Diesel particulate matter induces dose-dependent cytotoxicity, multi-faceted inflammation, MAPK activation, and oxidative stress in RAW 264.7 macrophages: an integrated transcriptomic and protein–protein interaction network analysis

Abstract Diesel particulate matter (DPM), a Group 1 carcinogen classified by the International Agency for Research on Cancer, is a major component of traffic-related air pollution. Although macrophages are central to the response to inhaled particles, the integrated network-level cellular response to DPM remains incompletely characterized. A combined three analytical methods was applied to RAW 264.7 macrophages exposed to DPM (NIST SRM2975) for 24 h: (i) dose–response phenotyping across twelve CCK-8 concentrations (0–750 μg/mL) and multi-modal characterization (Western blot, RT-qPCR, Griess assay, DCFH-DA ROS imaging, TNF-α ELISA) at sub-cytotoxic to near-IC50 doses; (ii) 3′ mRNA-sequencing at 100 μg/mL with edgeR-based differential expression (|fold change|≥ 1.5) and Gene Ontology, KEGG, and Reactome enrichment; and (iii) STRING-based protein–protein interaction (PPI) network analysis with Markov Cluster (MCL) modular clustering in Cytoscape. DPM reduced viability dose-dependently (IC50 ≈ 135–140 μg/mL) and induced concerted activation of iNOS, COX-2, MAPK phosphorylation, pro-inflammatory cytokines (TNF-α, IL-1β), antioxidant enzymes (SOD2, HO-1, Catalase), nitrite, ROS, and secreted TNF-α. Transcriptomics identified 750 differentially expressed genes (331 up; 419 down), with enrichment in cytokine production, NF-κB/MAPK signaling, inflammasome, and oxidative stress. PPI analysis resolved ten up- and ten down-regulated functional modules, with key hubs Ccl2, Gpx4, Ifit3/Stat1, Dna2/Rad52, and Csf1r. Beyond classical inflammation, these integrated findings reveal coordinated network-level perturbations consistent with ferroptosis vulnerability, genome instability, paradoxical immune dysregulation, and potential macrophage identity loss — hypothesis-generating axes for future mechanistic validation.