Fig. 5.

Network pharmacology analysis and verification. Analyzing existing databases to investigate the impact of diosgenin (DG) on the sirtuin 6/nuclear factor erythroid 2-related factor 2 (Sirt6/Nrf2) signaling pathway. (A) Venn diagram of DG active ingredients and targets related to diabetic wounds (DWs), DG, and ferroptosis. (B) Primary clusters extracted from the protein-protein interaction (PPI) network illustrate the main functional gene clusters of potential targets. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of core targets of DG against DWs. (D) Gene Ontology (GO) functional enrichment analysis of core targets of DG against DWs. (E) A ribbon model representing protein residues and illustrating the three-dimensional (3D) binding mode. DG docking with Sirt6 and Nrf2 showed binding affinities of −7.6 kcal/mol and −9.2 kcal/mol, respectively. A space-filling model shows DG embedded in the Sirt6 and Nrf2 binding pockets. (F−H) Human umbilical vein endothelial cells (HUVECs) were incubated with phosphate-buffered saline (PBS) or DG (20 μM) for 24 h, and cellular thermal shift assay (CETSA) (F) and quantitative analysis was performed to detect the thermal stability of Sirt6 (G) and Nrf2 (H) proteins. Data are presented as the mean ± standard deviation (SD) from 5 independent experiments (n = 5). Statistical significance is denoted as ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001; ns indicates no significant difference. MAPK: mitogen-activated protein kinase; BP: biological process; CC: cellular component; MF: molecular function.