Chikusetsusaponin V improves vascular endothelial dysfunction by binding to ITGB1 and mediating PURA-eNOS activation in mice with diabetes mellitus

Background Vascular endothelial dysfunction is a core pathological feature of diabetes-associated vascular complications, primarily driven by impaired nitric oxide (NO) bioavailability due to dysregulated endothelial nitric oxide synthase (eNOS) activity. While pharmacological strategies targeting the eNOS pathway have shown promise, the discovery of natural compounds capable of restoring endothelial function through novel molecular targets remains an important research direction. Purpose The aim of this study was to investigate the targets and molecular mechanisms of Chikusetsusaponin V (CsV) in regulating vascular endothelial function in diabetes mellitus (DM). Methods The DM models were constructed using db/db mice and high-glucose cultured human aortic endothelial cells (HAECs). The vasodilatory function was investigated using techniques such as metal wire microvascular tension measurement and ultrasonography after intervention with CsV. Modern techniques such as affinity chromatography, mass spectrometry, molecular docking and microscale thermophoresis (MST) test were used to identify the direct targets of CsV. The key pathways and downstream transcription factors involved in the improvement of vasodilatory function of CsV were screened using RNA-Seq. Subsequent validation was performed using a dual luciferase assay. Knockdown adenoviral vectors, quantitative polymerase chain reaction (qPCR), and Western blot (WB) were used to elucidate the molecular mechanism of CsV in regulating the vasodilatory function in DM. Results The results showed that CsV down-regulated blood glucose and increased the expression of eNOS and p-eNOS (S1177), which improved vasodilatation and reduced vascular stiffness in DM mice. Additionally, CsV increased NO release and tube-forming capacity of HAECs. CsV can directly bind to the membrane protein ITGB1. In vivo and in vitro experiments showed that administration of CsV after knockdown of ITGB1 or inhibiting PI3K significantly down-regulated the expression of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, NR4A3, PURA, and p-eNOS (S1177)/eNOS, decreased NO levels, and affected angiogenesis and endothelial cell function. In contrast, overexpression of ITGB1 produced the opposite results. Moreover, knockdown NR4A3 inhibited NR4A3/PURA/eNOS expression and affected endothelial function, whereas overexpression produced the opposite result. Conclusions CsV improves endothelial function in DM vasculature by targeting ITGB1, enhancing eNOS phosphorylation via the PI3K/AKT pathway, and promoting PURA/eNOS expression through PI3K/AKT/mTOR/NR4A3. These findings support CsV's potential use for improving diabetes vascular function. CsV Targets ITGB1 to Improve Endothelial Function via activating PI3K/AKT/mTOR/NR4A3/eNOS Pathway - present study vs. previous study. Download: Download high-res image (224KB) Download: Download full-size image