Background: Eicosapentaenoic acid (EPA) effectively modulates immune responses and reshapes the gut microbiota. Therefore, this study aims to investigate the role of EPA in modulating the composition of gut microbial and restoring mitochondrial and efferocytic function in atherosclerosis (AS).

Methods: Male apolipoprotein E knockout (ApoE^-/-) mice were fed a high-fat diet to induce AS and then subjected to EPA treatment. Fecal microbiota transplantation (FMT) was performed using fecal samples from untreated AS mice. Histopathological changes in the aortic root were assessed using hematoxylin and eosin staining and Oil Red O staining approaches. Serum lipid profiles were quantified using corresponding biochemical assays. Mitochondrial membrane potential and reactive oxygen species (ROS) levels were evaluated using 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining, respectively. Efferocytosis was analyzed using Diff-Quick staining, and the composition and functions of the gut microbiota were analyzed using 16S rRNA gene sequencing.

Results: EPA treatment attenuated AS progression and improved serum lipid profiles by reducing total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) while increasing high-density lipoprotein cholesterol (HDL-C). Furthermore, EPA treatment restored mitochondrial membrane potential, reduced ROS production, and enhanced macrophage efferocytosis in the aorta. Microbial analysis revealed that EPA reshaped the composition of gut microbiota by enriching beneficial bacterial taxa and altering metabolic pathways, including those related to carbohydrate metabolism and xenobiotic degradation. Notably, FMT from AS mice effectively reversed the protective effects of EPA on mitochondrial function, ROS levels, and efferocytosis.

Conclusion: This study demonstrates that EPA alleviates atherosclerotic pathology by modulating the composition of gut microbiota, restoring mitochondrial function, and enhancing efferocytosis. The findings support the therapeutic potential of EPA as a microbiota-targeting intervention for cardiovascular disease.