Effects of short-term waterfall forest aerosol air exposure on rat lung proteomics

Abstract

Background: Chronic exposure to airborne microparticles has been shown to increase the incidence of several chronic diseases. Previous studies have found that waterfall forest aerosols contribute to a diminished immune stress response in patients with asthma. However, the specific effects of short-term waterfall forest aerosol exposure on lung proteins have not been fully elucidated.

Methods: This study used liquid chromatography-tandem mass spectrometry (LC-MS) to analyze changes in protein expression in the lungs of rats exposed to short-term waterfall forest aerosol environments. Specific protein markers were identified using bioconductivity analysis screening and validated using immunohistochemistry.

Results: Waterfall forest aerosol environment exposure on day 5 downregulated the expression of the classical inflammatory pathway nuclear factor κB (NF-κB) signaling pathway. As the waterfall forest aerosol environment increased due to the duration of exposure, it was involved in oxidative phosphorylation and then hormone signaling in lung cells from the very beginning. In contrast, at day 15 of exposure, there is an effect on the regulation of the immune-related high-affinity IgE receptor pathway. In addition, iron-sulfur Rieske protein (Uqcrfs1), mitochondrial Tu translation elongation factor (Tufm) and ribosomal protein L4 (Rpl4) were identified as possible bioindicators for the evaluation of air quality.

Conclusions: These results provide a comprehensive proteomic analysis that supports the positive contribution of a good air quality environment to lung health.

Keywords: Huangguoshu waterfall; forest aerosol; mitochondria; proteomics; signaling pathway.

Figure 1

A flow chart showing the experimental procedure. N5: normal environment 5-day group; N10: normal environment 10-day group; N15: normal environment 15-day group; W5: waterfall forest aerosol 5-day group; W10: waterfall forest aerosol 10-day group; W15: waterfall forest aerosol 15-day group. SD, Sprague Dawley; HE, hematoxylin and eosin; RT-qPRC, real-time quantitative polymerase chain reaction.

Figure 2

Rat rearing conditions in different environments. (A) Conventional laboratory environment; (B) waterfall forest environment.

Figure 3

Hematoxylin and eosin staining shows the histopathological changes in rat lungs in the different environments. N5: normal environment 5-day group; N10: normal environment 10-day group; N15: normal environment 15-day group; W5: waterfall forest aerosol 5-day group; W10: waterfall forest aerosol 10-day group; W15: waterfall forest aerosol 15-day group.

Figure 4

Identification of the differential proteins in the different environments. (A) A Venn diagram of the proteins in the different environments and under different duration of exposure. (B) Quantitative statistical analyses of lung proteins in rats. The red bars represent expression of up-regulated proteins and the blue bars represent expression of down-regulated proteins. In the significant difference protein screen, the expression FC suggests the differences in expression. The number of proteins that are >10-fold up- or down-regulated is marked in darker colors. N5: normal environment 5-day group; N10: normal environment 10-day group; N15: normal environment 15-day group; W5: waterfall forest aerosol 5-day group; W10: waterfall forest aerosol 10-day group; W15: waterfall forest aerosol 15-day group. FC, fold change.

Figure 5

Heat maps of proteins at different exposure times. (A) A heat map of the differentially expressed proteins in the lungs after 5 days of exposure to waterfall forest aerosol compared to the conventional environmental group. (B) A heat map of the differentially expressed proteins in the lungs after 10 days of exposure to waterfall forest aerosol compared to the conventional environmental group. (C) A heat map of the differentially expressed proteins in the lungs after 15 days of exposure to waterfall forest aerosol compared to the conventional environmental group. N5: normal environment 5-day group; N10: normal environment 10-day group; N15: normal environment 15-day group; W5: waterfall forest aerosol 5-day group; W10: waterfall forest aerosol 10-day group; W15: waterfall forest aerosol 15-day group.

Figure 6

KEGG analysis of the enriched pathways. (A) KEGG signaling pathway enrichment on day 5 of different environmental exposure. (B) KEGG signaling pathway enrichment on day 10 of different environmental exposure. (C) KEGG signaling pathway enrichment on day 15 of different environmental exposure. The 20 most relevant signaling pathways were selected for each time period and plotted on a bubble diagram using R language. The larger the value of rich factor in the horizontal coordinate, the greater the enrichment. The redder the bubble color, the smaller the P value. The larger bubble shape suggests that the pathway is enriched with more differential metabolites. ECM, extracellular matrix; KEGG, Kyoto Encyclopedia of Genes and Genomes; GnRH, gonadotropin-releasing hormone; VEGF, vascular endothelial growth factor; TNF, tumor necrosis factor; HIF-1, hypoxia-inducible factor 1; AMPK, adenosine monophosphate-activated protein kinase.

Figure 7

GO enrichment analysis of differentially expressed proteins at different environmental exposure time points. (A) GO enrichment analysis at day 5 in the different environments. (B) GO enrichment analysis after 10 days of exposure in different environments. (C) GO enrichment analysis at day 15 after different environmental exposure. The horizontal line represents the functional classification of enriched GO terms into three main categories: biological process, molecular function, and cellular component. The vertical coordinate represents the number of different proteins under each functional category. The color bar represents the importance of the enriched GO functional classification. The color gradient represents the magnitude of the P value (P<0.05). The label at the top of the bar indicates the enrichment factor (rich factor ≤1), which represents the ratio of the number of differentially expressed proteins indicating that they are annotated as a particular GO functional class to the number of identified proteins annotated as that GO functional class. GO, Gene Ontology; BP, biological process; MF, molecular function; CC, cellular component.

Figure 8

Protein interaction networks at different time nodes for different environmental exposure. (A) The protein interaction network for differential expression in rat lungs on day 5. (B) The protein interaction network for differential expression in rat lungs on day 10. (C) The protein interaction network for differential expression in rat lungs on day 15. Nodes represent differentially expressed proteins and lines represent protein-protein interactions. Different colored regions represent the relevant biological functional range that the differentially expressed proteins are enriched in.

Figure 9

Histopathological changes in the rat lungs. Immunohistochemical staining; magnification, ×200. N15: normal environment 15-day group; W15: waterfall forest aerosol 15-day group. Uqcrfs1, ubiquinol-cytochrome C reductase iron-sulfur subunit 1; Tufm, mitochondrial Tu translation elongation factor; Rpl4, ribosomal protein L4.

Figure 10

WGCNA of lung proteins on day 5 of waterfall forest environmental exposure. (A) Module classification; ME stands for module, which is an analysis of the expression patterns of multiple proteins by means of weighted co-expression network analysis, and identification of highly synergistic protein modules (modules), which are analyzed by protein expression, as detailed in the online table (available at https://cdn.amegroups.cn/static/public/atm-22-4813-1.xls). The first row of each color bar represents the correlation coefficient with different groups of horizontal coordinates, and the value in parentheses is the p-value. The higher the correlation, the darker the color, and the lower the correlation, the lighter the color. (B) A schematic representation of reciprocal proteins. N5: normal environment 5-day group; N10: normal environment 10-day group; N15: normal environment 15-day group; W5: waterfall forest aerosol 5-day group; W10: waterfall forest aerosol 10-day group; W15: waterfall forest aerosol 15-day group. ME, module eigengene; WGCNA, weighted gene co-expression network analysis.