Potential hazard assessment of 6PPD-quinone in the context of ulcerative colitis: network toxicology, machine learning, transcriptomic analysis, and preliminary In vivo validation.

BACKGROUND: The ubiquitous tire-derived pollutant 6PPD-quinone (6PPD-Q) poses potential systemic health risks, yet its toxicological impact on the intestinal tract, particularly in the context of ulcerative colitis (UC), remains largely unknown. This study aimed to investigate whether 6PPD-Q aggravates DSS-induced colitis and to identify candidate molecular events using integrative computational and experimental approaches. METHODS: An integrative strategy combining network toxicology, machine learning, public bulk- and single-cell transcriptomic analyses, molecular simulations, and preliminary in vivo validation was applied. Molecular docking and molecular dynamics (MD) simulations were conducted to evaluate protein-ligand binding stability. For in vivo validation, a DSS-induced colitis mouse model was utilized. Furthermore, qRT-PCR and Western blot analyses were performed to evaluate colonic transcriptional alterations and tight-junction protein expression, respectively. Statistical analyses were conducted using Student's t-test or one-way ANOVA, with P < 0.05 considered statistically significant. RESULTS: Through multidimensional screening, we identified five core regulatory genes (MAPKAPK2, ANXA5, CFB, NR1H4, and PLIN2) potentially associated with 6PPD-Q and UC-related molecular alterations. Molecular docking and molecular dynamics simulations supported plausible predicted interactions between 6PPD-Q and the prioritized proteins, with MAPKAPK2 showing the most favorable docking score and a relatively stable simulated trajectory. In vivo experiments demonstrated that 6PPD-Q exposure significantly exacerbated DSS-induced colonic shortening, macroscopic lesions, and histopathological damage. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed increased colonic mRNA expression of Mapkapk2, Anxa5, and Cfb and decreased expression of Nr1h4 and Plin2 in the DSS plus 6PPD-Q group, consistent with the directions predicted by the bioinformatics analyses. Based on these findings, a proposed Adverse Outcome Pathway (AOP) framework was constructed, linking 6PPD-Q exposure with candidate molecular targets, putative PI3K-Akt/MAPK signaling perturbations, intestinal immune dysregulation, and aggravated colonic injury. CONCLUSIONS: This study provides an integrative mechanistic framework for investigating the potential intestinal effects of 6PPD-Q under inflammatory conditions. By integrating computational target prioritization with in vivo phenotypic and transcriptional evidence, the study identifies candidate molecular events that may contribute to 6PPD-Q-exacerbated intestinal inflammation and provides testable hypotheses for subsequent toxicological investigation.