[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; CellROXR 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.