Indirubin inhibits RSL3-induced ferroptosis and ameliorates acute lung injury by activating the Wnt/β-catenin signaling pathway

Acute lung injury (ALI) is a severe clinical condition with high morbidity and mortality, yet effective pharmacotherapies remain limited. Ferroptosis, a form of regulated cell death driven by iron‑dependent lipid peroxidation, has been implicated in the pathogenesis of ALI. Indirubin (ID), a natural bisindole alkaloid with established anti‑inflammatory properties, has been reported to activate the Wnt/β‑catenin signaling pathway; however, its role in ferroptosis‑associated ALI remains unexplored. This study aimed to investigate whether indirubin alleviates RSL3‑induced ferroptosis in ALI and to elucidate the involvement of the Wnt/β‑catenin pathway. A cellular model of ALI was established by treating human alveolar epithelial cells with RSL3, a specific GPX4 inhibitor. Cells were treated with indirubin or the ferroptosis inhibitor ferrostatin‑1 (Fer‑1). Protein expression levels of GPX4, SLC7A11, Wnt5a and β‑catenin were assessed by Western blotting; transcriptomic profiling was performed by RNA‑sequencing to identify differentially expressed genes and enriched pathways; ferroptosis‑associated ultrastructural changes were evaluated by transmission electron microscopy (TEM); and molecular docking was performed using MOE software to investigate the direct binding interaction between indirubin and β‑catenin. RSL3 treatment markedly downregulated GPX4 and SLC7A11 expression and suppressed the Wnt/β‑catenin pathway, as evidenced by decreased Wnt5a and β‑catenin levels. Co‑treatment with indirubin dose‑dependently activated the Wnt/β‑catenin pathway, which was accompanied by restored expression of GPX4 and SLC7A11. Transcriptomic analysis confirmed that indirubin enhanced Wnt/β‑catenin signaling components and corrected RSL3‑induced gene expression alterations in ferroptosis‑related pathways. TEM showed that RSL3‑induced mitochondrial shrinkage and increased membrane density were ameliorated by indirubin, and molecular docking revealed that indirubin directly binds to β‑catenin, providing a structural basis for pathway activation. Our findings demonstrate that indirubin attenuates RSL3‑induced ferroptosis and ameliorates ALI by activating the Wnt/β‑catenin signaling pathway, suggesting that indirubin represents a promising therapeutic candidate for acute lung injury.