Alpha-lipoic acid (α-LA) is a potent antioxidant that can prevent apoptosis associated with cisplatin-induced ototoxicity through ROS. Ferroptosis is defined as an iron-dependent cell death pathway that has recently been highlighted and is associated with the accumulation of intracellular lipid droplets (LDs) due to an inflammatory process. Herein, we investigated the impact of α-LA on ferroptosis and analyzed the characteristics of LDs in auditory hair cells treated with cisplatin using high-resolution 3D quantitative-phase imaging with reconstruction of the refractive index (RI) distribution. HEI-OC1 cells were treated with 500 μM α-LA for 24 h and then with 15 μM cisplatin for 48 h. With 3D optical diffraction tomography (3D-ODT), the RI values of treated cells were analyzed. Regions with high RI values were considered to be LDs and labelled to measure the count, mass, and volume of LDs. The expression of LC3-B, P62, GPX4, 4-hydroxynonenal (4-HNE), and xCT was evaluated by Western blotting. HEI-OC1 cells damaged by cisplatin showed lipid peroxidation, depletion of xCT, and abnormal accumulation of 4-HNE. Additionally, the count, mass, and volume of LDs increased in the cells. Cells treated with α-LA had inhibited expression of 4-HNE, while the expression of xCT and GPX4 was recovered, which restored LDs to a level that was similar to that in the control group. Our research on LDs with 3D-ODT offers biological evidence of ferroptosis and provides insights on additional approaches for investigating the molecular pathways.
Keywords: ferroptosis; holotomography; lipid droplet; ototoxicity; α-lipoic acid.