Defects in the intestinal epithelial barrier functions characterize inflammatory conditions such as Inflammatory Bowel Disease (IBD). Overexpression of pro-inflammatory cytokines such as TNF-α, IL-1B, IL-6 and INF-γ trigger epithelial damage. These cytokines are due to upregulation of claudin-2 (CLDN2) that form a pore channel, resulting in redistribution of TJs and an alteration of barrier permeability. Recently, we demonstrated that miR-195-5p is able to regulate CLDN2 and indirectly also CLDN1 in intestinal epithelial cells. Now, we aimed to investigate the modulation of miR-195-5p on the expression of CLDN2 and other TJs under inflammatory conditions induced by TNF-α. We demonstrated that miR-195-5p also modulated the expression of CLDN2 levels after stimulation with TNF-α. In addition, we discovered the role of miR-195-5p in the integrity of the intestinal barrier and in promoting the restoration of the intestinal epithelial. Moreover, we established that replacement of miR-195-5p attenuated the colonic inflammatory response in DSS-induced, colitis and it reduced colonic permeability. In conclusion, our data revealed the role of miR-195-5p in intestinal inflammation in ulcerative colitis, suggesting a potential pharmacological target for new therapeutic approaches.

Inflammasomes are multiprotein complexes expressed by immune cells in response to distinct stimuli that trigger inflammatory responses and the release of pro-inflammatory cytokines. Evidence suggests a different role of inflammasome NLRP3 in IBD. NLRP3 inflammasome activation can be controlled by post-translational modifications such as ubiquitination through BRCC3. The aim of this study was to investigate the effect of miR-369-3p on the expression and activation of NLRP3 inflammasomes via BRCC3 regulation. After bioinformatics prediction of Brcc3 as a gene target of miR-369-3p, in vitro, we validated its modulation in bone marrow-derived macrophages (BMDM). The increase in miR-369-3p significantly reduced BRCC3 gene and protein expression. This modulation, in turn, reduced the expression of NLRP3 and blocked the recruitment of ASC adaptor protein by NLRP3. As a result, miR-369-3p reduced the activity of Caspase-1 by the inflammasome, decreasing the cleavage of pro-IL-1β and pro-IL-18. These results support a novel mechanism that seems to act on post-translational modification of NLRP3 inflammasome activation by BRCC3. This may be an interesting new target in the personalized treatment of inflammatory disorders, including IBD.

Antioxidants are privileged candidates for the development of adjuvants able to improve the efficiency of pharmacological therapies, particularly for chronic inflammatory syndromes. During the last 20 years, anti-TNFα (tumor necrosis factor alpha) monoclonal antibodies infusion has been the biological therapy most frequently administered but there is still large space for improvement in disease remission rates and maintenance. In this context, nutritional bioactive compounds contained in dietary patterns or included as supplements, may act as adjuvants for the induction and maintenance of IBD (inflammatory bowel diseases) remission. To verify this possibility, a single-center preliminary study (SI-CURA, Soluzioni Innovative per la gestione del paziente e il follow up terapeutico della Colite UlceRosA) was designed and carried out to evaluate whether a daily administration of purple corn supplement could improve the response to Infliximab (IFX) infusion of IBD patients with both Crohn’s disease (CD) and ulcerative colitis (UC). A cohort of 47 patients was enrolled in the study. Biological samples were collected before the first and the third IFX infusion. All patients received nutritional guidelines, 27 of them received commercial red fruit tea with low anthocyanins content, while 20 received a purple corn supplement with a high anthocyanin content. Results show that the administration of an antioxidant-enriched purple corn supplement could improve IFX-mediated disease remission in terms of circulating inflammatory markers. Comparison between CD and UC patients revealed that, at this anthocyanin dosage, the purple corn extract administration improved the IFX response in CD but not in UC patients. Our results may pave the way for a new metacentric study of CD patients, recruiting a wider cohort and followed-up over a longer observational time.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation associated with an increased intestinal permeability. Several studies have shown that microRNAs (miRNAs) are involved in the IBD pathogenesis. Here, we aimed to functionally characterize the role of miRNAs in the regulation of intestinal permeability and barrier function. We identified 18 dysregulated miRNAs in intestinal epithelial cells (IECs) from the ulcerative colitis (UC) mice model and control mice. Among them, down-regulated miR-195-5p targeted claudin-2 (CLDN2) and was involved in impaired barrier function. CLDN2 expression levels were increased in UC mice models and negatively correlated with miR-195-5p expression. We demonstrated that gain-of-function of miR-195-5p in colonic epithelial cell lines decreased the CLDN2 levels. This modulation, in turn, downregulated claudin-1 (CLDN1) expression at protein level but not that of occludin. Our data support a previously unreported role of miR-195-5p in intestinal tight junctions’ regulation and suggest a potential pharmacological target for new therapeutic approaches in IBD.

The immunoproteasome is a multi-catalytic protein complex expressed in hematopoietic cells. Increased expression of immuno-subunits followed by increased proteasome activities is associated with the pathogenesis of IBD. Therefore, the identification of molecules that could inhibit the activities of this complex has been widely studied. microRNAs are small molecules of non-coding RNA that regulate the expression of target genes. Our purpose was to demonstrate that miR-369-3p is able to reduce the expression of the PSMB9 subunit and consequently modulate the catalytic activities of immunoproteasome. After bioinformatics prediction of the gene target of miR-369-3p, we validated its modulation on PSMB9 expression in the RAW264.7 cell line in vitro. We also found that miR-369-3p indirectly reduced the expression of other immunoproteasome subunits and that this regulation reduced the catalytic functions of the immunoproteasome. Increased levels of PSMB9 were observed in colon samples of acute IBD patients compared to the remission IBD group and control group. Our data suggest that miR-369-3p may be a future alternative therapeutic approach to several compounds currently used for the treatment of inflammatory disorders including IBD.

Background: Intrahepatic cholangiocarcinoma (iCCA) is a highly malignant tumor characterized by an intensive desmoplastic reaction due to the exaggerated presence of the extracellular (ECM) matrix components. Liver fibroblasts close to the tumor, activated by transforming growth factor (TGF)-β1 and expressing high levels of α-smooth muscle actin (α-SMA), become cancer-associated fibroblasts (CAFs). CAFs are deputed to produce and secrete ECM components and crosstalk with cancer cells favoring tumor progression and resistance to therapy. Overexpression of Notch signaling is implicated in CCA development and growth. The study aimed to determine the effectiveness of the Notch inhibitor, Crenigacestat, on the surrounding microenvironment of iCCA. Methods: We investigated Crenigacestat’s effectiveness in a PDX model of iCCA and human primary culture of CAFs isolated from patients with iCCA. Results: In silico analysis of transcriptomic profiling from PDX iCCA tissues treated with Crenigacestat highlighted “liver fibrosis” as one of the most modulated pathways. In the iCCA PDX model, Crenigacestat treatment significantly (p < 0.001) reduced peritumoral liver fibrosis. Similar results were obtained in a hydrodynamic model of iCCA. Bioinformatic prediction of the upstream regulators related to liver fibrosis in the iCCA PDX treated with Crenigacestat revealed the involvement of the TGF-β1 pathway as a master regulator gene showing a robust connection between TGF-β1 and Notch pathways. Consistently, drug treatment significantly (p < 0.05) reduced TGF-β1 mRNA and protein levels in tumoral tissue. In PDX tissues, Crenigacestat remarkably inhibited TGF-β signaling and extracellular matrix protein gene expression and reduced α-SMA expression. Furthermore, Crenigacestat synergistically increased Gemcitabine effectiveness in the iCCA PDX model. In 31 iCCA patients, TGF-β1 and α-SMA were upregulated in the tumoral compared with peritumoral tissues. In freshly isolated CAFs from patients with iCCA, Crenigacestat significantly (p < 0.001) inhibited Notch signaling, TGF-β1 secretion, and Smad-2 activation. Consequently, Crenigacestat also inactivated CAFs reducing (p < 0.001) α-SMA expression. Finally, CAFs treated with Crenigacestat produced less (p < 005) ECM components such as fibronectin, collagen 1A1, and collagen 1A2. Conclusions: Notch signaling inhibition reduces the peritumoral desmoplastic reaction in iCCA, blocking the TGF-β1 canonical pathway.