Regulation of Ferroptosis in Human Macrophage by Nitric Oxide Donors.

Ferroptosis is a programmed form of cell death in which iron-dependent lipid peroxidation is the main feature. Macrophages are the major cells of the immune system, they function in a pro-oxidative environment, so the study of their susceptibility to ferroptosis and the search for approaches to its regulation are important. Materials and Methods: RSL3 and ML-162, inhibitors of glutathione peroxidase 4 (GPX4), and erastin, an inhibitor of cystine/ glutamate transport, were used to induce ferroptosis in THP-1 macrophages. The progression of ferroptosis was monitored using three independent methods: reduction of Alamar blue by live cells, measurement of lactate dehydrogenase in the medium, and the LIVE/DEAD assay. Ferroptotic cell death was proven by using the specific inhibitor ferrostatin-1 and by detecting lipid oxidation in cells using the BODIPY 581/591 C11 fluorescent probe. Results: RSL3 and ML-162 dose-dependently induced ferroptosis in cells. THP-1 macrophage ferroptosis is a slow process and begins ~5 h after inducer addition. Erastin was a weak ferroptosis inducer; however, it enhanced ferroptosis induced by GPX4 inhibitors. We compared the ability of two NO donors with different half-lives to affect THP-1 macrophage ferroptosis: DEA NONOate (2 min) and DTPA NONOate (3 h). Donors were added either once after the inducer at a concentration of 100-120 μM or repeatedly until reaching the final concentration. DEA had no effect on THP-1 macrophage ferroptosis, whereas DPTA completely inhibited ferroptosis. Conclusion: DTPA, being an NO donor with a half-life of 3 h at 37°С, can be used to inhibit ferroptosis in THP-1 macrophages, which develops within 17-19 h. Therefore, there are mechanisms of prolongation of NO action in cells that should be studied to use NO donors for regulation of cellular ferroptosis.