Lacticaseibacillus rhamnosus GG Counteracts Rotavirus-Induced Ion Secretion and Enterocyte Damage by Inhibiting Oxidative Stress and Apoptosis Through Specific Effects of Living and Postbiotic Preparations

Abstract

Background: Administration of Lacticaseibacillus rhamnosus GG (LGG) to children with gastroenteritis is recommended by universal guidelines. Rotavirus (RV) causes diarrhea through combined cytotoxic and enterotoxic effects. Aim of this study was to evaluate the mechanisms of efficacy of LGG in an in-vitro model of RV diarrhea in its viable form (LGG) and conditioned medium (mLGG).

Methods: Ion secretion corresponding to the NSP4 enterotoxic effect, was evaluated by short circuit current (Isc) and the cytotoxic effect by transepithelial electrical resistance (TEER) in Ussing chambers, upon exposure to RV in Caco-2 enterocyte monolayers treated or not with living probiotic or its culture supernatant. Mechanisms of enterotoxic and cytotoxic damage were evaluated including oxidative stress measured by reactive oxygen species, apoptosis evaluated by DAPI and nuclear staining, NFkβ immunofluorescence.

Results: RV induced Isc increase and TEER decrease, respectively indicating ion secretion and epithelial damage, the two established pathways of diarrhea. Both probiotic preparations reduced both diarrheal effects, but their potency was different. Live LGG was equally effective on both enterotoxic and cytotoxic effect whereas mLGG was highly effective on ion secretion and showed minimal protective effects on cytoskeleton, apoptosis and NFkβ.

Conclusions: LGG counteracts RV-induced diarrhea by inhibiting both cytotoxic and enterotoxic pathogenic mechanisms. Namely, LGG inhibits chloride secretion by specific moieties secreted in the medium with a direct pharmacologic-like action. This is considered a postbiotic effect. Subsequently, live bacteria exert a probiotic effect protecting the enterocyte structure.

Keywords: Lacticaseibacillus rhamnosus GG; diarrhea; enterocyte damage; gastroenteritis; oxidative stress; postbiotics; probiotics; rotavirus.

Figure 1

Effects of LGG on enterotoxic effect induced by RV. Caco-2 cell monolayers were infected with RV and preincubated with LGG or mLGG as described in the Methods and then, the short-circuit current (Isc) was evaluated in Ussing chambers togheter with unifected cells (CTRL). *p < 0.05 vs CTRL; ^#p < 0.05 vs RV; ^§p < 0.05 vs LGG+RV.

Figure 2

Effect of LGG on oxidative stress induced by RV. (A) ROS production was evaluated in Caco-2 cell monolayers infected with RV preincubated with LGG or mLGG as described in the Methods. [*p< 0.05 vs CTRL; #p<0,05 vs RV]. (B) Caco2 cells were infected with RV following preincubation in the absence of LGG or mLGG and the fluorescence of the ROS probe was evaluated one hour following infection. Magnification: 400X (C) Caco-2 cells monolayers were infected with RV and glutathione was evaluated one hour following infection and levels of GSH (gray) and GSSG (white) were measured. LGG and mLGG preincubation are present during the activation phase of the virus as described in the Methods. [*p < 0.05 vs control; ^#p < 0.05 vs RV]. In all experiments, NAC was used as antioxidant factor.

Figure 3

Effect of LGG on cytotoxic damage induced by RV. (A) Occludin, as marker of tight junction structure, was evaluated with immunofluorescence in Caco-2 cell monolayers infected with RV preincubated with LGG or mLGG as described in the Methods Magnification: 400X. (B) Caco-2 cells monolayers were infected with RV following preincubation in the of LGG or mLGG and transepithelial electrical resistence (TEER) was evaluated as described in the Methods together with uninfected cells; [*p < 0.05 vs CTRL; ^#p < 0.05 vs RV]. (C) Phalloidin, as marker of cytoskeleton architecture, was used with immunofluorescence in Caco-2 cell monolayers infected with RV preincubated with LGG or mLGG as described in the Methods Magnification: 400X.

Figure 4

Effect of LGG on apoptosis induced by RV. (A) Apoptotic nuclei were evaluated in Caco-2 cell monolayers infected with RV preincubated with LGG or mLGG as described in the Methods Magnification: 400X. (B) Caco2 cells were infected with RV following preincubation in the absence of LGG or mLGG and caspase-3 activity as apoptotic marker was evaluated as described in Methds [*p < 0.05 vs CTRL; ^#p < 0.05 vs RV].

Figure 5

Effect of LGG on RV-induced activation of NF-kB pathway in Caco-2 cells. (A) Activated NF-kB p65 subunit (upper panel) was evaluated in RV-infected Caco-2 cells with or without the addition of mLGG or LGG and compared to total p65 levels in a western blot experiment. (B) NF-kB p65 subunit was detected in RV-infected cells with or without the addition of mLGG or LGG with immunofluorescent method as described in “Method” section and nuclei were stained by Hoerst. Red arrows indicate the p65 nuclear localization. Data are representative of 3 separate experiments.