Original uncropped Western blot images.

<div><p>Background</p><p>Acute respiratory distress syndrome (ARDS) / acute lung injury (ALI) is a serious medical disease characterized by pulmonary dysfunction and inflammation. This study aims to determine the main molecular modules linked to ARDS and investigate the role of Fibulin-1 (FBLN1) in regulating ferroptosis in ARDS.</p><p>Methods</p><p>Weighted Gene Co-expression Network Analysis (WGCNA) was employed on the GSE263867 dataset to find key modules associated with ALI. Differentially expressed genes (DEGs) and protein-protein interaction (PPI) networks were analyzed. MLE-12 cells were treated with lipopolysaccharide (LPS) to induce ferroptosis. <i>In vitro</i> studies were conducted to investigate the effects of FBLN1 and Transforming Growth Factor Beta 1 (TGF-β) overexpression on cell viability, oxidative stress markers, and ferroptosis-related proteins.</p><p>Results</p><p>WGCNA identified the turquoise module as significantly negatively correlated with ARDS. Five key overlapping genes (<i>GRIA1</i>, <i>OGN</i>, <i>COL14A1</i>, <i>FBLN1</i>, and <i>COL6A3</i>) were significantly downregulated in ARDS samples. LPS treatment induced ferroptosis in MLE-12 cells, indicated by increased malondialdehyde (MDA), lipid reactive oxygen species (ROS), and ferrous iron (Fe²⁺) levels, and decreased cell viability and glutathione (GSH) levels. FBLN1 overexpression partially reversed these effects. Additionally, FBLN1 inhibited the TGF-β/Smad signaling pathway, as shown by decreased TGF-β and p-Smad protein levels. TGF-β overexpression exacerbated LPS-induced oxidative stress and ferroptosis, reducing cell viability and GSH levels. FBLN1 overexpression counteracted this effect, suggesting antagonistic roles for FBLN1 and TGF-β in regulating ferroptosis.</p><p>Conclusion</p><p>This study highlights <i>FBLN1</i> as a critical regulator of ferroptosis in ARDS. Targeting the TGF-β/Smad pathway to modulate <i>FBLN1</i> expression offers a potential therapeutic strategy to alleviate oxidative stress and mitigate pulmonary injury in inflammatory lung diseases.</p></div>