Chlorpyrifos exposure induces inflammatory and immune dysregulation: An integrative in silico and in vitro analysis

Chlorpyrifos (CPF) is a widely used organophosphate insecticide that has been associated with chronic inflammatory and immune-related health effects. However, the molecular mechanisms underlying CPF-induced immune-inflammatory dysregulation remain incompletely defined. In this study, an integrated in silico and in vitro strategy was employed to elucidate the signaling pathways involved in CPF-mediated macrophage inflammatory activation. Network-based target prediction and pathway enrichment analysis identified MAPK14, which encodes p38 MAPK, and RELA, which encodes the NF-κB p65 subunit, as central regulatory nodes potentially involved in CPF-mediated inflammatory responses. These computational findings guided subsequent mechanistic validation in RAW264.7 macrophages. Consistent with the in silico predictions, CPF exposure selectively increased the activation of MAPK14 (which encodes the p38 MAPK) and RELA (which represents p65) in RAW264.7 macrophages, whereas other MAPK family members showed no obvious activation. Molecular docking and molecular dynamics simulations further demonstrated stable interactions between CPF and both predicted targets, supporting their potential involvement in CPF-responsive signaling regulation. Functionally, CPF treatment increased phosphorylation of p38 MAPK and p65, promoted the production of inflammatory mediators, and upregulated co-stimulatory and antigen-presenting molecules in macrophages. Collectively, these findings suggest that CPF is associated with macrophage-mediated inflammatory activation involving the p38 MAPK and NF-κB signaling pathways. This study provides mechanistic insight into CPF-associated immune-inflammatory dysregulation and supports the relevance of macrophage inflammatory activation in environmental CPF exposure.