Bioinformatics screening and experimental validation of candidate key genes in hyperoxia-induced acute lung injury

Hyperoxia-induced acute lung injury (HALI) represents a serious consequence of prolonged oxygen exposure, characterized by oxidative stress, inflammatory responses, and impaired pulmonary function, whereas its underlying molecular mechanisms remain incompletely elucidated. The present work sought to delineate candidate genes and associated signaling pathways involved in HALI through integrated transcriptomic analysis and experimental validation. The microarray dataset GSE237260, including six control and six hyperoxia-exposed mouse lung samples, was analyzed to identify differentially expressed genes (DEGs). A total of 981 DEGs were identified, and random forest analysis identified 20 characteristic genes, among which Sult1a1, Fibin, Ednrb, Tcim, and Ptpru were significantly overexpressed. Functional enrichment analysis indicated associations with hypoxia, inflammatory responses, mTORC1 signaling, p53 signaling, and TNF-α signaling via NF-κB. Hyperoxia-induced mouse and cellular models using A549 cells and human pulmonary microvascular endothelial cells (HPMECs) were established for experimental validation. Hyperoxia exposure induced inflammatory infiltration, pulmonary edema, impaired gas exchange, reduced cell viability, increased apoptosis, and elevated reactive oxygen species levels. Sult1a1, Fibin, and Ednrb were consistently upregulated in vivo and in vitro. Hyperoxia also increased the phosphorylation of mTOR, NF-κB p65, and S6K, whereas pharmacological inhibition of mTORC1 or NF-κB-related signaling and Ednrb knockdown attenuated cellular injury and reduced the phosphorylation of these signaling proteins. Conversely, Ednrb overexpression aggravated hyperoxia-induced cellular injury and increased the phosphorylation of mTOR, NF-κB p65, and S6K. Collectively, these findings identify candidate key genes and signaling pathways associated with HALI and provide experimental evidence supporting a potential involvement of Ednrb in hyperoxia-related cellular injury and mTORC1/NF-κB-related signaling.