Euphorbia hypericifolia Attenuates Citrinin-Induced Oxidative Stress and Maintains Tight Junction Integrity in Porcine Intestinal Epithelial Cells

Citrinin (CTN), a mycotoxin commonly found in contaminated food and animal feed, impairs intestinal barrier integrity through oxidative stress and cytotoxicity. However, its link to ferroptosis, an iron-dependent form of regulated cell death, remains unclear. This study investigated whether CTN induces ferroptosis in intestinal epithelial cells and evaluated the protective role of <i>Euphorbia hypericifolia</i> (EH) against CTN-induced oxidative damage and tight junction (TJ) disruption. Using IPEC-J2 cells exposed to CTN, intracellular ferrous ion (Fe²⁺) levels, reactive oxygen species (ROS) accumulation, and TJ integrity were assessed using FerroOrange and DCFH-DA staining, RT-qPCR, immunofluorescence, and WST-1 assays. Additionally, a high-throughput screen of 459 natural products identified EH extract as a top candidate in mitigating CTN toxicity. The CTN treatment significantly elevated intracellular Fe²⁺ and ROS levels, downregulated antioxidant genes (notably <i>CAT</i>), and disrupted <i>ZO-1</i> expression and TJ morphology in IPEC-J2 cells, all hallmarks of ferroptosis-like cell death. Co-treatment with EH extract effectively reversed these effects, restoring antioxidant gene expression, reducing Fe²⁺ and ROS accumulation, and preserving TJ structure. Phytochemical profiling of EH extract revealed several bioactive compounds potentially responsible for its protective effects. These findings suggest that CTN induces ferroptosis-related cytotoxicity in IPEC-J2 cells, but EH alleviates this toxicity by modulating oxidative stress and iron homeostasis, supporting its potential use as a natural feed additive for intestinal protection.