [chitin glucan (CG) 1.5 g/d in green], TNBS sensitized rats treated with chitin-glucan at 3.0 g/d (CG 3.0 g/d in orange). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. CG: Chitin glucan.

Figure 4

Improvement of clinical and histological scores in animals with colitis receiving chitin-glucan treatment. A: Stool consistency in mice with colitis [Dextran sodium sulfate (DSS), black] was improved by chitin glucan (CG) treatment at a concentration of 1.5 g/d (DSS + CG: 1.5 g, grey), or 3 g/d (DSS + CG: 3.0 g, white); B: Disease activity index (DAI) in mice with colitis (DSS, black) was improved by CG treatment at 1.5 g/d (DSS + CG: 1.5 g, grey), or 3.0 g/d (DSS + CG: 3.0 g, white); C: Regeneration score in mice with colitis (DSS-induced, black) was improved by CG treatment at 1.5 g/d (DSS + CG: 1.5 g, grey), or 3.0 g/d (DSS + CG: 3.0 g, white); D: Histologial score in mice with colitis (DSS, black) was improved by CG treatment at 1.5 g/d (DSS + CG: 1.5 g, grey), or 3.0 g/d (DSS + CG 3.0 g, white). ^aP < 0.05. DSS: Dextran sodium sulfate; CG: Chitin glucan; DAI: Disease activity index.

Figure 5

Modulation of analgesic-related receptors in HT-29 cells incubated with chitin-glucan at different concentrations, with or without lipopolysaccharide stimulation. A: Modulation of analgesic-related receptors in non-stimulated HT-29 cells incubated for 3 h in medium alone (medium), chitin-glucan at 500 microgram per milliliter [chitin glucan (CG) 500, grey], or chitin-glucan at 1000 microgram per milliliter (CG 1000, dark grey); B: Modulation of analgesic-related receptors in HT-29 cells stimulated during 24 h with lipopolysaccharide (LPS) and incubated 3 additional hours in medium alone (LPS), chitin-glucan at 500 microgram per milliliter (LPS + CG: 500, white), or chitin-glucan at 1000 microgram per milliliter (LPS + CG: 1000, black). ^aP < 0.05, ^bP < 0.01. MOR: Mu-opioid receptor; CB2: Cannabinoid receptor; CG: Chitin glucan; LPS: Lipopolysaccharide.

Figure 6

Modulation of inflammatory-related cytokines in HT-29 cells incubated with chitin-glucan at different concentrations, with or without lipopolysaccharide stimulation. A: Modulation of inflammatory-related cytokines in non-stimulated HT-29 cells incubated for 3 h in medium alone (medium, white), chitin-glucan at 500 microgram per milliliter [chitin glucan (CG): 500, grey)], or chitin-glucan at 1000 microgram per milliliter (CG 1000, dark grey); B: Modulation of inflammatory-related cytokines in HT-29 cells stimulated during 24 h by lipopolysaccharide (LPS) and incubated 3 additional hours in medium alone (LPS, white), chitin-glucan at 500 microgram per milliliter (LPS + CG 500, grey), or chitin-glucan at 1000 microgram per milliliter (LPS + CG 1000, black). ^bP < 0.01. IL-1b: Interleukin-1 beta; IL-8: Interleukin-8; IL-10: Interleukin-10; CG: Chitin glucan; LPS: Lipopolysaccharide.

Figure 7

Modulation of intestinal barrier-related molecules in HT-29 cells incubated with chitin-glucan at different concentrations, with or without lipopolysaccharide stimulation. A: Modulation of intestinal barrier-related molecules in non-stimulated HT-29 cells incubated for 3 h in medium alone (medium, white), chitin-glucan at 500 microgram per milliliter [chitin glucan (CG): 500, grey)], or chitin-glucan at 1000 microgram per milliliter (CG 1000, dark grey); B: Modulation of intestinal barrier-related molecules in HT-29 cells stimulated during 24 h by lipopolysaccharide (LPS) and incubated 3 additional hours in medium alone (LPS, white), chitin-glucan at 500 microgram per milliliter (LPS + CG: 500, grey), or chitin-glucan at 1000 microgram per milliliter (LPS + CG: 1000, black). ^bP < 0.01. MUC5AC: Mucin-5AC; ZO-2: Zonula occludens-2; CG: Chitin glucan; LPS: Lipopolysaccharide.

Figure 8

Molecular modelling of interaction between lipopolysaccharide and chitin-glucan. A: Trapping of lipopolysaccharide (LPS) (O antigen moiety colored in yellow and lipid A moiety colored in magenta) by chitin-glucan (CG) (chitin moiety colored in orange and glucan moiety colored in green); B: Folding of CG into a hairpin showing the cavity that hosts LPS in its middle (chitin moiety colored in orange and glucan moiety colored in green); C: Interactions of LPS (O antigen moiety colored in magenta and lipid A moiety colored in blue) with CG (chitin moiety colored in orange and glucan moiety colored in green) via a network of 18 hydrogen bonds (yellow dashed line).

Figure 9

Molecular modelling of interaction between lipoteichoic acid and chitin-glucan. A: Trapping of lipoteichoic acid (LTA) (colored in yellow) by chitin-glucan (CG) (chitin moiety colored in orange and glucan moiety colored in green); B: Folding of CG showing a small hole occupied by the shortest lipidic side chain of LTA (chitin moiety colored in orange and glucan moiety colored in green); C: Interactions of LTA (colored in magenta) with CG (chitin moiety colored in orange and glucan moiety colored in green) via four hydrogen bonds (yellow dashed line).

Figure 10

Molecular modelling of interaction between phospholipomannan and chitin-glucan. A: Trapping of phospholipomannan (PLM) (colored in yellow) by chitin-glucan (CG) (chitin moiety colored in orange and glucan moiety colored in green); B: Folding of CG showing the large cavity that hosts PLM in its middle (chitin moiety colored in orange and glucan moiety colored in green); C: Lipophilic potential surfaces (according to the lipophilicity scale from blue for the minimum until brown for the maximum) of PLM trapped by CG (chitin moiety colored in orange and glucan moiety colored in green); D: Interactions of PLM (colored in magenta) with CG (chitin moiety colored in orange and glucan moiety colored in green) via a network of 13 hydrogen bonds (yellow dashed line).