Network pharmacology analysis and experimental validation elucidate the protective mechanisms of BYHWD against hypoxia/reoxygenation-induced endothelial cell injury

FIGURE 1. (A) Serum CRE and BUN levels of renal IRI model mice 2 days after treatment with BYHWD. Significant differences are indicated by asterisks (*p < 0.05, **p < 0.01, ****p < 0.0001). (B) HE, Masson, PAS staining of renal tissue from IRI model mice at 2 days after treatment with BYHWD. Con: Control group; Sham: Sham operation group; IRI: Ischemia-reperfusion group; 1. HE staining: Reveals tubular epithelial cell vacuolar degeneration (black arrows) and pyknosis/karyorrhexis (indicating necrosis). Renal interstitial edema with inflammatory cell infiltration is also present (asterisks). 2. Masson’s staining: Demonstrates widened interstitium with collagen fiber/extracellular matrix deposition (blue arrows), representing an acute interstitial response to injury. 3.PAS staining: Shows loss of the brush border and glycogen in tubular epithelial cells (triangles), along with disruption and discontinuity of the tubular basement membrane (red arrows). IRI + L: BYHWD low-dose group; IRI + H: BYHWD high-dose group; 1. HE staining: Compared with the IRI group, the treatment groups exhibit a significant reduction in tubular epithelial cell vacuolar degeneration and necrosis, along with markedly…

FIGURE 2. (A) Venn diagram of traditional Chinese medicine-related disease actions. (B) Network diagram of drug-ingredient-target interaction. Green circles represent TCM (7 in total); Blue rhombuses represent targets (210 in total); Orange hexagons represent overlapping active ingredients of TCM (8 in total A1, Huangqi, Taoren; A2, Huangqi, Honghua; A3, Huangqi, Honghua; A4, Danggui, Chishao, Honghua, Taoren; A5, Danggui, Chishao, Honghua; A6, Chishao, Honghua; A7, Chishao, Honghua; A8, Chishao, Chuanxiong). The larger the shape, the more genes targeted by the active ingredient. (C) PPI Network and Key Targets of BYHWD Treatment on AKI. In the visualization representation of protein-protein interaction (PPI) networks, circular topological units represent target genes. The geometric size and chromatic gradient of nodes are positively correlated with their topological importance (quantitatively expressed through degree centrality, betweenness centrality, etc.). Edge attributes are characterized by chromatic differences to represent interaction types and strength, where darker lines indicate strong associations that are experimentally validated or statistically significant. (D)…

FIGURE 3. Molecular docking visualization. (A–E) represent the molecular docking results of luteolin, quercetin, ellagic acid, baicalin, β-carotene, and VEGFA, respectively. Among them, the molecular docking visualization of β-carotene and VEGFA shows no hydrogen bonds, which may be due to the large hydrophobic region of β-carotene. This could potentially form hydrophobic interactions with the hydrophobic part of VEGFA, thereby enhancing the binding energy.

FIGURE 4. (A) Changes in the survival rate of HUVECs after 24 h of stimulation with serum containing different drug concentrations. (B) Trypan blue staining of HUVEC cells under light microscopy (×200). Significant differences are indicated by asterisks (**p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 5. A: Detection of apoptosis in HUVEC groups using Annexin V/PI dual staining combined with flow cytometry. B: Flow cytometry analysis of oxidative stress levels in HUVEC cells. Significant differences are indicated by asterisks (**p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 6. Western blot analysis of NGAL expression in renal tissue from IRI model mice at 2 days after treatment with BYHWD. Con, Control group; Sham, Sham operation group; IRI, Ischemia-reperfusion group; IRI + L, BYHWD low-dose group; IRI + H, BYHWD high-dose group. Significant differences are indicated by asterisks (*p < 0.05, **p < 0.01).

FIGURE 7. (A) Changes in the expression of BAX and BCL-2 proteins in various cell groups. (B) Western blot analysis of MCP-1, CD-31, and α-SMA protein expression changes in each group of cells. (C) Western blot analysis of VEGFRII protein expression in cells from each group. (D) Changes in protein expression of p-PI3K, PI3K, p-AKT, AKT, p-FOXO1, and FOXO1 in each group of cells. Significant differences are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).