A Medium-Throughput System for In Vitro Oxidative Stress Assessment in IPEC-J2 Cells

Abstract

The feed industry continuously seeks new molecules with antioxidant capacity since oxidative stress plays a key role in intestinal health. To improve screening of new antioxidants, this study aims to set up an assay to assess oxidative stress in the porcine small intestinal epithelial cell line IPEC-J2 using plate-reader-based analysis of fluorescence. Two oxidants, H2O2 and menadione, were tested at 1, 2 and 3 mM and 100, 200 and 300 µM, respectively. Trolox (2 mM) was used as the reference antioxidant and the probe CM-H2DCFDA was used to indicate intracellular oxidative stress. Cell culture, reactive oxygen species (ROS) production and assessment conditions were optimized to detect a significant ROS accumulation that could be counteracted by pre-incubation with trolox. Menadione (200 µM) reproducibly increased ROS levels, H2O2 failed to do so. Trolox significantly decreased intracellular ROS levels in menadione (200 µM)-exposed cells in a consistent way. The system was further used to screen different concentrations of the commercially available antioxidant ELIFE®. Concentrations between 100 and 200 ppm protected best against intracellular ROS accumulation. In conclusion, the combination of CM-H2DCFDA fluorescence analysis by a plate-reader, trolox as a reference antioxidant and 200 µM of menadione as a stressor agent, provides a replicable and reliable medium-throughput setup for the evaluation of intracellular oxidative stress in IPEC-J2 cells.

Low birth weight female piglets show altered intestinal development, gene expression, and epigenetic changes at key developmental loci

“…To investigate the potential causality of DNA methylation changes with respect to gene expression, the noncancerous intestinal cell line IPEC‐J2 was subjected to pharmacological inhibition of DNMT1 with 5‐aza‐2′‐deoxycytidine. This cell line was selected since it presents advantages over other cancerous and noncancerous cell lines including morphology and protein expression 63‐67 . As expected, the treated group showed a statistically significant decrease in methylation in all the investigated genes.…”

“…This cell line was selected since it presents advantages over other cancerous and noncancerous cell lines including morphology and protein expression. [63][64][65][66][67] As expected, the treated group showed a statistically significant decrease in methylation in all the investigated genes. The expression of the digestive enzymes and differentiation markers assessed in the in vivo experiment was quantified in IPEC-J2 cells.…”

Ochratoxin A Induces Oxidative Stress in HepG2 Cells by Impairing the Gene Expression of Antioxidant Enzymes

“…ROS are produced by the cell during normal aerobic metabolism and possess a dual function on the cell. While lower ROS levels are required for several physiological mechanisms such as host defense [ 46 ], excessive levels of ROS negatively affect cell viability via lipid peroxidation and protein and nucleic acid oxidation [ 47 ]. Several studies observed increased/overproduced free radicals by exposing OTA in both in vitro and in vivo [ 17 , 20 , 36 , 37 , 48 , 49 , 50 ] and it has been linked to OTA toxicity.…”

Protective Effects of Grape Seed Oligomeric Proanthocyanidins in IPEC-J2–Escherichia coli/Salmonella Typhimurium Co-Culture

“…IPEC-J2 cells are capable of expressing tight junction proteins, as well as synthetizing cytokines, defensins, toll-like receptors, and mucins, and they can be used for studying the antioxidant activity of different substances. The cell line is not only important for investigations targeting the GI health of pigs, but can also serve as a model of the human GI epithelium [ 25 ]. Among the non-human cell lines, IPEC-J2 can model human epithelial cells most closely, as the porcine and human GI tracts are similar in many aspects (e.g., size, weight, anatomy, and physiology).…”