Dietary anthocyanins in gut health promotion: regulation of NF-κB and Nrf2 dependent gene transcription

Inflammatory Bowel Diseases (IBDs) show complex etiology, resulting from a genetically determined susceptibility, with more than 160 SNPs implicated, interacting with environmental factors, including diet and gut microbiota. Specific dietary interventions are likely to be appropriate. Chronic intestinal inflammatory disorders are characterized by excessive release of proinflammatory mediators, intestinal barrier dysfunction, and excessive activation of NF-kB cascade. Polarized basolateral intestinal secretion of inflammatory mediators, followed by activation of NF-kB signaling and inflammatory pathways in endothelial cells, efficiently triggering extravasation of neutrophils from the vasculature, thus contributing to the development and maintenance of intestinal inflammation. Proper regulation of NF-κB activation at the epithelial interface is crucial for the maintenance of physiological tissue homeostasis. Several studies report an important role of anthocyanins in the modulation of inflammation and oxidative stress and in reducing the risk of developing chronic diseases, such as IBDs. In this study an in vitro model of acute intestinal inflammation was employed using intestinal Caco-2 cells exposed to TNF-α, a proinflammatory cytokine playing a central role in intestinal inflammation of IBDs. The protective effects of cyanidin-3-O-glucoside (C3G), an anthocyanin widely distributed in mediterranean diet, were then evaluated. Caco-2 cells exposure to TNF-α activated NF-kB proinflammatory pathway and induced IL6, TNF-α, IL8, and COX-2 expression. C3G (20-40 µM) pretreatment for 24h dose-dependently prevented TNF-α-induced changes, and improved intracellular redox status. Furthermore, by using a co-culture in vitro system, the effects of TNF-α-activated intestinal cells on endothelial cells dysfunction, as well as the protective effects of C3G, were evaluated. TNF-α-activated Caco-2 cells induced endothelial dysfunction with increased E-selectin and VCAM-1 mRNA, leukocyte adhesion, and NF-kB levels in HUVECs, effects all inhibited by C3G pretreatment of Caco-2. Moreover, C3G, also without any kind of stimulus, increased the translocation of the transcription factor Nrf2 into the nucleus, thus activating antioxidant and detoxifying genes. However, SNPs in genes encoding NF-kB subunits (e.g. NFKB1 rs28362491), in Nrf2 promoter region, or in genes regulated by these transcription factors (e.g. HO-1 rs2071746) may affect both the risk of developing IBDs as well as the responsiveness to anthocyanins treatment. Finally, this model demonstrated that selective inhibition of the NF-kB pathway in epithelial cells represents the main mechanism by which C3G exerts these protective effects. In conclusion, C3G showed anti-inflammatory properties through the inhibition of NF-kB signalling in Caco-2 cells and these beneficial effects appear to be due to its ability to activate cellular protective responses modulated by Nrf2. These data suggest that anthocyanins could contribute, as complementary or preventive approaches, to the management of chronic gut inflammatory diseases.