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 Euphorbia hypericifolia (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 CAT), and disrupted ZO-1 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

Citrinin (CTN) is a mycotoxin that adversely affects livestock by contaminating stored grains, leading to significant health and economic impacts. This study investigates the toxicological effects of CTN on porcine small intestinal epithelial cells (IPEC-J2) and explores potential mitigation strategies using natural products and chemical inhibitors. Our study demonstrates that CTN induces cytotoxicity through the TGF-β signaling pathway, triggering apoptosis and G2/M phase cell cycle arrest. We examined cell viability, cell cycle progression, and gene expression changes in IPEC-J2 cells treated with CTN, 4′-Hydroxydehydrokawain (4-HDK), and LY-364947, a TGF-β receptor inhibitor. LY-364947 treatment confirmed that CTN-induced toxicity is mediated through TGF-β signaling. Although 4-HDK alleviated CTN-induced cytotoxicity by improving cell viability and reducing apoptosis, its direct involvement in TGF-β inhibition remains unclear. These results suggest that CTN disrupts intestinal epithelial cell homeostasis via TGF-β activation, whereas 4-HDK may exert protective effects through an alternative mechanism. Our study provides novel insights into CTN-induced toxicity mechanisms and highlights the therapeutic potential of 4-HDK as a mitigator of mycotoxin-induced cellular damage.