Abstract

Background This experiment aimed to explore the protective effects and mechanism of N-carbamoyl glutamate (NCG) against hydrogen peroxide (H 2 O 2 )-induced oxidative damage in intestinal porcine epithelial cells (IPEC-J2), providing a theoretical basis for the application of NCG in alleviating intestinal oxidative stress in weaned piglets. In this experiment, an oxidative stress model in IPEC-J2 cells were established using 400 µM H₂O₂, with an optimal NCG concentration (80 µM) determined via CCK-8 viability assay. Cells were divided into four groups: untreated group (Control), treated with 400 µM H 2 O 2 group (H 2 O 2 ), treated with 80 µM NCG group (NCG), and NCG pre-treatment (80 µM) followed by H 2 O 2 (400 µM) group (NCG + H 2 O 2 ). Results The results demonstrated that, compared with the H2O2 group, NCG pretreatment significantly increased the levels of reduced glutathione (GSH) and the activity of superoxide dismutase (SOD) in oxidative stress-induced IPEC-J2 cells, while decreasing malondialdehyde (MDA) and reactive oxygen species (ROS) levels ( P  < 0.05). NCG pretreatment upregulated the relative mRNA expression levels of TFAM and PGC-1α in oxidatively stressed cells and attenuated the H 2 O 2 -induced reduction in mitochondrial membrane potential (MMP) ( P  < 0.05). Furthermore, NCG pretreatment significantly downregulated Keap1 expression, enhanced Nrf2 expression, and concurrently increased the relative mRNA expression levels of antioxidant enzyme-related genes, including CAT , SOD1 , and SOD2 ( P  < 0.05). Additionally, NCG pretreatment markedly increased the relative mRNA expression level of Claudin-1 in oxidative stress-exposed cells and alleviated the H 2 O 2 -induced alterations in inflammatory gene expression, as evidenced by reduced mRNA levels of TNF-α and IL-6 mRNA expression in IPEC-J2 cells ( P  < 0.05). Conclusions In summary, NCG can enhance the antioxidant capacity of IPEC-J2 cells under oxidative stress via the Keap1/N