Evaluation and Characterization of Acute respiratory distress syndrome in tree shrews through TMT proteomic method

Abstract

Acute respiratory distress syndrome (ARDS), a common cause of acute fatal respiratory, is characterized by severe inflammatory lung injury as well as hallmarks of increased pulmonary vascular permeability, neutrophil infiltration, and macrophage accumulation. Tree shrew, a squirrel-like small animal model, has been confirmed to have more similar traits to human ARDS with one-hit intratracheal instillation of LPS in our previous study. In this study, we characterized protein profile changes induced by intranasal LPS challenge in the tree shrew model through tandem mass tag (TMT)-based quantitative proteomics and type II alveolar epithelial cells through pathological analysis. In total, 4070 proteins (p < 0.05) were identified from lung tissues of the LPS-induced group and PBS group. Among the differential expression proteins (DEPs) detected by t-test (≥|1.5-fold|), 529 DEPs were identified, of which 304 were upregulated, and 225 were downregulated. The most important pathways involved in the process of ARDS had been identified by enrichment analysis: oxidative stress, apoptosis, inflammatory responses, and vascular endothelial injury. In addition, proteins have been reported in animal models or clinical patients also detail investigated for further analysis, such as ceruloplasmin (CP), hemopexin (HPX), sphingosine kinase 1 (SphK1), lactotransferrin (LTF), and myeloperoxidase (MPO) were upregulated in induced tissues and confirmed by western blot analysis. Overall, this study not only reveals a comprehensive proteomic analysis of the ARDS tree shrew model but also provides novel insights into multi-pathways responses induced by the LPS challenge of tree shrews. We highlight shared and unique proteomic changes in the lungs of ARDS tree shrews and identify novel pathways for acute lung injury, which may promote the model into basic research and translational research.

Fig. 1. LPS induces lung hyper-responsiveness in tree shrews.

(A) Immunofluorescence of images of the PBS and LPS groups. Representative images of the two groups at magnifications of 100 × and 400 × , stained immunofluorescence with aquaporin 5 (AQP5) antibody and surfactant protein C (SP-C) antibody and DAPI. Alveolar type I epithelial cells were identified with red fluorescence, while alveolar type II epithelial cells were identified with green fluorescence. The DAPI stained the nucleus blue. The LPS group showed obvious increased alveolar type II epithelial cells. (B) Semi-quantize analysis of the slides. The number of alveolar type II epithelial cells was calculated based on slides. (C) Differential expression heatmap between PBS and LPS, respectively. Three biological replicates are shown. (D) Heatmap of genes corresponding to differentially expressed proteins (DEPs). The heatmap displays 775 differentially expressed genes (DEGs) selected through statistical testing (t-test, p.adjust <  0.01), highlighting the most significant differential genes. To emphasize significance, only the top 30 genes with the smallest p.adjust values are shown in the figure, representing the most differentially expressed genes. The read indicated upregulated genes, and blue showed downregulated genes. ***p <  0.001 vs PBS.

Fig. 2. Functional enrichment analysis of DEPs.

(A) A chord dendrogram of GO cluster plot. GO cluster plot showing a chord dendrogram of the clustering of the expression spectrum of significantly changed genes. (B) A circular dendrogram of GO cluster plot. GO cluster plot showing a circular dendrogram of the clustering of the expression spectrum of DEPs. (C) GO term enrichment dot plot, triangle diagram, and square of the (C) DEPs and (D) upregulated genes. The y-axis represents GO‑enriched terms. The x-axis represents the fold of enrichment.

Fig. 3. KEGG analysis of DEPs.

(A) Top 30 enriched of KEGG enrichment. (B) KEGG pathway analysis of upregulated DEPs. (C) The circos diagram of the DEPs and its corresponding KEGG signaling pathways.

Fig. 4. Constructed protein-protein interaction (PPI) networks and conducted analysis.

(A) Construction of PPI networks based on STRING database data. The circle size represents the MCODE score, where a higher score indicates higher connectivity and dense interactions within the module. These modules typically represent functionally related groups of proteins. A lower score indicates looser connections within the module, potentially signifying weaker functional relationships among these nodes. (B) Selection of high-scoring modules from the STRING network analysis. (C) Identification of key areas involving oxidative stress, immunity, apoptosis, endothelial damage, and ARDS case reports, with 1.5-fold DEPs. A Venn diagram shows the overlapping human proteins across the five categorized groups. (D) A heatmap plotted using the pheatmap R package (version 1.42.2). It displays relative expression levels with intensities of red and blue, indicating the relative increase and decrease of these proteins. (E) Integrated PPI network analysis of the five categorized groups. PPI network analysis was performed using Cytoscape based on the STRING database with high confidence for each category group. Constructed oxidative stress PPI network, consisting of 99 nodes and 199 edges. Constructed apoptosis PPI network, consisting of 192 nodes and 314 edges. Constructed endothelial damage PPI network, consisting of 35 nodes and 71 edges. Constructed inflammatory response PPI network, consisting of 160 nodes and 282 edges. Constructed ARDS case report-related PPI network, consisting of 63 nodes and 129 edges.

Fig. 5. Confirmation of DEPs by western blot.

(A) Immunoblotting analysis of proteins (CP, HPX, SphK1, LTF, and MPO) in LPS-induced and PBS tree shrews. The β-tubulin protein was used as a control. (B) Heatmaps (hierarchical clustering) of the 5 proteins between the PBS and LPS groups, respectively. Three biological replicates are shown. (C) Quantitative analysis of A using ImageJ software. * p <  0.05, ***p <  0.001 vs PBS.

Fig. 6. Key pathways and protein profiles in ARDS tree shrews.

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