A probiotic multi-strain mixture combined with hydroxyectoine improves intestinal barrier function by alleviating inflammation in lipopolysaccharide stimulated differentiated Caco-2 cells

Many studies have highlighted the role of probiotics in re-establishing the gut microbiota balance and preventing intestinal barrier dysfunction. In fact, they can also contribute to the upregulation of anti-inflammatory genes and the downregulation of pro-inflammatory genes, which are known to contribute to the development of the inflammatory bowel disease (IBD) syndrome. The present study aims to investigate the effect of the compatible solute hydroxyectoine (HOE), to be used as a cryopreservant but also for its intrinsic biological properties, to obtain a new formula containing three probiotic strains (Limosilactobacillus fermentum (L. fermentum), Levilactobacillus brevis SP-48 (L. brevis), and Bifidobacterium lactis HN019 (B. lactis)), and evaluate the latter for its ability to prevent lipopolysaccharide (LPS)-induced inflammation in an in vitro bi-dimensional model of the intestinal barrier using a Caco-2 cell monolayer. The mRNA expression levels of the inflammatory cytokines (IL-6, IL-1 beta, and TNF-alpha) were analyzed by real-time PCR. Changes in the modulation of (TLR-4 and NF-kappa B) proteins were assessed by western blotting, and the effect of the HOE/PRO formula on the intestinal epithelial barrier function was also assessed using an immunofluorescence microscope for the tight junction protein zonula occludens-1 (ZO-1). This study found that this novel probiotic formulation containing HOE is capable of decreasing LPS-induced cytokines, as confirmed by the results of RT-PCR and ELISA and preserving the integrity of tight junctions as demonstrated by the relevant expression of ZO-1. HOE/PRO was shown to be effective in reducing the expression of TLR-4 and NF-kappa B. The latter plays a key role as an inflammation modulator as shown through experiments run with the THP-1/NF-kappa B reporter gene. Collectively, our data indicate that the HOE/PRO formula is a good candidate for potential preventive and/or therapeutic implementation in IBD.