The protective effects and mechanism of N-carbamoyl glutamate against H2O2-induced oxidative damage in IPEC-J2 cell

This experiment aimed to explore the protective effects and mechanism of N-carbamoyl glutamate (NCG) against hydrogen peroxide (H2O2)-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 H2O2 group (H2O2), treated with 80 µM NCG group (NCG), and NCG pre-treatment (80 µM) followed by H2O2 (400 µM) group (NCG + H2O2). 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 H2O2-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 H2O2-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). In summary, NCG can enhance the antioxidant capacity of IPEC-J2 cells under oxidative stress via the Keap1/Nrf2 signaling pathway and simultaneously upregulate the mRNA expression levels of tight junction proteins and inflammatory cytokines, thereby reinforcing cellular immune defense mechanisms and preserving the integrity of the intestinal epithelial barrier.