Perturbations of the intestinal epithelial barrier function lead to an increase in intestinal permeability with the consequent entry of harmful substances from the external environment. This condition of intestinal hyperpermeability, also known as leaky gut syndrome, has been correlated to some intestinal pathologies such as inflammatory bowel diseases, therefore interest in plant substances capable of improving the function of the epithelial barrier is growing recently. It has been shown that polyphenols, secondary metabolites contained in different types of fruits and vegetables, are able to improve intestinal health and that this effect is at least partly due to their ability to modulate the AMPK/SIRT1 pathway. Therefore, the aim of this work was to evaluate the in vitro effect on intestinal epithelial barrier function of a standardized and commercially available extract obtained from the leaves of Cynara cardunculus L. (CCLE), rich in polyphenols such as chlorogenic acid, luteolin and their derivatives, which represent a waste product of the food industry. Caco-2 cells were seeded and treated with CCLE (5, 10 or 15 μg/mL) from seeding to day 18. For the in vitro evaluation of epithelial monolayer formation, trans-epithelial electrical resistance (TEER) was monitored throughout the differentiation period. At day 18, fluorescein permeability and alkaline phosphatase activity were measured, and calcium switch assay was carried out. Western blot analysis was used to evaluate tight junctions (TJs) expression (claudin-1, occludin, and ZO-1). In addition, p-AMPK protein expression was evaluated with western blot analysis, whereas Real-Time PCR technique was employed to investigate SIRT1 mRNA expression. A series of experiments were conducted exposing Caco-2 cells to Compound C (Comp. C), a selective AMPK inhibitor. CCLE treatment stimulated epithelial barrier function, significantly increasing TEER values at day 18 and reducing paracellular permeability to fluorescein. These functional improvements were related to the ability of CCLE to stimulate the differentiation of intestinal epithelial monolayers as demonstrated by the increase in alkaline phosphatase activity and TJs proteins expression. Furthermore, the Ca2+ switch assay confirmed the important role played by CCLE in the assembly and stabilization of TJs leading to an improvement of the intestinal epithelial barrier function. Finally, thanks also to the use of Comp. C, it was confirmed that the beneficial effects of CCLE on the stimulation of epithelial cell differentiation and on the intestinal barrier function were related to the ability of the extract to activate the AMPK/SIRT1 pathway. In conclusion, these data demonstrate the power of CCLE to improve the intestinal barrier function and support the hypothesis that this beneficial effect is due to the ability of the polyphenols contained in the extract to activate the AMPK/SIRT1 pathway. Finally, these data promote the use of agricultural waste products, such as the leaves of Cynara cardunculus L., to obtain extracts rich in bioactive polyphenols potentially useful for the health of the intestinal epithelial barrier.
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