Single-cell RNA sequencing identifies cellular heterogeneity in endothelial and epithelial cells associated with nitrogen dioxide-induced acute lung injury

Inhalation of nitrogen dioxide (NO₂), a representative irritant gas, can trigger acute lung injury (ALI), typically characterized by increased permeability and dysfunction of the blood-air barrier. However, the exact mechanisms underlying NO₂ inhalation-induced ALI (NO₂-ALI) remain poorly understood. Using single-cell RNA sequencing (scRNA-seq), we identified significant alterations in endothelial and epithelial cells during NO₂-ALI. Notably, leucine-rich alpha-2-glycoprotein 1 (Lrg1) and uncoupling protein 2 (Ucp2), which have been implicated in ALI progression, were significantly upregulated in endothelial cells following NO₂ exposure (P < 0.05 compared to control). General capillaries (GCs) potentially function as stem cells, facilitating endothelial cell repair and recruiting neutrophils to amplify inflammatory responses. Furthermore, a novel subpopulation of epithelial cells, identified as lymphocyte antigen 6 A⁺ (Ly6a) alveolar cells, showed a significant increase in abundance (P < 0.05 compared to control) and played a pivotal role in alveolar epithelial cell differentiation after NO₂ inhalation. Overall, these findings shed insights into the pathogenic roles of endothelial and epithelial cells in NO₂-ALI.