Disulfiram Alleviates MTX-Induced Pulmonary Fibrosis by Inhibiting EMT in Type 2 Alveolar Epithelial Cells.

Abstract

Purpose: Methotrexate (MTX)-induced pulmonary fibrosis is associated with high morbidity and mortality, with limited treatment options available. This study investigates whether disulfiram (DSF) can mitigate MTX-induced pulmonary fibrosis and explores the underlying mechanisms.

Methods: Eight-week-old male mice were divided into control, DSF, MTX, and MTX+DSF groups and treated for 8 weeks. Weight, food, and water intake were monitored. Post-treatment, lung tissues were analyzed using HE and Masson staining, and electron microscopy. Real-time qPCR and ELISA were employed to assess inflammatory markers such as IL-1β and TNF-α in lung tissues and serum. PCR, ELISA, and Western blot were used for fibrotic markers including Col1α1, α-SMA, and hydroxyproline. Type 2 alveolar epithelial cell line MLE12 cells were similarly grouped, followed by RNA sequencing and bioinformatics analysis to elucidate the mechanisms by which DSF exerts anti-MTX-induced pulmonary fibrosis effects. ELISA and Western blot were used to measure E-cadherin and α-SMA expression.

Results: DSF significantly reduced MTX-induced alveolar septal thickening, pulmonary fibrosis, and inflammatory cell infiltration. It also decreased the expression of inflammatory factors IL-1β and TNF-α, as well as the expression of Col1α1, α-SMA, and others. RNA-seq revealed that DSF induces changes in multiple signaling pathways associated with pulmonary fibrosis, particularly in extracellular matrix-related genes. ELISA and Western blot showed decreased E-cadherin and increased α-SMA in the MTX group, which was partially restored with DSF treatment.

Conclusion: DSF alleviates MTX-induced pulmonary fibrosis by reducing epithelial-mesenchymal transition (EMT) in type 2 alveolar epithelial cells. Disulfiram shows potential as a therapeutic agent for MTX-induced pulmonary fibrosis.