Abstract: Objective To investigate the effect of a methionine-restricted diet on the progression of atherosclerosis (AS) in ApoE-/- mice and to elucidate the underlying molecular mechanisms. Methods A total of twenty-four 8-week-old male ApoE-/- mice were randomly allocated into three groups (n=8 per group): a control group receiving a standard chow diet (0.86% methionine), a high-fat diet (HFD) group (0.86% methionine), and a methionine-restricted (MR) group receiving an HFD with 0.12% methionine. Following an 8-week dietary intervention, the atherosclerotic plaque area in the aortic sinus was quantified using Oil Red O staining. The expression levels of markers associated with inflammation and oxidative stress were evaluated by immunohistochemistry, quantitative real-time PCR (RT-qPCR), and Western blotting. Furthermore, transcriptome sequencing (RNA-seq) was employed to identify differentially expressed genes (DEGs) and their enriched signaling pathways. Results Compared to the HFD group, dietary methionine restriction significantly attenuated the atherosclerotic plaque burden in the aortic sinus (P<0.05). Histological analyses revealed that methionine restriction markedly reduced macrophage infiltration (CD68), reactive oxygen species (ROS) levels (DHE), and the expression of adhesion molecules (Icam1, Vcam1) and cell proliferation markers (Pcna, Ki67) within the plaques (all P<0.05). These findings were corroborated by RT-qPCR and Western blot analyses, which demonstrated that the MR diet downregulated the mRNA and protein expression of genes associated with inflammation, adhesion, and proliferation in aortic tissues. Transcriptome profiling identified 5 425 DEGs regulated by methionine restriction. Gene enrichment analysis indicated that these DEGs were predominantly involved in signaling pathways related to cell adhesion, NF-κB, and PPAR. Notably, this was characterized by a downregulation of pro-inflammatory genes and an upregulation of antioxidant genes, such as Sod2. Conclusion Dietary methionine restriction mitigates the progression of atherosclerosis in ApoE-/- mice by concurrently inhibiting key pathological processes, including vascular inflammation, oxidative stress, and cellular proliferation, via multi-target mechanisms.

Key words: Methionine restriction, Atherosclerosis, ApoE-/- mice