THERAPEUTIC EFFICACY OF PROBIOTICS AND SHORT CHAIN FATTY ACIDS IN GASTROINTESTINAL DISEASE: EVALUATION OF METABOLIC, HORMONAL AND INFLAMMATORY PARAMETERS

In these three years we studied probiotics and postbiotics efficacy and their mechanism/s in preventing or limiting gastrointestinal diseases, such as hepatic steatosis and ulcerative colitis. On the basis of strength anatomical correlation between liver and gut, we wanted to observe if microorganisms present in gut and their postbiotic derivatives, the short chain fatty acids, were able to prevent or cure not only local intestinal disease but also to limit extraintestinal and systemic pathologies, i.e. liver steatosis and insulin-resistance. Our results showed a protective and curative effect of probiotics and short chain fatty acids in limiting the onset and the progression of these pathologies. We demonstrate several mechanisms of action for these effects: probiotics and postbiotics restore alteration in gut microflora, in particular, Lactobacillus paracasei B21060 can compete with pathogen bacteria avoiding them to attach intestinal epithelium and stimulating immune response. Both probiotics and short chain fatty acids, in particular butyrate, are able to influence directly colonic functions improving innate immune defense mechanisms. In particular, Lactobacillus paracasei B21060 and butyrate-based compounds improved not only tight-junction structure reducing intestinal permeability but also stimulated mucin and defensine production increasing antimicrobial properties of intestinal epithelial cells. Alteration of tight-junctions or mucin production together with changes in microbiota composition, can be considered as predisposing factors to development of gastrointestinal disease. Specially in western life style, bad alimentary habits taken together with smoke or alchol consumption, represent a favourable situation for the onset of these morbidity. In this context, daily assumption of probiotics or directly of their fermentation products as butyrate, could be considered a valid approach for prevention or treatment of gastrointestinal pathologies. In our models of Non-alcoholic fatty liver disease (NAFLD) and ulcerative colitis (UC), both a synbiotic based on Lactobacillus paracasei B21060 and butyrate prevented the beginning of the principal events which underlie progression of these conditions such as insulin-resistance and intestinal inflammation. The enhancement of epithelial barrier integrity by the synbiotic, through an increase in tight junction proteins expression (ZO-1 and occludin), determines a reduction of DAMPs, PAMPs, and FFA afflux to liver. These effects are related to reduction of hepatic TLR4 expression, and its accessory molecule CD14, TLR2 and TLR9 and NF-κB-activation with a decrease in inflammatory gene transcription. The reduced expression of inflammatory cytokines (TNF- α and IL-6) leads to an improvement of insulin signaling (reduction of IRS-1 phosphorylation in Ser 307 and down-regulation of SOCS3), accompanied by an increase in insulin sensitivity. The synbiotic increases PPAR-α expression in liver, contributing to the reduction of inflammation and the increase in fatty acid catabolism. Consistently, in adipose tissue the synbiotic increases PPAR-γ expression and adiponectin transcription, contributing to reduce inflammation. Finally, the therapeutic/preventive role of the synbiotic in hepatic steatosis could be related to its ability to limit inflammatory liver damage, insulin signaling impairment, and imbalance of TLR patterns induced by high fat diet. Our results support probiotics as innovative, preventive and therapeutic strategy for NAFLD, using synbiotic preparations containing selected strain with clear and demonstrated beneficial immunomodulatory effects. Among probiotics, L. paracasei B21060, can be considered a potential approach, limiting the main pathogenetic events involved in the onset of insulin-resistance and steatosis induced by high fat diet. This synbiotic, alone or in combination with other therapies, could be useful in the treatment of fatty liver in children who are hardly able to follow a program of hypocaloric diet and regular physical activity. Also sodium butyrate and its palatable chemical derivative N-(1-carbamoyl-2-phenylethyl) butyramide (FBA) were able to attenuate steatosis and liver injury with reduction of inflammatory responses via suppression of Toll-like receptors and NF-kB pathways. In a murine model of ulcerative colitis, we used dextran sodium sulphate at 2.5% since it is able to reproduce the complexity and event’s cascade typical of moderate to active colitis development. Our data demonstrated that the synbiotic-based Lactobacillus paracasei B21060 was able to prevent immune cell infiltration and to reduce tissue damage in colon, modulating inflammatory and anti-inflammatory cytokines and adipokines, and preventing tight-junction loss. We assessed also the role of inflammosome in this model and its modulation by synbiotic. Moreover the marked changes in defensin-β and mucin-1 expression demonstrated the capability of this synbiotic formulation to maintain the intestinal mucosal homeostasis regulating mucus and antimicrobial peptides secretion by colonic mucosa. In this work we clearly showed that this synbiotic formulation was able to enhance intestinal barrier function and epithelial repair capability in response to DSS induced colitis in mice, suggesting not only a potential therapeutic role for this agent in this pathology, but also the possibility that a supplement of these lactobacilli might prevent the relapse of ulcerative colitis. In the same experimental model of colitis, postbiotics such as butyrate and FBA improved gut permeability avoiding bacterial translocation and impairing immune cell recruitment. These anti-inflammatory effects were evident by reduction in neutrophil infiltration and reduced HDAC9 transcription in colonic mucosa. These events improved intestinal immune homeostasis and tolerance vs. commensal bacteria. In addition, butyrate-based compounds restored colonocyte ability to up-take butyrate after fiber digestion: this occurred with unaltered PPAR-γ levels but blocking NF-kB activation, leading to colonocyte protection from inflammatory process. Efficacy of butyrate to limit early molecular events underlying inflammatory process linked to intestinal damage, suggests its potential clinical utility as a preventive and therapeutic strategy for ulcerative colitis. Since butyrate derivative does not have the characteristic odour of rancid cheese, this derivative may represent a viable therapeutic alternative to butyrate, favouring a better compliance and a greater effectiveness