Anti-inflammatory effect of diosmectite in hapten-induced colitis in the rat

Abstract

1. Diosmectite is a natural silicate effectively used in the treatment of infectious diarrhoea. Its antidiarrhoeal properties involve adsorption of toxins and bacteria and modifications of the rheological characteristics of gastrointestinal mucus. Hence, the aim of this study was to test the intestinal anti-inflammatory activity of diosmectite. 2. Diosmectite (500 mg x kg(-1) day(-1), p.o.) was administered as a post-treatment to rats with chronic trinitrobenzene sulphonic acid colitis. Colonic status was checked 1 and 2 weeks after colitis induction by macroscopic, histological and biochemical examination. 3. Diosmectite post-treatment resulted in amelioration of the morphological signs (intestinal weight, macroscopic damage, necrosed area, histology) and biochemical markers (myeloperoxidase activity, glutathione levels, MUC2 expression, inducible nitric oxide synthase and interleukin-1beta (IL-1beta) and leukotriene B(4) synthesis), as well as in the reduction of the severity of diarrhoea. The effect of the clay was comparable to that of sulphasalazine (50 mg x kg(-1) day(-1)). 4. 5. Diosmectite exhibited a dose-dependent capacity to adsorb proteins in vitro as well as a dose-dependent inhibitory effect on the basolateral secretion of IL-8 by lipopolysaccharide (LPS)-stimulated HT29 cells. Diosmectite had a dose-dependent inhibitory effect on IL-1beta production by LPS-stimulated THP-1 cells. 6. The effect of diosmectite on MUC2 was post-transcriptional, since mRNA levels were unaffected. However, diosmectite is able to upregulate MUC2 mRNA levels in HT29-MTX cells. 7. Diosmectite has anti-inflammatory activity administered as a post-treatment. Possible mechanisms include adsorption of luminal antigens, increase of colonic mucin levels and possibly a direct modulatory action of cytokine production by mucosal cells.

Figure 1

Histological sections of TNBS colitic rats at 1 week. (a) Control group. (b). TNBS group. (c) Diosmectite group. Bar: 200 μm.

Figure 2

Biochemical parameters of TNBS colitic rats. The colonic levels of MPO, LTB₄ and glutathione are shown. ⁺P<0.05 vs group C; ^*P<0.05 vs group T.

Figure 3

Colonic expression of IL-1β, iNOS, MUC and MUC4. Representative Western blots and densitometric analysis (+, positive control; C, control group; T, TNBS group; D, diosmectite group). ⁺P<0.05 vs control group; ^*P<0.05 vs TNBS group.

Figure 4

mRNA levels of mucins and TFF3 in TNBS colitis. RT–PCR analysis and quantification are shown. ⁺P<0.05 vs control group.

Figure 5

Effect of diosmectite on HT29 and THP-1 cells. (Upper panel) Dose–response curve of the effect of apically added diosmectite on LPS-stimulated IL-8 secretion by HT29 cells. ^*P<0.05 vs no addition of diosmectite. The effect of the clay was not attributable to a deleterious effect on the cells, because the levels of LDH in the culture medium were similar in the control and diosmectite groups (data not shown). Values are mean±s.e.m. No significant effects were detected. (Bottom panel) Effect of diosmectite on IL-1β expression in THP-1 cells. D: diosmectite. The clay reduced the extracellular and intracellular levels of the cytokine. The positive control (left lane) was the supernatant from activated THP-1 cells from a different experiment.

Figure 6

Effect of diosmectite on mucin mRNA levels in HT29-MTX cells. The gels shown are representative of at least quadruplicate experiments. Diosmectite strongly augments MUC2 expression at 1 mg ml⁻¹.

Figure 7

Binding curves of protein substrates to diosmectite. (a) Protein concentrates. (b) Purified enzymes. Proteins were incubated in HT29 culture medium with increasing concentrations of the clay at 4°C. The amount of protein cleared by centrifugation at 7000 × g was considered to bind diosmectite. Values are mean±s.e.m.