New findings on the antagonism of the environmental chemical toxicity 2-ethylhexyl diphenyl phosphate: Glycyrrhizic Acid as an Nrf2 activator targets Nrf2/ROS/STAT3 signalling crosstalk to alleviate thymic injury in chicks

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.