Supplementation With 2'-FL and scGOS/lcFOS Ameliorates Rotavirus-Induced Diarrhea in Suckling Rats

Abstract

Rotavirus (RV) is considered to be the most common cause of gastroenteritis among infants aged less than 5 years old. Human milk bioactive compounds have the ability to modulate the diarrheic process caused by several intestinal pathogens. This study aimed to evaluate the potential protective role of a specific human milk oligosaccharide, 2'-fucosyllactose (2'-FL), a mixture of the prebiotic short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides 9:1 (GOS/FOS) and their combination (2'-FL+GOS/FOS) on RV-induced diarrhea in suckling rats. The nutritional intervention was performed from the second to the sixteenth day of life by oral gavage and on day 5 an RV strain was orally administered to induce infection. Fecal samples were scored daily to assess the clinical pattern of severity, incidence and duration of diarrhea. Blood and tissues were obtained at day 8 and 16 in order to evaluate the effects on the epithelial barrier and the mucosal and systemic immune responses. In the assessment of severity, incidence and duration of diarrhea, both 2'-FL and GOS/FOS displayed a beneficial effect in terms of amelioration. However, the mechanisms involved seemed to differ: 2'-FL displayed a direct ability to promote intestinal maturation and to enhance neonatal immune responses, while GOS/FOS induced an intestinal trophic effect and an RV-blocking action. The combination of 2'-FL and GOS/FOS showed additive effects in some variables. Therefore, it could be a good strategy to add these compounds in combination to infant formulas, to protect against human RV-induced diarrhea in children.

Keywords: 2′-FL; diarrhea; prebiotic; rotavirus; scGOS/lcFOS; suckling rats.

Figure 1

Effect of GOS/FOS, 2′-FL and their combination on histomorphometric variables and the intestinal gene expression of maturation markers at the peak of diarrhea (day 8). (A) Representative images of histological sections of the jejunum stained with hematoxylin and eosin, 100X. (B) Villi height, villi width, villi area, crypts depth, villi height/crypts depth ratio and perimeter of the jejunum of suckling rats. (C) Relative gene expression of the maturation markers Pdmr (encoding for Blimp-1) and Fcgrt (encoding for FcRn) by real-time PCR. The relative gene expression was calculated with respect to REF, which corresponded to 100% of transcription. Results are expressed as mean ± S.E.M. (n = 4–8). *p < 0.05 compared to REF group; ^#p < 0.05 compared to RV group (by MWU test).

Figure 2

Clinical indices of diarrhea. (A,B) The severity of diarrhea is represented with the Diarrhea Index (DI), which is based on scoring fecal samples from 1 to 4 depending on the color, texture and abundance. Scores of DI ≥2 indicate presence of diarrhea, whereas scores < 2 indicate absence of diarrhea. (C,D) The incidence of diarrhea is represented with the percentage of diarrheic animals (%DA), which is based on the percentage of animals displaying DI scores ≥2 in each group. (A,C) Data is shown non-normalized or (B,D) normalized for the RV+GOS/FOS and RV+GOS/FOS+2′-FL groups taking into account basal values. (E) The fecal weight, as an objective indicator of the severity of diarrhea, was calculated as the mean value in the pre-diarrhea, diarrhea and post-diarrhea periods. Results are expressed as mean ± S.E.M. for severity (n = 3–24, depending on the number of fecal samples obtained each day) and unique values for incidence (derived from all animals each day). Statistical differences not shown in (A–D). *p < 0.05 compared to REF group; ^#p < 0.05 compared to RV group (by MWU test).

Figure 3

Viral shedding and in vitro blocking activity of the oligosaccharides used in this study. (A) The viral shedding in feces was assessed at the peak of viral elimination, corresponding to day 6. (B) The blocking activity of the oligosaccharides was tested by an in-house in vitro blocking assay using the ELISA technique. SA11 stock was diluted with PBS-Tween 1% to reach a concentration of 5 × 10⁴ viral particles/mL. Starting from the in vivo-administered concentration, different dilutions (from 1/2 to 1/16) of GOS/FOS, 2′-FL and GOS/FOS+2′-FL were preincubated with the virus at 1/1 ratio for 30 min. Free, non-coated viral particles were quantified by ELISA. The dotted line represents the addition of the results of the GOS/FOS and 2′-FL analyzed separately. Results are expressed as mean ± S.E.M. (n = 8 for viral shedding and n = 2 replicates from 2 experiments for the blocking assay). *p < 0.05 compared to REF group; ^#p < 0.05 compared to RV group (by MWU test).

Figure 4

Assessment of oligosaccharide supplementation on the intestinal barrier function. The effects on the intestinal barrier function was studied at the peak of diarrhea (day 8). (A) The alpha-1 antitrypsin (A1AT) concentration in the gut wash was analyzed by ELISA as a measure of the intestinal barrier disruption. (B) Relative expression of tight junction molecules Cldn2 and Ocln and the mucin Muc2 were quantified by real-time PCR. Relative gene expression was calculated with respect to REF, which corresponded to 100% of transcription. Results are expressed as mean ± S.E.M. (n = 4–8). *p < 0.05 compared to REF group; ^#p < 0.05 compared to RV group (by MWU test).

Figure 5

Assessment of cytokine gene expression in the small intestine. Cytokines were quantified in the gut wash by real-time PCR at the peak of diarrhea (day 8). Relative gene expression was calculated in respect to REF, which corresponded to 100% of transcription. Results are expressed as mean ± S.E.M. (n = 4–8). *p < 0.05 compared to REF group; ^#p < 0.05 compared to RV group (by MWU test).

Figure 6

Cecal SCFA composition at the end of the study (day 16). Total SCFA, acetic, propionic, isobutyric, butyric and isovaleric acid production was quantified by HS-GC-MS. Results are expressed as mean ± S.E.M. (n = 9). *p < 0.05 compared to REF group; ^#p < 0.05 compared to RV group (by MWU test).