Investigating the Role of Glycolysis in Xuefu Zhuyu Capsule-Promoted Angiogenesis in Endothelial Cells: A Study Based on Network Pharmacology, Molecular Docking, and In Vitro Validation

<b>Background</b>: Peripheral artery disease (PAD) represents a major global cause of mortality and disability. A primary therapeutic strategy involves promoting angiogenesis in ischemic limbs. The Xuefu Zhuyu Capsule (XFZYC) is widely used in China for treating PAD and demonstrates therapeutic potential; however, the mechanism underlying its pro-angiogenic effect remains unclear. <b>Methods</b>: The components of XFZYC were identified via TCMSP and HERB databases, with network pharmacology and molecular docking predicting its potential targets and pathways. For in vitro validation, drug-containing serum and blank control serum were prepared. Human Microvascular Endothelial Cells (HMEC-1) cells were treated with 1.25%, 2.5%, or 5% serum to determine the optimal concentration using tube formation assays and Western blot (WB) analysis of HIF-1α, HK2, and PFKFB3. The efficacy of XFZYC was further assessed through CCK-8, scratch wound healing, cell adhesion, and tube formation assays. Glycolytic metabolite levels and enzyme activities were measured by colorimetric assays and WB. <b>Results</b>: Network pharmacology screening identified 167 active components in XFZYC and 2967 potential targets. GO functional and KEGG pathway enrichment analyses suggested that XFZYC likely promotes the glycolytic pathway via the HIF-1 signaling pathway, specifically mediated by HK2 and PFKFB3. In vitro experiments confirmed that XFZYC enhanced HMEC-1 cell viability, migration, adhesion, and tube formation. Concurrently, it augmented the glycolytic capacity of HMEC-1 cells, manifested by increased glucose consumption, lactate production, enhanced activity of key glycolytic enzymes (HK, PFK, and PK), and upregulated protein expression of PFKFB3. Treatment with 3PO, a glycolytic inhibitor, significantly suppressed these drug-induced effects. <b>Conclusions</b>: XFZYC promotes angiogenesis in endothelial cells by modulating the glycolytic pathway, an effect primarily mediated through the upregulation of PFKFB3 expression. This study offers a preliminary exploration of the underlying mechanisms by which XFZYC may act in the treatment of PAD, thereby providing a new scientific perspective for further understanding its therapeutic effects.