Prolonged exposure to traffic-related particulate matter and gaseous pollutants implicate distinct molecular mechanisms of lung injury in rats

Abstract

Background: Exposure to air pollution exerts direct effects on respiratory organs; however, molecular alterations underlying air pollution-induced pulmonary injury remain unclear. In this study, we investigated the effect of air pollution on the lung tissues of Sprague-Dawley rats with whole-body exposure to traffic-related PM₁ (particulate matter < 1 μm in aerodynamic diameter) pollutants and compared it with that in rats exposed to high-efficiency particulate air-filtered gaseous pollutants and clean air controls for 3 and 6 months. Lung function and histological examinations were performed along with quantitative proteomics analysis and functional validation.

Results: Rats in the 6-month PM₁-exposed group exhibited a significant decline in lung function, as determined by decreased FEF_25-75% and FEV₂₀/FVC; however, histological analysis revealed earlier lung damage, as evidenced by increased congestion and macrophage infiltration in 3-month PM₁-exposed rat lungs. The lung tissue proteomics analysis identified 2673 proteins that highlighted the differential dysregulation of proteins involved in oxidative stress, cellular metabolism, calcium signalling, inflammatory responses, and actin dynamics under exposures to PM₁ and gaseous pollutants. The presence of PM₁ specifically enhanced oxidative stress and inflammatory reactions under subchronic exposure to traffic-related PM₁ and suppressed glucose metabolism and actin cytoskeleton signalling. These factors might lead to repair failure and thus to lung function decline after chronic exposure to traffic-related PM₁. A detailed pathogenic mechanism was proposed to depict temporal and dynamic molecular regulations associated with PM₁- and gaseous pollutants-induced lung injury.

Conclusion: This study explored several potential molecular features associated with early lung damage in response to traffic-related air pollution, which might be used to screen individuals more susceptible to air pollution.

Keywords: Gaseous pollutant; Lung injury; Molecular mechanism; Particulate matter; Proteomics; Traffic-related air pollution.

Fig. 1

Lung function examination. Forced expiratory flow at 25–75% of forced vital capacity (FEF_25–75%) and the ratio of forced expiratory volume at 20 ms and forced vital capacity (FEV₂₀/FVC) were measured for each rat with at least three acceptable measurements. Significant reductions in FEF_25–75% and FEV₂₀/FVC were observed in rats from the 6 M-PM₁ group; * p < 0.05

Fig. 2

Histological images of rat lung tissues. a, b, and c are representative images of the 3 M-CTL, 3 M-GAS, and 3 M-PM₁ groups, whereas d, e, and f are representative images of the 6 M-CTL, 6 M-GAS, and 6 M-PM₁ groups, respectively. The images show the alveoli (left panel) and airway (right panel) tissues. Bars in the airway figures indicate the thickness of the airway wall. Black arrowheads point to the infiltration of immune cells within the alveolar and bronchial walls. The black arrow in c indicates damage within the bronchial wall

Fig. 3

Workflow and DEPs in lung tissue proteomics analysis. a Experimental workflow for lung tissue proteomics analysis. The lung tissues from five rats in individual groups were lysed, digested, and pooled to generate six pooled peptide samples. Each pooled sample was labelled with one of the TMTs separately. The labelled peptides were then combined for reverse phase StageTip fractionation, followed by LC-MS/MS analysis and database searching for protein identification and quantitation. A total of 2673 proteins were identified, of which 2526 proteins were quantified. b The overlapping of DEPs in the 3 M-GAS and 3 M-PM₁ groups, progressive GAS and PM₁ exposures, and particle-specific DEPs under 3- and 6-month exposure

Fig. 4

Enriched pathways associated with subchronic and chronic exposures to GAS and PM₁. a The enriched pathways associated with subchronic (3-month, the two left columns), chronic (6-month, the two middle columns) to GAS and PM₁ as well as progressive exposure to GAS, PM₁ and CTL (the three right columns). b The enriched pathways specifically regulated by fine particles. Pathway enrichment analysis was performed using IPA. The size of the circle represents the p value for enrichment analysis. The colour of the circle indicates activation z-scores (red for upregulation and blue for downregulation)

Fig. 5

Proposed molecular mechanisms associated with lung injury under subchronic and chronic exposures to traffic-related GAS and PM₁. Boxes in the upper side of the protein gene symbol indicate the protein expression in PM₁ (upper) and GAS (lower) groups. Boxes in the left panel of the upper side show the protein ratios of 3 M-PM₁/CTL and 3 M-GAS/CTL, whereas those in the right panel represent progression from 3- to 6-month exposures (PM₁-6 M/3 M and GAS-6 M/3 M, respectively). The particle specific regulations are shown as boxes in the right side of the protein gene symbol (3 M-PM₁/GAS, 6 M-PM₁/GAS, respectively). Upregulated proteins are marked in red, downregulated proteins in green, and unchanged proteins in white. Protein expression enhanced by particles (PM₁/GAS) is marked in fuchsia, whereas suppression is marked in cyan

Fig. 6

Functional validation of selected proteins, oxidative stress, and IL-6 in lung tissues. a, b, and c present the statistical results of Western blot analysis for C3, Chp1, and Serpina3 proteins in lung tissues, respectively. Vinculin was used as the loading control. The expression level of each protein was further normalized to that in the reference sample running in every gel for between-gel comparison. d and e present the ELISA results of 8-isoprostane and IL-6, representing the status of oxidative stress and inflammation in lung tissues, respectively. The quantities of 8-isoprostane and IL-6 were normalized to the total protein lysate before comparison. * p < 0.05