Development of a Lipidomics-Based Cell Screening Platform for Indirect Antioxidants Targeting Oxidized Lipid Droplet Formation and Mitochondrial Membrane Abnormality

<b>Background/Objectives</b>: Oxidized lipid droplet formation and cardiolipin (CL) profile abnormality in mitochondrial membranes are associated with metabolic dysfunction-associated steatotic liver disease (MASLD). However, studies on cell models to easily and simultaneously assess the preventive effects on oxidized lipid droplet formation and CL abnormality by indirect antioxidants are limited. Here, we aimed to develop a lipidomics-based cell screening platform to simultaneously evaluate the preventive effects of indirect antioxidants on oxidized lipid droplet formation and mitochondrial membrane lipid abnormalities. <b>Methods</b>: We created a novel lipidomics-based cell screening platform using oxidized low-density lipoprotein (oxLDL) and a human liver-derived cell line (C3A), and screened indirect antioxidants to promote the expression of cellular antioxidant enzymes, preventing oxidized lipid droplet formation. <b>Results</b>: Mass spectrometry revealed that oxLDL increased the levels of cholesteryl ester hydroperoxides. Thus, oxidized lipid droplet formation was confirmed. Three indirect antioxidants (kaempferol, quercetin, and hesperetin) were examined in the lipidomics-based platform. Consequently, quercetin significantly decreased major lipids and lipid hydroperoxide species, particularly triglycerides and triglyceride hydroperoxides with five or more double bonds. Furthermore, fluorescence microscopy revealed that quercetin reduced the size of small oxidized lipid droplets; it also decreased monolysocardiolipin, which could be associated with mitochondrial dysfunction. <b>Conclusions</b>: Overall, we demonstrated that this method could be useful for screening indirect antioxidants with excellent preventive effects against oxidized lipid droplet formation and CL abnormality by simultaneously analyzing various lipids.