Quercetin reverses DEHP-induced ZBP1-mediated PANoptosis in thymus by targeting NF-κB to regulate the TNF-α/ROS signaling pathway

Quercetin (Que), a natural dietary flavonoid, possesses significant antioxidative and immunoregulatory properties. Di(2-ethylhexyl) phthalate (DEHP), a pervasive environmental endocrine-disrupting chemical, triggers oxidative stress (OS) and elicits immunotoxic effects in various species. However, whether Que can target NF-κB to alleviate DEHP-induced OS and PANoptosis in the chicken thymus remains unclear to date. To investigate the mechanism of thymus damage induced by DEHP and the intervention effect of Que, we established an in vivo chicken model treated with DEHP and/or Que. Based on the results of network pharmacology, molecular docking, molecular dynamics simulations, and Cell Thermal Shift Assay, we predicted that NF-κB is the key target for the protective effect of Que. Subsequently, through hematoxylin and eosin staining, CCK-8, immunofluorescence, Western blot, and Other techniques, we verified the effects of DEHP and/or Que on OS, the assembly of PANoptosome, and cellular PANoptosis in chicken thymus tissue and chicken lymphocyte cell line (MSB-1). Meanwhile, to clarify the key role of NF-κB in the above regulatory process, we conducted reverse validation experiments by introducing the NF-κB Activator Act1 in cellular experiments. The results demonstrated that DEHP exposure markedly induces the upregulation of NF-κB and TNF-α (P<.05), leading to a pronounced burst of Reactive Oxygen Species and consequent OS within the chicken thymus. This process further activates the ZBP1-mediated PANoptosis pathway, ultimately causing damage to chicken thymus tissue. Que intervention effectively suppresses NF-κB activation, blocking the transmission of the NF-κB/TNF-α/ROS signaling axis, thereby reversing DEHP-induced ZBP1-mediated PANoptosis in chicken thymus.