Dipotassium Glycyrrhizate Improves Intestinal Mucosal Healing by Modulating Extracellular Matrix Remodeling Genes and Restoring Epithelial Barrier Functions

Gut mucosal healing (MH) is considered a key therapeutic target and prognostic parameter in the management of inflammatory bowel disease (IBD). The dipotassium glycyrrhizate (DPG), a salt of the glycoconjugated triterpene glycyrrhizin, has been shown to inhibit the High Mobility Group Box 1 (HMGB1) protein, an allarmin strongly implicated in the pathogenesis of most inflammatory and auto-immune disorders. Here we discuss new insights on how DPG acts on MH comparing the acute phase and the recovery phase from experimental colitis in mice. We found that DPG strongly accelerates MH by differently regulating pro-inflammatory (CXCL1, CXCL3, CXCL5, PTGS2, IL-1β, IL-6, CCL12, CCL7) and wound healing (COL3A1, MMP9, VTN, PLAUR, SERPINE, CSF3, FGF2, FGF7, PLAT, TIMP1) genes as observed only during the recovery phase of colitis. Relevant issue is the identification of extracellular matrix (ECM) remodeling genes, VTN, and PLAUR, as crucial genes to achieve MH during DPG treatment. Furthermore, a noticeable recovery of intestinal epithelial barrier structural organization, wound repair ability, and functionality is observed in two human colorectal adenocarcinoma cell lines exposed to DPG during inflammation. Thus, our study identifies DPG as a potent tool for controlling intestinal inflammation and improving MH

Transcription Factor ZNF281: A Novel Player in Intestinal Inflammation and Fibrosis

Background and aims: Recent evidences reveal the occurrence of a close relationship among epithelial to mesenchymal transition (EMT), chronic inflammation and fibrosis. ZNF281 is an EMT-inducing transcription factor (EMT-TF) involved in the regulation of pluripotency, stemness, and cancer. The aim of this study was to investigate in vitro, in vivo, and ex vivo a possible role of ZNF281 in the onset and progression of intestinal inflammation. A conceivable contribution of the protein to the development of intestinal fibrosis was also explored.Methods: Human colorectal adenocarcinoma cell line, HT29, and C57BL/6 mice were used for in vitro and in vivo studies. Mucosal biopsy specimens were taken during endoscopy from 29 pediatric patients with Crohn's disease (CD), 24 with ulcerative colitis (UC) and 16 controls. ZNF281 was knocked down by transfecting HT29 cells with 20 nM small interference RNA (siRNA) targeting ZNF281 (siZNF281).Results: We show for the first time that ZNF281 is induced upon treatment with inflammatory agents in HT29 cells, in cultured uninflamed colonic samples from CD patients and in DSS-treated mice. ZNF281 expression correlates with the disease severity degree of CD and UC patients. Silencing of ZNF281 strongly reduces both inflammatory (IL-8, IL-1beta, IL-17, IL-23) and EMT/fibrotic (SNAIL, Slug, TIMP-1, vimentin, fibronectin, and α-SMA) gene expression; besides, it abolishes the increase of extracellular-collagen level as well as the morphological modifications induced by inflammation.Conclusions: The identification of transcription factor ZNF281 as a novel player of intestinal inflammation and fibrosis allows a deeper comprehension of the pathogenetic mechanisms underlying inflammatory bowel disease (IBD) and provide a new target for their cure

Endoplasmic reticulum stress and unfolded protein response are involved in paediatric inflammatory bowel disease

Endoplasmic reticulum stress and unfolded protein response have been recently associated with the development of inflammatory bowel diseases in adults. We aimed at assessing the involvement of these pathways also in paediatric inflammatory bowel disease by analysing the expression of the main genes involved in endoplasmic reticulum stress and correlating them with the degree of intestinal inflammation. Real-time PCR and Western blot analysis of the expression of the endoplasmic reticulum stress marker HSPA5 and of selected genes representing the three pathways of unfolded protein response (IRE-XBP1, PERK-ATF4, ATF6p90-p50) in inflamed and uninflamed biopsies from 28 inflammatory bowel disease paediatric patients and 10 controls. HSPA5, PDIA4, as well as unspliced and spliced XBP1 mRNAs were significantly increased in patients' inflamed colonic mucosa compared to uninflamed mucosa and controls. HSPA5, PDIA4, ATF6, and phospho-IRE proteins were also upregulated, indicating the activation of the IRE-XBP1 and ATF6p90-p50 branches of unfolded protein response. A positive significant correlation between interleukin-8 levels, as a marker of inflammation, and upregulated genes was found in the inflamed colonic mucosa. A deregulation of the genes involved in the endoplasmic reticulum stress and unfolded protein response pathways may be a key component of the inflammatory response in paediatric patients with inflammatory bowel disease

Innovative method to grow the probiotic Lactobacillus reuteri in the omega3‐rich microalga Isochrysis galbana

Microalgae are natural sources of valuable bioactive compounds, such as polyunsaturated fatty acids (PUFAs), that show antioxidant, anti-inflammatory, anticancer and antimicrobial activities. The marine microalga Isochrysis galbana (I. galbana) is extremely rich in ω3 PUFAs, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Probiotics are currently suggested as adjuvant therapy in the management of diseases associated with gut dysbiosis. The Lactobacillus reuteri (L. reuteri), one of the most widely used probiotics, has been shown to produce multiple beneficial effects on host health. The present study aimed to present an innovative method for growing the probiotic L. reuteri in the raw seaweed extracts from I. galbana as an alternative to the conventional medium, under conditions of oxygen deprivation (anaerobiosis). As a result, the microalga I. galbana was shown for the first time to be an excellent culture medium for growing L. reuteri. Furthermore, the gas-chromatography mass-spectrometry analysis showed that the microalga-derived ω3 PUFAs were still available after the fermentation by L. reuteri. Accordingly, the fermented compound (FC), obtained from the growth of L. reuteri in I. galbana in anaerobiosis, was able to significantly reduce the adhesiveness and invasiveness of the harmful adherent-invasive Escherichia coli to intestinal epithelial cells, due to a cooperative effect between L. reuteri and microalgae-released ω3 PUFAs. These findings open new perspectives in the use of unicellular microalgae as growth medium for probiotics and in the production of biofunctional compounds

NOD2 Is Regulated by MIR-320 in Physiological Conditions but this Control Is Altered in Inflamed Tissues of Patients with Inflammatory Bowel Disease

BACKGROUND: Large evidence supports the role of microRNAs as new important inflammatory mediators by regulating both the adaptive and innate immunity. In the present study, we speculated that miR-320 controls NOD2 (nucleotide-binding oligomerization domain) expression, because it contains multiple binding sites in the 3'-untranslated region of the gene. NOD2, the first gene associated to increased susceptibility to Crohn's disease, is a cytosolic receptor that senses wall peptides of bacteria and promotes their clearance through initiation of a proinflammatory transcriptional program. This study aims at demonstrating that NOD2 is a target of miR-320 as well as investigating the role of inflammation in modulating the miR-320 control on NOD2 expression and analyzing miR-320 expression in intestinal biopsies of children with inflammatory bowel disease. METHODS: The colonic adenocarcinoma cell line HT29 was used to assess the miR-320-mediated regulation of NOD2 expression. MiR-320 and NOD2 expression were analyzed in mucosal samples of 40 children with inflammatory bowel disease. RESULTS: During inflammation, NOD2 expression is inversely correlated with miR-320 expression in vitro and ex vivo. Exogenous miR-320 transfection in HT29 cells leads to a significant decrease of NOD2 expression, whereas the miR-320 inhibitor transfection leads to increase of NOD2 expression, nuclear translocation of nuclear factor κB, and activation of downstream cytokines. CONCLUSIONS: We show for the first time that NOD2 expression is under the control of miR-320. We also show in vitro and ex vivo that inflammation induces a decrease of miR-320 and the latter correlates negatively with NOD2 expressio

NOD2 induces autophagy to control AIEC bacteria infectiveness in intestinal epithelial cells

Objective The importance of autophagy in mechanisms underlying inflammation has been highlighted. Downstream effects of the bacterial sensor NOD2 include autophagy induction. Recently, a relationship between defects in autophagy and adherent/invasive Escherichia coli (AIEC) persistence has emerged. The present study aims at investigating the interplay between autophagy, NOD2 and AIEC bacteria and assessing the expression level of autophagic proteins in intestinal biopsies of pediatric patients with inflammatory bowel disease (IBD). Methods A human epithelial colorectal adenocarcinoma (Caco2) cell line stably over-expressing NOD2 was produced (Caco2NOD2). ATG16L1, LC3 and NOD2 levels were analysed in the Caco2 cell line and Caco2NOD2 after exposure to AIEC strains, by western blot and immunofluorescence. AIEC survival inside cells and TNF a , IL-8 and IL-1 b mRNA expression were analysed by gentamicin protection assay and real time PCR. ATG16L1 and LC3 expression was analyzed in the inflamed ileum and colon of 28 patients with Crohn’s disease (CD), 14 with ulcerative colitis (UC) and 23 controls by western blot Results AIEC infection increased ATG16L1 and LC3 in Caco2 cells. Exposure to AIEC strains increased LC3 and ATG16L1 in Caco2 overexpressing NOD2, more than in Caco2 wild type, while a decrease of AIEC survival rate and cytokine expression was observed in the same cell line. LC3 expression was increased in the inflamed colon of CD and UC children. Conclusions The NOD2-mediated autophagy induction is crucial to hold the intramucosal bacterial burden, especially towards AIEC, and to limit the resulting inflammatory response. Autophagy is active in inflamed colonic tissues of IBD pediatric patients

Krill oil reduces intestinal inflammation by improving epithelial integrity and impairing adherent-invasive Escherichia coli pathogenicity

Krill oil is a marine derived oil rich in phospholipids, astaxanthin and omega-3 fatty acids. Several studies have found benefits of krill oil against oxidative and inflammatory damage