**Background:** Exposure to diesel exhaust particles (DEPs), a major component of fine particulate matter (PM2.5), triggers pulmonary inflammation, oxidative stress, and behavioral disorders in mammals. Dietary intervention with polyphenolic compounds may offer a preventive strategy. Ellagic acid (EA), a polyphenol found in berries, grapes, and pomegranates, has known anti-inflammatory and antioxidant properties and has shown protective effects against various pulmonary toxicants. However, its efficacy against PM-induced pulmonary pathology and hyperactivity had not been fully characterized.
**Methods:** Seven-week-old male C57BL/6NCrlOri mice were acclimatized for 7 days and randomly assigned to four groups (n=8/group): CON (5% DMSO orally for 14 days, distilled water instillation on days 8-14), PMI (PM 5 mg/kg intratracheally on days 8-14), EL+PMI (EA 20 mg/kg orally for 14 days, PM 5 mg/kg on days 8-14), and EH+PMI (EA 100 mg/kg orally for 14 days, PM 5 mg/kg on days 8-14). PM instillation was performed 1 hour after EA treatment. Mice were euthanized on day 15. Bronchoalveolar lavage fluid (BALF) was collected for cell counting and differential staining (Diff-Quik). TNFα, IL-6, and H2O2 levels in BALF were measured by ELISA. Pulmonary mRNA expression of Tnfα, Il-1β, Il-6, Vegfα, and Ankrd37 was analyzed by qRT-PCR normalized to Gapdh. An open field test (OFT) was performed 1 hour after the last PM instillation, recording distance, duration, and velocity over 10 minutes. Serum corticosterone was measured by ELISA. Statistical analysis used one-way ANOVA with Tukey's post hoc test or Kruskal-Wallis with Dunn's post hoc test (p<0.05).
**Key Results:** PM instillation significantly increased delta body weight and relative lung weight, but EA treatment attenuated the delta body weight increase regardless of dose. PM exposure increased total BALF immune cells approximately 3-fold versus CON, with significant increases in neutrophils, macrophages, eosinophils, and lymphocytes. EA treatment did not reduce total cells, neutrophils, or macrophages but dose-dependently decreased eosinophils and lymphocytes. BALF TNFα and IL-6 protein levels were elevated approximately 2.6-fold and 19-fold, respectively, in the PMI group versus CON; EA did not prevent these increases. H2O2 levels were unchanged across groups. At the mRNA level, PMI increased pulmonary Tnfα (6.2-fold), Il-1β (3.1-fold), Il-6 (1.4-fold), Vegfα (4.2-fold), and Ankrd37 (2.1-fold) versus CON. EA treatment reduced Tnfα by 1.9-fold (EL+PMI) and 2.9-fold (EH+PMI); Il-1β by 1.6-fold and 2.1-fold; Il-6 by 0.8-fold and 0.9-fold; Vegfα by 1-fold and 1.7-fold; and Ankrd37 by 1.2-fold and 1.4-fold compared to PMI. In the OFT, PMI mice showed increased total, border, and center moving distances and mean speeds. EA (especially 100 mg/kg) reduced these hyperactivity measures. Time spent in the center was not significantly different, though PMI increased it by 30.4% (p=0.19) and EA groups decreased it by 75.5-85.6% versus PMI. Serum corticosterone did not differ among groups.
**Clinical Implications:** This pilot study demonstrates that oral EA pretreatment can attenuate PM-induced pulmonary inflammatory and hypoxic gene expression and normalize hyperlocomotive behavior in mice, despite not reducing BALF inflammatory cell counts or cytokine protein levels. The findings align with prior work showing that phenolic compounds like quercetin similarly prevent PM-induced hyperactivity. The authors acknowledge key limitations: the short exposure duration (7 days of PM), high PM concentration (5 mg/kg), and lack of effect on BALF protein-level inflammation suggest the model may not fully replicate chronic human exposure. The dissociation between mRNA and protein-level inflammation, and between hypoxic gene expression and H2O2 levels, requires further investigation. Future studies should use longer exposure periods with lower PM concentrations and explore NFκB and HIF1α pathway mechanisms. Nonetheless, the behavioral normalization by EA is a notable finding, as PM exposure in early adulthood induced hyperactivity, and dietary EA effectively reversed it without altering serum corticosterone. EA may represent a feasible dietary preventive strategy against PM-induced pathophysiology, but clinical translation requires optimized experimental models and mechanistic validation.