Ferroptosis induced by erastin (an inhibitor of cystine transport) and butionine sulfoximine (an inhibitor of glutathione biosynthesis) was prevented by the mitochondria-targeted antioxidants SkQ1 and MitoTEMPO. These effects correlate with the prevention of mitochondrial lipid peroxidation, which precedes cell death. Methylene blue, a redox agent that inhibits the production of reactive oxygen species (ROS) in complex I of the mitochondrial electron transport chain, also inhibits ferroptosis and mitochondrial lipid peroxidation. Activation of ROS production in complex I with rotenone in the presence of ferrous iron stimulates lipid peroxidation in isolated mitochondria, while ROS produced by complex III are ineffective. SkQ1 and methylene blue inhibit lipid peroxidation. We suggest that ROS formed in complex I promote mitochondrial lipid peroxidation and ferroptosis.

Lipid peroxidation plays an important role in various pathologies and aging, at least partially mediated by ferroptosis. The role of mitochondrial lipid peroxidation during ferroptosis remains poorly understood. We show that supplementation of exogenous iron in the form of ferric ammonium citrate at submillimolar doses induces production of reactive oxygen species (ROS) and lipid peroxidation in mitochondria that precede ferroptosis in H9c2 cardiomyocytes. The mitochondria-targeted antioxidant SkQ1 and the redox mediator methylene blue, which inhibits the production of ROS in complex I of the mitochondrial electron transport chain, prevent both mitochondrial lipid peroxidation and ferroptosis. SkQ1 and methylene blue also prevented accumulation of lipofuscin observed after 24 h incubation of cardiomyocytes with ferric ammonium citrate. Using isolated cardiac mitochondria as an in vitro ferroptosis model, it was shown that rotenone (complex I inhibitor) in the presence of ferrous iron stimulates lipid peroxidation and lipofuscin accumulation. Our data indicate that ROS generated in complex I stimulate mitochondrial lipid peroxidation, lipofuscin accumulation, and ferroptosis induced by exogenous iron.

Berberine is a plant isoquinoline alkaloid widely used in traditional medicine for the therapy of diabetes, cardiovascular and other diseases. Ferroptosis, a regulated form of cell death driven by lipid peroxidation, is thought to contribute to the pathogenesis of various diseases associated with excessive oxidative stress. The therapeutic actions of berberine are mediated, at least in part, by its antioxidant effects. Here, we report that the lipophilic berberine derivative 13-decyl berberine (C10Berb) is a mitochondria-targeted antioxidant that exhibits superior ferroptosis inhibition compared to native berberine in H9c2 cardiomyocytes and human fibroblasts. C10Berb efficiently accumulates in mitochondria, suppressing both mitochondrial lipid peroxidation, reactive oxygen species formation, and lipofuscin accumulation at concentrations markedly lower than berberine. Mechanistic studies indicate that the anti-ferroptotic effect of C10Berb is independent of AMPK or Nrf2 activation and is primarily due to its direct antioxidant activity in mitochondria. In isolated cardiac mitochondria, C10Berb potently inhibited lipid peroxidation induced by either reactive oxygen species produced in the electron transport chain or artificial free radical initiators. These results support the hypothesis that mitochondrial lipid peroxidation is critical for ferroptosis and highlight the potential of mitochondria-targeted berberine derivatives as promising therapeutic agents for conditions associated with ferroptotic cell death.