Network toxicology reveals key genes of amiodarone induced pulmonary fibrosis: based on machine learning and SHAP analysis

Figures

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  FIGURE 1. The Chemical Structure of AMD and Its Potential Protein Targets. (A) 2D planar chemical structure of AMD; (B) 3D minimum-energy conformation of AMD; (C) the Venn diagram of predicted human protein targets of AMD from three independent databases: ChEMBL, SEA, and SwissTargetPrediction.

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  FIGURE 2. DEGs Analysis of PF. (A,B) Comparison of PCA plots before (A) and after (B) batch correction demonstrates the effective mitigation of batch-specific clustering. (C) Volcano plot of differentially expressed genes (DEGs). Genes are plotted based on the magnitude of log2 FC and statistical p value. Red dots represent significantly upregulated genes, green dots represent significantly downregulated genes, and gray dots indicate non-significant genes. (D) Heatmap displays gene expression levels, with red and blue representing high and low expression relative to the mean, respectively. Rows represent individual genes, while columns represent sample origins.

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  FIGURE 3. WCGNA Analysis of PF. (A) Module-trait relationship heatmap, showing the module signature genes and key traits identified by WGCNA analysis. Each row represents a module color, and each column represents a trait. Positive correlations are shown in red, and negative correlations are shown in blue. (B) Hierarchical clustering dendrogram of genes and identified co-expression modules. Genes were clustered based on a dissimilarity matrix (1-TOM). (C) Gene significance across modules. (D) Scatter plot showing the strong positive correlation (cor = 0.86, p = 5.7e-46) between module membership and gene significance for the treatment phenotype. (E) Venn diagram summarizing DEGs, WGCNA (MEred), and their union.

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  FIGURE 4. Identification of hub genes in AIPF and enrichment analysis. (A) Intersection of AMD target genes and PF target genes. (B) Drug-disease-gene network diagram. (C) PPI network diagram of target genes. (D) GO enrichment. (E) KEGG enrichment.

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  FIGURE 5. Identification and validation of hub genes in AIPF based on ML and SHAP analysis. (A) Heatmap compares the AUC of various ML models to determine the best predictive model (glmboost + GBM). (B) SHAP summary plot visualizes the contribution of genes to the GBD model. (C) An attempt to decompose the feature contributions of representative samples confirms the global importance observed in SHAP analysis. (D) SHAP analysis results for the intersecting genes, shown as a SHAP value bar chart. (E) Violin plot depicts the expression distribution of core genes in AIPF and the control group. The eigenvalues of CTSK are positively correlated with SHAP values, while ADORA3, AGER, and FLT3, show negative correlations. (F) ROC curves validate the clinical data for core genes.

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  FIGURE 6. Molecular docking results. The molecular docking results of amiodarone with ADORA3 (A), AGER (B), CTSK (C), and FLT3 (D) are presented.

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  FIGURE 7. 50 ns molecular dynamics simulation analysis of the complex. (A) RMSD curve of the complex, (B) RMSD curve of the small molecule, (C) Rg curve of the protein, (D) SASA curve of the protein, (E) RMSF curve of the protein, (F) Hydrogen bond evolution curve of the complex, (G) Gibbs FEL of the AMD-complex.

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  FIGURE 8. Effects of AMD on BEAS-2B cell migration, invasion capacity, and hub gene expression. (A) CCK8 assay determines the optimal concentration for AMD intervention in BEAS-2B cell. (B) The wound healing assay demonstrated significantly accelerated wound closure after 24 h of AMD treatment (scale bar: 100 μm). (C) Transwell invasion/migration assay revealed significantly increased BEAS-2B cell density after 24-h AMD treatment (scale bar: 200 μm) (*p < 0.05, **p < 0.01).

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  FIGURE 9. mRNA and protein expression of AIPF hub genes. (A) qRT-PCR analysis revealed upregulation of CTSK mRNA expression and downregulation of ADORA3 and AGER mRNA expression following AMD treatment. (B) Western blot was used to detect the expression of target proteins in the Control group and AMD group. (C) Immunofluorescence validated key protein expression and localization: CTSK (green) showed enhanced granular expression in the cytoplasm; ADORA3 (green) exhibited reduced ring-like expression at the cell membrane; AGER (green) decreased in both membrane and cytoplasm; FLT3 (green) showed no significant change; nuclei were labeled with DAPI (blue) (scale bar: 50 μm) (*p < 0.05, **p < 0.01).