RAGE/NF-κB signaling mediates paraquat-induced alveolar-capillary barrier injury in a human alveolar microphysiological system

Paraquat (PQ), a highly toxic herbicide that causes fatal pulmonary injury, has no specific clinical antidotes, mainly due to incomplete understanding of its pathogenic mechanisms. Inhalational exposure to PQ is the primary route of toxicity among agricultural workers. However, conventional experimental models fail to accurately replicate the physiological characteristics of the alveolar-capillary barrier and the air-liquid interface (ALI). In this study, we developed an Alveolar Microphysiological System (AMS) that co-cultures alveolar epithelial cells (EPCs) and pulmonary microvascular endothelial cells (EDCs) with ALI, effectively mimicking PQ-induced lung injury. This injury is characterized by increased permeability of the alveolar-capillary barrier, compromised intercellular junction integrity, and ultrastructural alterations. Bulk RNA sequencing, public single-cell RNA sequencing (scRNA-seq), and functional validation assays revealed that PQ triggers divergent, cell-type-specific responses: activating the RAGE/NF-κB signaling pathway and pro-inflammatory programs in EPCs, while suppressing these pathways in EDCs. Furthermore, PQ treatment results in the aberrant expression of pro-inflammatory cytokines (e.g., IL-1α/β, TNF-α), chemokines (e.g., CCL3/5, CXCL8/10), and growth factors (e.g., PDGF, TGF-α) in EPCs, a pattern that significantly differs from the expression profile observed in EDCs. Additionally, FPS-ZM1, a specific inhibitor of RAGE, mitigated PQ-induced barrier dysfunction by restoring normalized AMS permeability, enhancing E-cadherin expression in EPCs, and improving adherens junction integrity in EDCs. Collectively, these findings suggest that the AMS serves as a valuable translational platform for developing therapeutic strategies. Moreover, the RAGE/NF-κB signaling pathway exhibits a cell-type-specific function in pulmonary injury induced by PQ exposure under air-liquid interface conditions, indicating that modulation of the RAGE/NF-κB axis may offer a promising therapeutic avenue for managing paraquat poisoning.