Single-cell transcriptomes reveal heterogeneity of chlorine-induced mice acute lung injury and the inhibitory effect of pentoxifylline on ferroptosis.

ABSTRACT

To investigate the effect of pentoxifylline (PTX) on Chlorine (Cl2)-induced acute lung injury (ALI) by single-cell RNA sequencing (scRNA-seq). Female BALB/c mice were exposed to Cl2 at 400 ppm for 15 min. H&E staining was used to observe the degree of lung injury. scRNA-seq was conducted to analysis of normal and Cl2-exposed mice lung tissues. Immunofluorescence was used to observe genes of interest. Thirty-two mice were randomly divided into four groups Control, Cl2, Cl2+Fer-1, Cl2+PTX. TEM, WB and ELISA were used to detect ferroptosis-related indicators. The 5, 8, 10, 12, 16, 20 clusters were epithelial cells and 4, 15, 18, 19, 21 clusters were endothelial cells. Pseudo-time analysis revealed the differentiation trajectory of epithelial cells and key regulatory genes (Gclc, Bpifa1, Dnah5 and Dnah9) during the process of injury. Cell-cell communication analysis identified several important receptor-ligand complexes (Nrp1-Vegfa, Nrp2-Vegfa, Flt1-Vegfa and Flt4-Vegfa). Ferroptosis were found up-regulated in epithelial and endothelial cells by GSVA analysis. Highly expressed genes to which closely related ferroptosis were found by SCENIC analysis. PTX could significantly decrease the levels of MDA and abnormal high expression of solute carrier family 7 member 11 (SLC7A11, the key transporter of cystine) as well as increase the expression of GSH/GSSG and glutathione peroxidase 4 (GPX4) (p < 0.05). This study revealed novel molecular features of Cl2-induced ALI. PTX may be a potential specific drug by inhibiting the process of ferroptosis in epithelial and endothelial cells.