Triterpenoid saponins from Ilex pubescens promote blood circulation in blood stasis syndrome by regulating sphingolipid metabolism and the PI3K/AKT/eNOS signaling pathway

Blood stasis syndrome (BSS) is a severe disorder involving disturbances in glycerophosphocholine metabolism. Ilex pubescens (IP) can regulate the levels of lipids, such as lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE); however, the main active constituent of IP and its corresponding mechanism in BSS treatment are still unclear.To explore the mechanisms by which triterpenoid saponins of IP (IPTS) promote blood circulation using system pharmacology-based approaches.Sprague-Dawley (SD) rat BSS model was prepared by oral administration of IPTS for 7 days followed by adrenaline hydrochloride injection before immersion in ice water. Coagulation parameters in plasma and thromboxane B2 (TXB2), endothelin (ET) and 6-keto-PGF1α in serum were measured. The possible influence on abdominal aortas was evaluated by histopathology assessment. Human vein endothelial cells (HUVECs) were incubated with ox-LDL, and the effects of IPTS on cell viability and LDH release were investigated. UPLC-QTOF-MS/MS was used for metabolic profile analysis of lipid-soluble components in rat plasma and intracellular metabolites in HUVECs. Network pharmacology was used to predict the relevant targets and model pathways of BSS and the main components of IPTS. Molecular docking, molecular dynamics (MD) simulation and biochemical assays were used to predict molecular interactions between the active components of IPTS and target proteins. RT-PCR was used to detect the mRNA level of target proteins. Western blotting and immunohistochemistry (IHC) were used to verify the mechanisms by which IPTS promotes blood circulation in BSS.IPTS improved blood biochemical function in the process of BSS and played a role in vascular protection and maintenance of the normal morphology of blood vessels. Furthermore, metabolite pathways involved in steroid biosynthesis and sphingolipid metabolism were significantly perturbed. Both metabolomics analysis and network pharmacology results showed that IPTS ameliorates vascular injury and that lipid accumulation may be mediated by PI3K/AKT signaling pathway activation. MD simulation and enzyme inhibitory activity results suggested that the main components of IPTS can form stable complexes with PI3K, AKT and eNOS and that the complexes have significant binding affinity. PI3K, AKT, p-AKT, and eNOS mRNA and protein levels were considerably elevated in the IPTS-treated group. Thus, IPTS protects the vasculature by regulating the PI3K/AKT signaling pathway, activating eNOS and increasing the release of NO.A possible mechanism by which IPTS prevents BSS is proposed: IPTS can promote blood circulation by modulating sphingolipid metabolism and activating the PI3K/AKT/eNOS signaling pathways.