2-Ethylhexyl diphenyl phosphate (EHDPHP) is a pervasive environmental pollutant known to induce oxidative damage in organisms
however, its precise mechanisms of toxicity remain unclear. Furthermore, limited research has been conducted on potential therapeutic agents to counteract EHDPHP toxicity. Glycyrrhizic acid (GA), a triterpenoid saponin compound with recognized antioxidant, anti-inflammatory, and immunomodulatory properties, represents a promising candidate for mitigating EHDPHP-induced oxidative injury. In chickens, the thymus is the main immune organ. This study aims to investigate the mechanism of EHDPHP-induced thymus damage and the role and mechanism of GA intervention in this process. A potential 'EHDPHP/GA-Target-Oxidative Stress (OS)' network was constructed using network biology. A model of EHDPHP-induced chicken thymic injury was established by continuous oral administration of EHDPHP (160 mg/kg) for 42 days. Moreover, the mechanism of action by which GA antagonizes EHDPHP-induced oxidative damage was explored using MDCC-MSB1 cells in vitro. Network biology analyses showed that 'EHDPHP/GA-OS' targets were mainly enriched in the adipocytokine and apoptotic signaling pathways. Molecular docking demonstrated the binding interactions of GA and EHDPHP with Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and signal transducer and activator of transcription 3 (STAT3) proteins. Both in vitro and in vivo experiments revealed that GA attenuated EHDPHP-induced damage to thymus and MDCC-MSB1 cells, as evidenced by reductions in oxidative stress markers (ROS, MDA, T-AOC, SOD, and GSH-Px), inflammation factors (NF-κB, IL-6, and TNF-α), and the apoptotic factor (Caspase 3) expression. GA treatment increased the expression of Nrf2 and HO-1 while reducing the expression of Keap1, JAK1, Phospho-JAK1 (P-JAK1), STAT3, and Phospho-STAT3 (P-STAT3). Furthermore, the protective effect of GA against EHDPHP-induced MDCC-MSB1 cell injury, as well as its inhibition of the JAK1/STAT3 pathway, was diminished by the Nrf2 inhibitor ML385. These findings suggest that GA exerts its protective effects through Nrf2 and mitigates EHDPHP-induced thymic injury in chickens by modulating the crosstalk between the Nrf2/ROS/STAT3 signaling pathways. Overall, this study highlights the novel role of GA in treating EHDPHP-induced injury and underscores its potential application in diseases treatable with Nrf2 activators.