[Wnt5a regulates SiO₂-induced ferroptosis in mouse alveolar macrophages by positive feedback]

Objective To investigate the potential role of Wnt5a signaling in SiO₂-induced ferroptosis in mouse alveolar macrophages. Methods C57BL/6 mice were treated by intratracheal instillation of crystalline silica (SiO₂) or saline, and plasma and bronchial alveolar lavage fluid (BALF) were harvested at 24 hours post injury. Immunofluorescence cytochemical staining was used to detect the expression of Wnt5a, p65 (NF-κB p65) and Toll-like receptor 4 (TLR4) in alveolar macrophages. ELISA was employed to detect the levels of inflammatory cytokines IL-6 and TNF-α in plasma and BALF. In vitro, the RAW264.7 cells were treated with 0, 50, 100, 150, 200 and 300 μg/mL SiO₂, while the levels expression of p65(p-NF-κB, p65), Wnt5a, cleaved caspase-1(c-caspase-1), gasdermin D, NLR family pyrin domain containing 3(NLRP3), glutathione peroxidase 4(GPX4), NADPH oxidase 1(NOX1) and transferrin were then determined by Western blot; CellROX^R fluorescent probe loading assay was used to detect the release of reactive oxygen species (ROS); real time quantitative PCR tested the mRNA levels of Wnt5a, IL-6, IL-10 and TNF-α. Wnt5a recombinant protein; small molecule antagonist BOX5 were used to activate and inhibit the Wnt5a signaling to investigate the role of Wnt5a in ferroptosis, respectively. Results The stimulation of SiO₂ could significantly activate Wnt5a and other inflammatory signaling pathways, meanwhile, release inflammatory factors such as IL-6 and TNF-α, and inhibit the expression of GPX4 protein. The Wnt5a recombinant protein and SiO₂ could synergistically inhibit the expression of GPX4, whereas BOX5 reduced SiO₂-induced GPX4 and Ferroptosis. Conclusion Wnt5a signaling plays a positive feedback role in SiO₂-induced ferroptosis in mouse alveolar macrophages.