Elucidating the mechanism of plasticizers associated with atherosclerosis through network toxicology, bulk RNA sequencing, and single-cell RNA sequencing analysis

PURPOSE: This study aims to screen and identify potential atherosclerosis (AS) biomarkers associated with common plasticizer exposure, providing a basis for future investigation into targeted therapeutic strategies. METHODS: AS-related datasets (from public databases) and common plasticizers (ATBC, DEP, DMP, DOP) were used. First, plasticizer-related genes (PRGs) and AS-related genes (ASRGs) were ascertained from public databases, respectively. Biomarkers were then determined through differential expression, machine learning, receiver operating characteristic (ROC), and gene expression analyses. To further explore the biological mechanisms underlying AS, functional enrichment, immune infiltration, molecular docking, molecular dynamics (MD) simulations, and single-cell RNA sequencing (scRNA-seq) analyses were conducted. PRINCIPAL RESULTS: In this study, AR and CCR2 were recognized as biomarkers for AS, with AR showing significantly lower expression in AS samples and CCR2 demonstrating significantly higher expression. These biomarkers were co-enriched in the "cytokine-cytokine receptor interaction" pathway. Additionally, 5 immune cell types with differential infiltration were ascertained, with regulatory T cells showing a strong correlation with both biomarkers. Molecular docking revealed favorable binding between the biomarkers and ATBC, with a particularly moderate binding energy of -7.1 kcal/mol between CCR2 and ATBC. MD simulations confirmed the stability of the CCR2-ATBC complex. Finally, scRNA-seq analysis ascertained vascular smooth muscle cells (VSMCs) and T lymphocytes as key cell types in AS, with dynamic expression patterns of AR and CCR2 observed during their differentiation. MAJOR CONCLUSION: Our study identifies AR and CCR2 as biomarkers linking plasticizer exposure to AS. These findings elucidate potential molecular mechanisms and offer promising directions for future experimental validation and therapeutic exploration.