In vitro intestinal epithelial cell and fibroblast models to investigate the anti-inflammatory and anti-fibrotic effects of high concentration multi-strain probiotic formulations

The oral administration of probiotics has been shown to exert positive effects on chronic inflammatory disorders, such as inflammatory bowel disease (IBD). IBD is a recurrent inflammatory disease of the gastrointestinal tract. Several studies have demonstrated that hypoxia plays a key role in IBD. Hypoxia responses are modulated and affected by oxygen-sensitive transcription factors such as hypoxia-inducible factor (HIF). Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in inducing adaptive mechanisms to hypoxia and is negatively regulated by prolyl hydroxylase 2 (PHD2). Moreover, HIF-1α is stabilized through PI3K/AKT signaling regardless of oxygen levels. Considering the crucial role of the HIF pathway in intestinal mucosal physiology and its relationships with gut microbiota, the first objective of this study aimed to evaluate the ability of the lysate from the multi-strain probiotic formulation SLAB51 to affect the HIF pathway and to counteract the inflammatory stimulus of lipopolysaccharide (LPS) in a model of in vitro human intestinal epithelium (intestinal epithelial cells, IECs). The exposure of IECs to SLAB51 lysate under normoxic conditions led to a dose-dependent increase in HIF-1α protein levels, associated with higher glycolytic metabolism and L-lactate production. Probiotic lysate significantly reduced PHD2 levels and HIF-1α hydroxylation, thus leading to HIF-1α stabilization. SLAB51 lysate ability to increase HIF-1α levels was also associated with the activation of PI3K/AKT pathway and with the inhibition of NF-κB, nitric oxide synthase 2 (NOS2), and IL-1β increase elicited by LPS treatment. Our results suggest that the probiotic treatment, by stabilizing HIF-1α, can protect from LPS-induced inflammatory response through a mechanism involving PI3K/AKT signaling. An important complication associated to IBD is intestinal fibrosis. To date, no specific anti-fibrotic therapy exists to prevent or reverse intestinal fibrosis. Only few studies are available regarding the effects of probiotics on IBD-associated intestinal fibrosis. Therefore, the other aim of the present project has been to investigate the potential anti-fibrotic effect of two different high-concentration multi-strain probiotic formulations (VSL#3® and Vivomixx®) on CCD-18Co human intestinal fibroblasts activated by TGF-β1. CCD-18Co cells activated in vitro by TGF-β1 represent a model of intestinal fibrosis widely used in experimental studies. In particular, the cell growth and viability, Collagen-I, α-SMA, pSmad2/3 expression levels, and TGF-β1 levels were evaluated. The results suggested that Vivomixx® probiotic lysate, but not VSL#3® lysate, was able to counteract the pro-fibrotic effects of TGF-β1 on CCD-18Co cells. The mechanism underlying the observed effect appeared to be related to inhibition of the TGF-β1/Smad pathway. In conclusion, the results provide valuable evidence of Vivomixx®’s role as a promising therapeutic strategy against intestinal fibrosis in Crohn’s patients.