Baicalein Ameliorates ALOX15-mediated Lipid Peroxidation in Mitochondria and Protects Cardiomyocytes against Palmitate Acid-induced Ferroptosis through the ACSL4/ALOX15/GPX4 Axis

Abstract Background: During prediabetes, increased free fatty acid levels induce negative actions on cardiomyocytes, but effective approaches to prevent these negative actions and the development of diabetic cardiomyopathy are limited. Saturated fatty acids such as palmitate have been shown to contribute to the development of diabetic cardiomyopathy (DbCM), with ferroptosis being recognized as a potential mechanism of palmitate-induced cardiac injury. ALOX15 is a driving factor of ferroptosis and also contributes to inflammation and oxidative stress in DbCM. Baicalein is a natural inhibitor of ALOX15, but the effects of baicalein on ferroptosis in DbCM remain unknown. Aims and Objectives: The overall objective of the present study was to elucidate the potential effects of baicalein on ferroptosis in cardiomyocytes when exposed to elevated free fatty acid levels. Materials and Methods: H9c2 cardiomyocytes were treated with palmitic acid at different concentrations to induce lipid peroxidation and ferroptosis-related responses. The cells were subsequently incubated with baicalein to evaluate its protective effects. Lipid peroxidation and intracellular reactive oxygen species (ROS) levels were measured. Production of 12- and 15-hydroxyeicosatetraenoic acid (15-HETE), as well as mitochondrial function indicators including mitochondrial membrane potential, cytochrome c release, and mitochondrial ROS, were assessed. Protein expression levels of GPX4, ACSL4, and ALOX15 were analyzed by Western blot. Statistical analysis was performed to determine concentration-dependent effects and treatment efficacy. Results: Our results revealed that 12-, 15-HETE production, lipid peroxidation and intracellular reactive oxygen species (ROS) levels increased in a concentration-dependent manner when treated with palmitic acid. Baicalein effectively inhibited the ALOX15 associated 12-, 15- HETEs production and decreased lipid peroxidation in H9c2 cells. Furthermore, baicalein also ameliorated mitochondrial dysfunction through restoring mitochondrial membrane potential and decreasing the release of cytochrome c, as well as mitochondrial ROS. Treatment with baicalein also increased GPX4 and reduced ACSL4 and ALOX15 protein expression in H9c2 cells. Conclusions: These results suggest that baicalein may protect cardiomyocytes against ferroptosis through an ACSL4-ALOX15-GPX4 axis.