Immune gene MMP9 as a potential key mediator in TCDD exposure-associated atherosclerosis: An integrated study based on network toxicology and experimental validation.

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic, recalcitrant persistent organic pollutant with ubiquitous environmental distribution. Atherosclerosis (AS) is a systemic vascular disorder characterized by chronic vascular inflammation, lipid accumulation, and atherosclerotic plaque formation, in which immunometabolic dysregulation serves as one of the central pathogenic mechanisms. Although an association between environmental TCDD exposure and AS has been preliminarily corroborated, the precise molecular mechanisms underlying TCDD exposure-associated initiation and progression of AS remain incompletely elucidated, especially the mediating role of immune genes. OBJECTIVES: This study sought to explore the potential molecular mechanisms linking TCDD exposure and atherosclerosis via an integrated strategy combining network toxicology and experimental validation. We aimed to screen and identify a core immune gene matrix metallopeptidase 9 (MMP9) and its related regulatory pathways potentially involved in this pathological process through systematic bioinformatic analysis, thereby providing novel theoretical insights and candidate targets for risk assessment, early diagnosis, and targeted intervention of environmentally associated atherosclerosis. METHODS: First, publicly available AS-related transcriptomic datasets and TCDD exposure-associated gene expression profiles were retrieved from the GEO database, subjected to batch correction, and analyzed in an integrated manner. Immune-related differentially expressed genes (Immune-DEGs) closely linked to both TCDD exposure and atherosclerosis were screened using network toxicology approaches. A diagnostic model was constructed using a combined machine-learning pipeline incorporating multiple foundational algorithms, and core immune targets were prioritized via SHapley Additive exPlanations (SHAP) analysis and Weighted Gene Co-expression Network Analysis (WGCNA). Functional enrichment and immune infiltration analyses were performed to characterize the biological functions of core immune targets and their associations with the atherosclerotic inflammatory microenvironment. Pseudotime analysis based on publicly available single-cell RNA sequencing data was conducted to delineate the dynamic expression pattern of MMP9 during atherosclerotic plaque evolution, with in silico knockout employed to evaluate its functional contribution. Furthermore, an in vitro cellular model of atherosclerosis was established using oxidized low-density lipoprotein (ox-LDL) to induce human umbilical vein endothelial cell (HUVEC) injury, and MMP9 mRNA and protein expression levels were validated by quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot (WB), respectively. Molecular docking and molecular dynamics simulations were performed to assess the binding affinity between TCDD and MMP9. RESULTS: Integrated bioinformatic analyses and machine learning screening identified MMP9 as a core immune gene potentially mediating TCDD exposure-associated atherosclerosis. Immune infiltration analysis revealed a positive correlation between MMP9 expression and pro-inflammatory cell infiltration within atherosclerotic lesions. Functional enrichment indicated that MMP9 is predominantly involved in inflammatory signaling activation, vascular matrix remodeling, and lipid metabolism regulation. Pseudotime analysis demonstrated that MMP9 was markedly upregulated in advanced atherosclerotic plaques, driving pathological extracellular matrix remodeling and enhanced plaque vulnerability. In silico MMP9 knockout significantly attenuated extracellular matrix degradation, reduced inflammatory infiltration, and preserved the structural stability of the fibrous cap. In the in vitro AS cellular model, MMP9 expression was significantly elevated. Molecular docking and dynamics simulations verified high-affinity binding between TCDD and MMP9, with a binding energy of -9.7 kcal/mol, supporting the formation of a stable complex. CONCLUSIONS: This study identifies the immune gene MMP9 as a potential pivotal molecular mediator in TCDD exposure-associated atherosclerosis. Bioinformatic evidence and molecular simulation results suggest that TCDD may specifically bind to MMP9 to modulate inflammatory responses and vascular matrix remodeling, thereby potentially exacerbating the initiation and progression of atherosclerosis.