Ellagic acid in the control of intestinal inflammation/oxidative stress-induced barrier permeabilization

FRAGA, CESAR G.; D. E. IGLESIAS; P. I. OTEIZA

Kobe

Congreso; 9th International Conference on Polyphenols and Health (ICPH); 2019

ICPH

background: Intestinal inflammation can have negative health consequences both locally and systemically. Production of cytokines and chemokines attracts and activates immune cells, perpetuating local inflammation and oxidative stress, and promotes intestinal epithelium permeabilization, leading to systemic inflammation and associated co-morbidities. Among plant bioactives, ellagic acid (EA), a polyphenol abundant in pomegranate, nuts and berries, has shown mitigating effects in animal models of inflammatory bowel disease.Objective: This work investigated the capacity of EA to mitigate tumor necrosis alpha (TNFα)-induced inflammation, oxidative stress, and loss of barrier integrity in Caco‐2 cells differentiated into an intestinal epithelial monolayer.Methods: Caco-2 cells were preincubated for 24 h with IFNg (10 ng/ml) to upregulate the TNFα receptor. Then, cells were incubated for 1 h in the absence or presence of variable amounts of EA added to the apical compartment, and subsequently with TNFα (10 ng/ml) added to the basolateral compartment. Parameters of inflammation (IL-6, IL-8, ICAM-1, NF-κB), oxidative stress (NOX1),and tight junction functionality (MLCK, tight junction proteins) were measured by ELISA, RT-PCR or Western blot. Monolayer permeability was assessed by measuring TEER.Results: TNFα triggered interleukin (IL)-6 release to the medium, which was inhibited by EA in a dose-dependent manner. According to the calculated IC50 for the inhibition of IL-6 release (17.3 μM), the EA concentrations used in subsequent experiments were 10, 20 and 40 μM. EA did not cause cytotoxicity within this range of concentrations. TNFα-stimulated IL-8 synthesis andrelease were also inhibited by EA. In addition, EA prevented TNFα-induced NF-κB activation and ICAM-1 overexpression. EA was also effective at inhibiting TNFα-stimulated oxidant production and increase in NADPH oxidase 1 (NOX1) expression, both indicators of oxidative stress. TNFα caused an increase in mitochondrial oxidant production and a decrease in mitochondrial membrane potential. These adverse effects of TNFα on mitochondrial function were prevented by EA.Moreover, TNFα led to myosin light chain kinase (MLCK) upregulation, to a decrease in tight junction proteins levels, and, as a consequence, to Caco-2 cell monolayer permeabilization. EA inhibited TNFα-induced barrier permeabilization, which is in part due to its capacity to downregulate MLCK via NF-κB inhibition and to the prevention of tight junction proteins loss.Conclusion: EA prevented TNFα-mediated Caco-2 cells inflammation, oxidative stress, and monolayer permeabilization mainly through the inhibition of NOX/NF-κB activation. Thus, EA can break the cycle of oxidative stress, NF-κB activation, and intestinal permeabilization. This suggests that consumption of EA, via foods or supplements, could contribute to mitigate the local and systemic adverse effects of intestinal inflammation and permeabilization.Supported by Pfizer Consumer Healthcare, Inc. and NIFA USDA (CA-D*-xxx-7244-H).Keywords: ellagic acid, inflammation, intestinal barrier permeabilization, NF-κB activation.