Bowel Inflammation and Nutrient Supplementation: Effects of a Fixed Combination of Probiotics, Vitamins, and Herbal Extracts in an In Vitro Model of Intestinal Epithelial Barrier Dysfunction

Abstract

The gut microbiota is a very important factor in the state of health of an individual, its alteration implies a situation of "dysbiosis," which can be connected to functional gastrointestinal disorders and pathological conditions, such as Inflammatory Bowel Disease (IBD), Irritable Bowel Syndrome (IBS), Ulcerative Colitis (UC) and Crohn's Disease (CD), and Colorectal Cancer (CRC). In this work, we studied the effect of a food supplement called ENTERO-AD containing a mix of probiotics (Lactobacillus acidophilus LA1, L. reuteri LR92, Bifidobacterium breve Bbr8), Matricaria Chamomilla, and B group vitamins (B1, B2, B6) on intestinal inflammation. The in vitro model used for the study is the Caco-2 cell, a culture derived from human intestinal adenocarcinoma; the inflammatory condition was achieved with treatment with Lipopolysaccharide (LPS) and the association between Tumor necrosis factor α/Interferon γ (TNF-α/IFN-γ) [1,2]. The effect of ENTERO-AD was evaluated by cell viability, measures of Transepithelial Electrical Resistance (TEER), paracellular permeability, and immunofluorescence. Results of the study have shown that ENTERO-AD has a favorable effect on Caco-2 cells in inflammatory conditions. It improves the integrity of Occludin and Zonula Occludens-1 (ZO-1) proteins, leading to an improvement in terms of TEER values and a reduction of paracellular permeability. This evidence underlines the protective effect of ENTERO-AD and its components in intestinal inflammation.

Keywords: Caco-2; Intestinal inflammation; TEER; probiotics.

Figure 1

Cell viability. Effect of ENTERO-AD 10x10³, 10x10⁴, 10x10⁵, 10x10⁶, 10x10⁷, 10x10⁸ CFU/mL of ENTERO-AD on the viability of the Caco-2 cell line after 48 hours of treatment. Data are expressed as Mean ± SD of three independent experiments. ***p<0.001; ****p<0.0001 treated vs control.

Figure 2

TEER measurements. a. Effect of LPS (250 μg/mL) and IFN-γ (10 ng/mL)/TNF-α (10 ng/mL) on the integrity of the intestinal epithelium barrier after 24 hours of treatment. b. Effect of ENTERO-AD 10x10⁴ and 10x10⁵ CFU/mL on the integrity of intestinal epithelium barrier after 24 hours of treatment. Data are expressed as % TEER compared to baseline (t₀) ± SD, obtained from three to four independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 treated vs control.

Figure 3

TEER measurements. a. Effect of LPS (250 μg/mL), ENTERO-AD (10x10⁴ CFU/mL), and relative association and b. LPS (250 μg/mL), ENTERO-AD (10x10⁵ CFU/mL), and relative association on the integrity of the intestinal epithelium barrier after 24 hours of treatment. c. Effect of IFN-γ (10 ng/mL)/TNF-α (10 ng/mL), ENTERO-AD (10x10⁴ CFU/mL) and relative association and d. IFN-γ (10 ng/mL)/TNF-α (10 ng/mL), ENTERO-AD (10x10⁵ CFU/mL), and relative association on the integrity of intestinal epithelium barrier after 24 hours of treatment. Data are expressed as % TEER from baseline (t₀) ± SD, obtained from three to four independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 treated vs control; #p<0.05, ##p<0.01, ###p<0.001, ####p<0.001 treated vs inflammation.

Figure 4

Paracellular Permeability. a. Effect of ENTERO-AD (10x10⁴ and 10x10⁵ CFU/mL), b. LPS (250 μg/mL) and associations with ENTERO-AD, c. IFN-γ (10 ng/mL)/TNF-α (10 ng/mL) and associations with ENTERO-AD on paracellular permeability. Data are expressed as ratio to control and are the mean ± SD of three to four independent experiments. *p<0.05, **p<0.01 treated vs control; #p<0.05 treated vs inflammation.

Figure 5

Immunofluorescence. Effect of ENTERO-AD (10x10⁴ and 10x10⁵ CFU/mL), LPS (250 μg/mL), IFN-γ/TNFα (10 ng/mL), and relative associations on Occludin morphology in the Caco-2 cell line at 48 hours of treatment. Marking of Occludin (green, Alexa Fluor 488). Acquisition using LSM 800 confocal microscope and ZEN 2.1 software with 60x objective. Images are representative of three independent experiments.

Figure 6

Immunofluorescence. Effect of ENTERO-AD (10x10⁴ and 10x10⁵ CFU/mL), LPS (250 μg/mL), IFN-γ/TNFα (10 ng/mL), and relative associations on ZO-1 morphology in the Caco-2 cell line at 48 hours of treatment. Marking of ZO-1 (red, Cy5). Acquisition using LSM 800 confocal microscope and ZEN 2.1 software with 60x objective. Images are representative of three independent experiments.