Inflammation is a natural defense process of the innate immune system, associated with the release of proinflammatory cytokines such as interleukin-1β, interleukin-6, interleukin-12 and TNFα; and enzymes including iNOS through the activation and nuclear translocation of NF-κB p65 due to the phosphorylation of IκBα. Regulation of intracellular Ca2+ is considered a promising strategy for the prevention of reactive oxygen species (ROS) production and accumulation of DNA double strand breaks (DSBs) that occurs in inflammatory-associated-diseases. Among the metabolites of ellagitannins that are produced in the gut microbiome, urolithin A (UA) has received an increasing attention as a novel candidate with anti-inflammatory and anti-oxidant effects. Here, we investigated the effect of UA on the suppression of pro-inflammatory molecules and NF-κB activation by targeting TLR4 signalling pathway. We also identified the influence of UA on Ca2+ entry, ROS production and DSBs availability in murine bone-marrow-derived macrophages challenged with lipopolysaccharides (LPS). We found that UA inhibits IκBα phosphorylation and supresses MAPK and PI3K activation. In addition, UA was able to reduce calcium entry, ROS production and DSBs availability. In conclusion, we suggest that urolithin A is a promising therapeutic agent for treating inflammatory diseases through suppression of NF-κB and preserving DNA through maintaining intracellular calcium and ROS homeostasis.
Background/Aims: Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions, which is in part accomplished by Na+/H+ exchangers, such as NHE1. The carrier is sensitive to oxidative stress. Growth of tumor cells could be suppressed by the polyphenol Ellagic acid, which is found in various fruits and vegetables. An effect of Ellagic acid on transport processes has, however, never been reported. The present study thus elucidated an effect of Ellagic acid on cytosolic pH (pHi), NHE1 transcript levels, NHE1 protein abundance, Na+/H+ exchanger activity, and lactate release. Methods: Experiments were performed in Ishikawa cells without or with prior Ellagic acid (20 µM) treatment. NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance by Western blotting, pHi utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, cell volume from forward scatter in flow cytometry, reactive oxygen species (ROS) from 2’,7’-dichlorodihydrofluorescein fluorescence, glucose uptake utilizing 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose, and lactate concentration in the supernatant utilizing a colorimetric (570 nm)/ fluorometric enzymatic assay. Results: A 48 hour treatment with Ellagic acid (20 µM) significantly decreased NHE1 transcript levels by 75%, NHE1 protein abundance by 95%, pHi from 7.24 ± 0.01 to 7.02 ± 0.01, Na+/H+ exchanger activity by 77%, forward scatter by 10%, ROS by 82%, glucose uptake by 58%, and lactate release by 15%. Conclusion: Ellagic acid (20µM) markedly down-regulates ROS formation and NHE1 expression leading to decreased Na+/H+ exchanger activity, pHi, glucose uptake and lactate release in endometrial cancer cells. Those effects presumably contribute to reprogramming and growth inhibition of tumor cells.
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