Ferroptosis-inducing effects of cromolyn in microglia through NCOA4-mediated ferritinophagy

Cromolyn has anti-inflammatory and neuroprotective effects. However, its influence on microglial cell viability and death pathways remains largely unexplored. This study aimed to elucidate the cellular and molecular mechanisms underlying the effects of cromolyn exposure on microglial viability, with a particular focus on ferroptosis and ferritinophagy. HMC3 microglial cells were treated with cromolyn for 24, 48, and 72 h. Cell viability, nuclear morphology, cell cycle, MDA, GSH, and intracellular iron levels were assessed. Western blot analysis evaluated the expression of ferroptosis-related (GPX4, ACSL4, SLC7A11) and autophagy-associated (NCOA4, FTH1) proteins. Functional validation was performed using ferroptosis and autophagy inhibitors, and NCOA4-silencing. Cromolyn induced time- and dose-dependent cytotoxicity (IC₅₀ at 48 h = 9.4 µM), with prominent G0/G1 cell cycle arrest and nuclear abnormalities emerging at 48 h. At 72 h, excessive cell death limited mechanistic analyses. Cromolyn triggered ferroptosis via the GPX4-regulated pathway, evidenced by increased MDA, iron accumulation, and altered expression of GPX4, ACSL4, and SLC7A11. This ferroptotic response was mechanistically linked to NCOA4-mediated ferritinophagy, leading to GPX4 suppression and lipid peroxidation. NCOA4 knockdown rescued cell viability, restored FTH1 levels, and reduced lipid peroxidation. Our findings suggest, for the first time, that cromolyn may regulate microglial survival through an NCOA4-dependent ferritinophagy-ferroptosis axis. Given the dual roles of microglia in neuroinflammation and neurodegeneration, these data highlight both the therapeutic potential and risks of cromolyn in neurodegenerative disorders.