Laboratory of Applied Veterinary Morphology, Department of Veterinary Sciences, Faculty of Biomedical, Pharmaceutical and Veterinary Sciences, University of Antwerp, 2610 Wilrijk, Belgium.
Int J Mol Sci. 2020 Oct 1;21(19):7263. doi: 10.3390/ijms21197263.
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, HO 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, HO 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.
饲料行业一直在寻找具有抗氧化能力的新分子，因为氧化应激在肠道健康中起着关键作用。为了改进新抗氧化剂的筛选，本研究旨在建立一种使用基于平板读数器的荧光分析来评估猪小肠上皮细胞系 IPEC-J2 中氧化应激的测定方法。两种氧化剂 HO 和亚甲蓝分别在 1、2 和 3 mM 以及 100、200 和 300 µM 下进行了测试。Trolox（2 mM）用作参考抗氧化剂，探针 CM-H2DCFDA 用于指示细胞内氧化应激。优化了细胞培养、活性氧 (ROS) 产生和评估条件，以检测可被 Trolox 预孵育拮抗的显著 ROS 积累。亚甲蓝（200 µM）可重现性地增加 ROS 水平，而 HO 则不能。Trolox 以一致的方式显著降低了暴露于亚甲蓝（200 µM）的细胞中的细胞内 ROS 水平。该系统进一步用于筛选市售抗氧化剂 ELIFE 的不同浓度。在 100 至 200 ppm 之间的浓度可最佳地防止细胞内 ROS 积累。总之，平板读数器的 CM-H2DCFDA 荧光分析与 Trolox 作为参考抗氧化剂以及 200 µM 亚甲蓝作为应激剂的组合，为评估 IPEC-J2 细胞中的细胞内氧化应激提供了一种可重复且可靠的中高通量设置。
