Probiotic Lactobacillus strains attenuate oxysterols-induced alteration of human intestinal membrane permeability

The intestinal membrane is an important structure which carries out central functions such as nutrient absorption and excretion and secretion of several products and acts as a barrier to protect the human body from potentially harmful compounds arriving from diet; foods are sources of both potentially dangerous and potentially protective molecules. When a noxious stimulus occurs, it can alter membrane balance and functionality, mostly by altering the tight junctions, increasing its permeability, or causing a shift on microbiota composition and thus sustaining inflammation. Inflammation and oxidative stress have been linked to the loss of intestinal integrity, a crucial event in the initiation and progression of pathological intestinal disorders such as inflammatory bowel diseases (IBD) and cancer. Oxysterols are cholesterol oxidative products, which have been reported to act negatively on intestinal membrane, causing an increase in its permeability and a local inflammation. On the other hand, several studies reported probiotics and short chain fatty acids (SCFAs), produced by the microbiota, as able to exert anti-inflammatory properties and to improve gut barrier permeability. In this context, the aim of this research project was to evaluate the capacity of two probiotic strains of Lactobacillus (Lactobacillus plantarum 299v® (DMS 9843) and Lactobacillus casei DG® (CNCMI1572), used as bacterial extract or live culture, to protect intestinal epithelium against the alteration of permeability induced by oxysterols and to investigate the mechanism of action in relation to tight junctions modulation and cellular signaling. A preliminary study was also conducted to investigate any modifications induced by oxysterols or probiotics on the metabolic activity of the gut resident bacteria. To achieve this objective, monolayers of differentiated Caco-2 cells have been used as in vitro model of intestinal barrier, and a batch culture system, to mimic the colonic environment. The alteration of cell monolayers permeability, treated with oxysterols alone or together with the bacterial extract or live culture was evaluated through the measurement of transepithelial electrical resistance (TEER), in relation to the modulation of tight junctions, occludin, zonulin and JAM-A, linked to MAPKs, p38 and ERK1/2 activation. Batch culture system have been inoculated with a human faecal sample in a basal media added with oxysterols and/or the bacterial extract or the live culture of the two different strains of the lactobacillus in order to evaluate, possible changes in microbiota SCFAs production. Our results provide, for the first time, evidence of the ability of Lactobacillus spp. probiotics to protect intestinal cells against the pro-inflammatory effect of oxysterols, in vitro. We observed a protective effect turned toward one key inflammatory mechanism such as the alteration of the intestinal permeability, caused by the oxysterols-induced TJs disruption, due, at least in part, to the modulation of MAPK/p38 pathway. The similar efficacy exerted by the bacterial extracts and the pure cultures, suggest a promising effect of both probiotic strains tested as bacterial extracts in the treatment of intestinal inflammation, avoiding the stimulation of the immune system, which is a side effect of the use of the commercialised pure cultures. Regarding batch cultures, we did not detect any significant interaction among oxysterols, probiotics and intestinal microbiota metabolic activity in the experimental condition used; this part of the research project is a preliminary study which we aim to enrich with further investigations. Taken together our data strengthen the link between diet and intestinal inflammation and encourages us to continue studying probiotics as a useful tool in the prevention and management of the most common intestinal pathologies linked to the inflammatory process.