Identification of key genes and immune infiltration mechanisms in limb ischemia-reperfusion injury: a bioinformatics and experimental study

Figure 1. Technical route flow chart of this study.

Figure 2. “Decision-making” process and tradeoffs for dataset selection in limb IRI analysis.

Figure 3. Boxplot after data normalization.

Figure 4. Principal component analysis plot (A) and clustering analysis plot (B).

Figure 5. Volcano plot of differential gene expression.

Figure 6. Bar chart of GO enrichment (A) and KEGG enrichment (B) analysis.

Figure 7. 10-fold cross-validation in Lasso regression to obtain IRI characteristic genes (A); cross-validation error calculation in the random forest algorithm (B); Intersection of characteristic genes obtained from both machine learning algorithms (C).

Figure 8. Changes in hind limb skin and muscle post-ischemia and reperfusion. Pre-modeling (A) shows rosy skin with normal temperature and no swelling, after 4 hours of ischemia (B) the skin appears cyanotic, cold, and significantly swollen, after 4 hours of reperfusion (C) normal rat muscle is firm, smooth, and evenly red without swelling or atrophy, while the IRI group gastrocnemius muscle is markedly swollen with uneven dark red appearance and hemorrhagic spots.

Figure 9. HE staining of rat gastrocnemius tissue (×100 in A, B, ×200 in C, D) (A, C Control Group; B, D IRI Group).

Figure 10. Relative mRNA expression of key genes in A (∗ indicates P<0.01, ∗∗ indicates P<0.001); Comparison of relative immune cell expression levels in B (Note: ∗ indicates P<0.01); Bar graph of immune cell infiltration proportions in C; Heatmap of immune cell correlation in D.

Figure 11. Protein expression of WNT5A, PLC, IP3R, and CAMK II in skeletal muscle tissue. (A) Western blot images. (B) Quantification of relative protein levels normalized to Tubulin. (Data are presented as mean ± SEM. *P<0.05 vs Control;# P<0.05 vs IRI;^ P<0.05 vs Box5).

Figure 12. mRNA expression of WNT5A, PLC, IP3R, and CAMK II in skeletal muscle tissue. (Data are presented as mean ± SEM. *P<0.05 vs Control;# P<0.05 vs IRI;^ P<0.05 vs Box5).