Identifying potential biomarkers for early evaluating mechanical compression injuries to skeletal muscle through proteomic analysis: A rat model

Figures

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  Fig 1. Schematic diagram of the process for establishing skeletal muscle mechanical compression injury models with varying degrees of severity.

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  Fig 2. Experimental design.

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  Fig 3. H&E staining of the tibialis anterior muscle in rats at 3, 7, and 14 days after mechanical compression injury of varying degrees. (A) Typical H&E staining images of tibialis anterior muscle 3 days post-injury. (B) Percentage of injured myofibers at 3 days post-injury. (C) Representative images of muscle regeneration at 7 days post-injury. (D) Quantitative analysis of the number of regenerated muscle fibers in each group at 7 days post-injury. (E) Representative images of muscle regeneration at 14 days post-injury. (F) Quantitative analysis of the number of regenerated muscle fibers in each group at 14 days post-injury. (G) Quantitative analysis of the diameter of regenerated muscle fibers in each group at 7 days post-injury. (H) Quantitative analysis of the diameter of regenerated muscle fibers in each group at 14 days post-injury. All data are presented as mean ± standard deviation. N = 5.

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  Fig 4. Histopathological staining of the tibialis anterior muscle in rats at 28 days after mechanical compression injury of varying degrees. (A) Representative images of muscle regeneration, as shown by H&E staining. (B) Quantitative analysis of the average myofiber size in each group. (C) Representative image of fibrosis in each group, as shown by Masson trichrome staining. (D) Quantitative analysis of the fibrotic area in each group. (E) Representative image of Sirius Red staining at 28 days post-injury under bright field and polarized light microscopy. (F) Quantitative analysis of collagen fiber area under bright field microscopy. (G) Quantitative analysis of type I and type III collagen fiber area under polarized light. All data are presented as mean ± standard deviation. N = 5.

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  Fig 5. Protein identification results. (A) The total number of peptides and proteins identified. (B) Bar graph showed the number of differentially expressed proteins in pairwise comparisons among the three groups. (C-E) Volcano plots of pairwise comparisons among the three groups. (C) Comparison between the mild injury group and the control group. (D) Comparison between the severe injury group and the control group. (E) Comparison between the severe injury group and the mild injury group. Red dots represented significantly upregulated proteins, blue dots represented significantly downregulated proteins, and gray dots represented proteins with no significant difference. The names of the top 5 upregulated and downregulated proteins (ranked by absolute Log2 Ratio) were labeled in the plot.

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  Fig 6. Thirty-nine proteins could distinguish different degrees of muscle damage. (A) The union of the intersection between the differentially upregulated proteins in pairwise comparisons MvsC and SvsM, and the intersection between the differentially downregulated proteins in pairwise comparisons MvsC and SvsM, yielded a total of 39 proteins. (B) a quantitative heatmap of the intersecting proteins.

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  Fig 7. GO and KEGG analysis of DEPs. (A) Cellular component of DEPs. (B) Biological processes of DEPs. (C) Molecular function of DEPs. (D) KEGG pathways. The x-axis represented the fold enrichment of functional enrichment after Log2 transformation, with larger values indicating higher enrichment levels. The color of the dots indicated the significance of enrichment P value, with darker blue indicating stronger enrichment significance. The size of the dots represented the number of differential proteins, with larger dots indicating a greater number of differential proteins.

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  Fig 8. Protein-protein interaction network construction. The color represented the MCC value of DEPs, with darker colors indicating higher MCC values. The size of the node represented the degree, where larger nodes indicated higher degrees. Generally, proteins with higher degrees or MCC values were more likely to be key proteins.