PM2.5 induces ferroptosis in chronic obstructive pulmonary diseases via the GSK-3β/NRF2 pathway

Background: Recent studies have shown that fine particulate matter (PM2.5) exposure is a key harmful risk factor for chronic obstructive pulmonary disease (COPD) and PM2.5-associated ferroptosis plays an important role during the process of airway oxidative stress. Our preliminary study revealed that PM2.5 reduces the expression of phosphorylated glycogen synthase kinase (GSK)-3β in airway epithelial cells, the overactivity of the GSK-3β/Nuclear Factor erythroid 2-Related Factor 2 (NRF2) pathway is related to ferroptosis. Accordingly, we explored whether PM2.5 could induce ferroptosis in airway epithelial cells and promote the development of COPD via the GSK-3β/NRF2 pathway. Methods: The effect of GSK-3β/NRF2-mediated ferroptosis was assessed using an in vivo model of 20 μg/μl PM2.5-induced COPD by tracheal infusion and 50 μg/ml PM2.5-exposed airway epithelial cells in vitro. Then we performed qRT-PCR to detect mRNA expression; Western blotting, immunofluorescence and immunohistochemical staining to detect protein expression; flow cytometry and spectrophotometry to measure the levels of intracellular lipid peroxidation; small animal spirometry to examine the lung function in mouse, and hematoxylin and eosin (H&E) staining to measure the average alveolar septa in mouse lung sections. Results: We found that PM2.5 decreased the ferroptosis marker mRNA expression of NRF2, SLC7A11 and GPX4, and also decreased the protein expression of p-GSK-3β, NRF2, SLC7A11 and FTH-1, increased the protein expression of NCOA4, then increased the level of lipid peroxidation and MDA in human airway epithelial cells. Further, PM2.5 reduced the expression of p-GSK-3β, NRF2, SLC7A11 and GPX4 in the lungs, subsequently induced lung injury and impaired lung function of mice. Treatment with ferroptosis inhibitors FER-1 and GSK-3β inhibitor TDZD-8 reversed this effect. Conclusion: Our findings suggested that PM2.5 induced ferroptosis of airway epithelial cells, contributing to airway oxidative stress via the GSK-3β/NRF2 signaling pathway in vivo and in vitro, which could be a therapeutic target for PM2.5-induced COPD.