Mechanisms of antidiarrhoeal effects by diosmectite in human intestinal cells

Abstract

Background: Rotavirus (RV) induces diarrhoea through a sequence of enterotoxic and cytotoxic effects. The former are NSP4-dependent, induce calcium-dependent chloride secretion and involve oxidative stress. Diosmectite (DS) is a natural clay that has been recommended as an active therapy for diarrhoea, but the mechanism of its effect is not clear. Electrical parameters may be used to measure the direct enterotoxic and cytotoxic effects in polar epithelial intestinal cells. To investigate the effects of DS on RV-induced enterotoxic and cytotoxic damage. Caco-2 cells were used as a model of RV infection to evaluate chloride secretion, epithelial integrity, oxidative stress and viral infectivity in Ussing chambers.

Results: Diosmectite reduced the expression of NSP4 and oxidative stress, resulting in a strong inhibition of chloride secretion. Preincubating RV with DS reduced the cytotoxic effect. Finally, the viral load was reduced by DS but not by control clay. This result suggests that DS specifically affects the early events of RV infection protecting the enterocyte, whereas it does not restore already-established cell damage.

Conclusion: These findings indicate that DS exerts an anti-diarrhoeal effect by inhibiting viral replication and the expression of NSP4. Both ion secretion and cell damage induced by RV are strongly inhibited consequent to the antiviral effect, which explains its clinical efficacy.

Keywords: Chloride secretion; Diosmectite; Oxidative stress; Rotavirus infection.

Fig. 1

Binding of RV to DS. DS was incubated with RV, and the suspension was then probed with fluorescein isothiocyanate (FITC) conjugated anti-RV antibody as described in the “Methods”. DS alone or TMG incubated with RV was used as negative controls. Images are representative of three separate experiments

Fig. 2

Effect of DS on RV infectivity. Caco-2 cells were infected with RV with or without DS for 3 days. a Quantitative analysis of RV-infected Caco-2 cells. Caco-2 cell monolayers were infected with RV preincubated in the presence (filled triangle) or absence (open circle) of DS as described in the “Methods”, and the RV-infected cells (RV+) were counted. TMG (times) was used as the negative control together with uninfected cells (filled circle) [*p < 0.05 vs DS-treated RV]. b Viral titer of the culture medium of RV-infected Caco-2 cells alone (grey) or preincubated with DS (white) [*p < 0.05 vs RV at 24 h;^#p < 0.05 vs RV at the same time]

Fig. 3

Effects of DS on the enterotoxic effect induced by RV. Caco-2 cell monolayers were infected with RV preincubated in the presence (open triangle) or absence (filled circle) of DS for 2 h as described in the “Methods”, and then the short-circuit current (Isc) was evaluated in Ussing chambers together with uninfected cells (open square). This is a single representative experiment

Fig. 4

DS prevents the enterotoxic effect induced by RV. a Maximal effect by RV on Isc. Three independent experiments were pooled to analyse the statistical significance [*p < 0.05 vs control; **p < 0.05 vs RV]. b DS totally inhibited the enterotoxic effect of RV on the Isc in a dose-dependent manner [*p < 0.05 vs RV alone]. c NSP4 expression was substantially inhibited by pretreatment with DS

Fig. 5

Effect of DS on oxidative stress induced by RV. a Caco2 cells were infected with RV following preincubation in the absence or presence of DS, and the fluorescence of the ROS probe was evaluated 1 h following the infection. Magnification: 400×. b Time-course of ROS production. Caco-2 cell monolayers were infected with RV preincubated in the presence (filled circle) or absence (filled square) of DS as described in the “Methods”, and the ROS intracellular levels were evaluated together with uninfected cells (open circle) [*p < 0.05 vs the control at the same time]. c Caco-2 cell monolayers were infected with RV, glutathione was evaluated 1 h following the infection, and the levels of GSH (grey) and GSSG (white) were measured. DS was present during the activation phase of the virus, as described in the “Methods”. *p < 0.05 vs control;^#p < 0.05 vs RV

Fig. 6

Effect of DS on cytotoxic damage induced by RV. a Caco-2 cells monolayers were infected with RV following preincubation in the presence or absence of DS for 48 h. Caco-2 cell monolayers were infected with RV following preincubation in the presence (filled triangle) or absence of DS (open square) and the transepithelial electrical resistance (TEER) was evaluated as described in the “Methods” together with uninfected cells (open circle); *p < 0.05 vs the control at the same time. b Effect of DS on RV-induced cytotoxic damage compared with an increasing viral load. *p < 0.05 vs control; ^#p < 0.05 vs RV 25 and 50 MOI