Dl-3-n-butylphthalide ameliorates diabetic foot ulcer by inhibiting apoptosis and promoting angiogenesis

Figure 1 Pharmacological targets of Dl-3-n-butylphthalide in diabetic foot ulcers. A: Twenty-six intersection genes of Dl-3-n-butylphthalide against diabetic foot ulcers; B and C: Protein-protein interaction network of the 19 melatonin targets; D and E: Gene ontology and Kyoto Encyclopedia of genes and genomes enrichment analysis of the 26 Dl-3-n-butylphthalide targets. DFU: Diabetic foot ulcers; NBP: Dl-3-n-butylphthalide; GO: Gene ontology; KEGG: Kyoto Encyclopedia of genes and genomes.

Figure 2 Wound healing of different time points in diabetic foot ulcers mice. A: Images of mice wounds at different time points; B: The healing rate of foot wounds at different time points in mice; C: The wound tissues of mice were stained with hematoxylin-eosin and Masson 14 days after surgery; D: The epidermal thickness of mice 7 days after surgery; E: Immunofluorescence staining showed capillary density analysis of wound tissues in mice. Data expressed as individual values with mean ± SE. ^bP < 0.01. DFU: Diabetic foot ulcers; NBP: Dl-3-n-butylphthalide; HE: Hematoxylin-eosin.

Figure 3 Dl-3-n-butylphthalide promotes cellular proliferation under high glucose conditions. A-C: Cell Counting Kit 8 assay was used to assess the effect of Dl-3-n-butylphthalide on cell proliferation; D: Edu assay showed the cell proliferation of human umbilical vein endothelial cells; E: Edu assay showed the cell proliferation of human keratinocytes cells; F: Edu assay showed the cell proliferation of human dermal fibroblasts; G: Quantification of Edu corporation assay in human umbilical vein endothelial cells, human keratinocytes cells, and human dermal fibroblasts. Data expressed as individual values with mean ± SE. ^dP < 0.0001, ^cP < 0.001, ^bP < 0.01, ^aP < 0.05, ^nsP > 0.05. HUVEC: Human umbilical vein endothelial cells; HaCaT: Human keratinocyte cells; HDF: Human dermal fibroblasts; HG: High glucose; NBP: Dl-3-n-butylphthalide.

Figure 4 Dl-3-n-butylphthalide promotes cellular migration under high glucose conditions. A: The scratch assay showed the cell migration of human umbilical vein endothelial cells; B: Cell migration of human keratinocytes cells; C: Cell migration of human dermal fibroblasts; D: Quantitative analysis of migration cells in a scratch assay; E: Transwell migration experiment detected the migration abilities of Human umbilical vein endothelial cells; F: Transwell migration experiment of human keratinocytes cells; G: Transwell migration experiment of human dermal fibroblasts; H: Quantification of the number of cells that migrated in Transwell assays. Data expressed as individual values with mean ± SE. ^dP < 0.0001, ^cP < 0.001, ^bP < 0.01, ^aP < 0.05. HG: High glucose; NBP: Dl-3-n-butylphthalide; HUVEC: Human umbilical vein endothelial cells; HaCaT: Human keratinocyte cells; HDF: Human dermal fibroblasts.

Figure 5 Dl-3-n-butylphthalide reduces cell apoptosis under high glucose conditions. A: Flow cytometry detection of cell apoptosis of Human umbilical vein endothelial cells; B: Flow cytometry detection of human keratinocytes cells; C: Flow cytometry detection of human dermal fibroblasts; D: Statistical results of apoptotic cells in flow cytometry; E: TUNEL staining was detected the cell apoptosis of human umbilical vein endothelial cells; F: TUNEL staining of human keratinocytes cells; G: TUNEL staining of human dermal fibroblasts; H: Statistical analysis of the percentage of apoptotic cells according to the result of the TUNEL assay; I: Apoptosis-related protein expression was examined by Western blot analysis. Data expressed as individual values with mean ± SD. ^dP < 0.0001, ^cP < 0.001, ^bP < 0.01, ^aP < 0.05. HG: High glucose; NBP: Dl-3-n-butylphthalide; HUVEC: Human umbilical vein endothelial cells; HaCaT: Human keratinocyte cells; HDF: Human dermal fibroblasts.

Figure 6 Dl-3-n-butylphthalide exerts anti-apoptotic and anti-oxidative stress effects by modulating the advanced glycation end products-receptor of advanced glycation end products signaling pathway under high glucose conditions. A: Western blot analysis of the protein expression of advanced glycosylation end-product specific receptor; B: Flow cytometry analysis of reactive oxygen species levels; C and D: Kit-based detection of superoxide dismutase and malondialdehyde levels. Data expressed as individual values with mean ± SD. ^dP < 0.0001, ^cP < 0.001, ^bP < 0.01, ^aP < 0.05. AGER: Advanced glycosylation end-product specific receptor; HG: High glucose; NBP: Dl-3-n-butylphthalide.

Figure 7 Molecular docking analysis and mRNA expression analysis of core genes. A: Molecular docking revealed the binding of Dl-3-n-butylphthalide to its targets. Blue solid line: Hydrogen bond; Gray dashed line: Hydrophobic interaction; Yellow dashed line: Electrostatic interaction; B: The quantitative real-time polymerase chain reaction analysis of the mRNA levels of heme oxygenase 1, caspase 3, B cell leukemia/lymphoma 2, brain derived neurotrophic factor and nuclear factor erythroid 2 L2. Data expressed as individual values with mean ± SD. ^bP < 0.01, ^aP < 0.05. HMOX: Heme oxygenase; CASP3: Caspase 3; BCL2: B cell leukemia/lymphoma 2; BDNF: Brain derived neurotrophic factor; NFE2: Nuclear factor erythroid 2; HG: High glucose; NBP: Dl-3-n-butylphthalide.