Abstract
Oxidative stress in the small intestinal epithelium can lead to barrier malfunction. In this study, the effect of rosmarinic acid (RA), quercetin (Que), gallic acid (GA), lipoic acid (LA), ethoxyquin (ETQ) and Se-methionine (SeMet) pre-treatments using 2 mM Trolox as a control on the viability and the generation of intracellular reactive oxygen species (iROS) of oxidatively (H2O2) stressed intestinal porcine epithelial cells (IPEC-J2) was investigated. A neutral red assay showed that RA (50–400 µM), Que (12.5–200 µM), GA (50–400 µM), ETQ (6.25–100 µM), and SeMet (125–1000 µM) pre-treatments but not LA significantly increased the viability of H2O2-stressed IPEC-J2 cells (p < 0.05). A 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) fluorescent probe showed that RA (100–600 µM), Que (25–800 µM), ETQ (3.125–100 µM) and SeMet (500–2000 µM) pre-treatments significantly reduced iROS in IPEC-J2 monolayers (p < 0.05). Moreover, RA and Que were most effective in reducing iROS. Therefore, the effects of RA and Que on barrier functioning in vitro were examined. RA and Que pre-treatments significantly decreased fluorescein isothiocyanate (FITC)-conjugated dextran-4 (4 kDa) permeability and transepithelial electrical resistance (TEER) of an IPEC-J2 cell monolayer (p < 0.05). These in vitro results of RA and Que hold promise for their use as antioxidants in pig feed.
Highlights
Cellular lipids, proteins, and DNA structures can be damaged when the redox balance of the cell is disturbed by failure of the endogenous antioxidant system or the presence of excessive amounts of reactive oxygen species (ROS) [1]
For several concentrations of rosmarinic acid (RA), the viability rose over 100%
Depending on the experimental set-up, it is not uncommon that the viability increases over 100%, as test samples are compared to a control sample and not to an internal control, as such washing steps and the specific cell density at the start of the experiment could explain these differences [12]
Summary
Proteins, and DNA structures can be damaged when the redox balance of the cell is disturbed by failure of the endogenous antioxidant system or the presence of excessive amounts of reactive oxygen species (ROS) [1]. Elevated levels of ROS in the mucosa of the jejunum are induced by changes in diet [2], and alter microbiota compositions [3,4]. Oxidation–reduction imbalance in the intestinal epithelium has an impact on its functionality [5]. The small intestine is in the primary site of nutrient absorption and part of a first line of defense in the body against dietary-derived oxidants [6,7]. It is important to investigate how different types of antioxidants can be protective.
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