Integrated identification and mechanism exploration of bioactive ingredients from Salvia miltiorrhiza to induce vascular normalization

BACKGROUND: The clinical management of ischemic disease and cancer is complex, with disruptions in local vascular function and tumor angiogenesis contributing to blood stasis, which complicates treatment strategies. Salvia miltiorrhiza, a natural product, is known to restore vascular structure and function. However, its specific roles in concurrently addressing ischemic disease and cancer within the same organism remain poorly understood. PURPOSE: This study aimed to explore the material basis, pharmacological effects, and underlying mechanisms of Salvia miltiorrhiza extract (SME) in promoting blood flow recovery in ischemic hindlimbs and inducing tumor vascular normalization. METHODS: The pharmacological effects of SME were evaluated in a mouse model combining ischemic hindlimbs and tumors. Mice were administered low (SME-L) or high (SME-H) doses of SME daily, and the gastrocnemius muscle mass and tumor vascular structure were assessed. Laser Doppler perfusion imaging (LDPI) was used to monitor hindlimb blood flow recovery and tumor vascular perfusion. The pharmacokinetics of the key bioactive constituents in SME were characterized by liquid chromatography-mass spectrometry (LC-MS). Interactions between SME's active compounds and predicted targets were investigated using molecular docking, microscale thermophoresis (MST), and luciferase reporter assays. The synergistic effects of the primary components, Tanshinone I (Tan I) and Salvianolic acid A (Sal A), were analyzed through tube formation assays, enzyme-linked immunosorbent assays (ELISA), immunofluorescence staining, and western blot. RESULTS: Phytochemical profiling revealed that SME contains several active compounds, including Danshensu, Sal A, Sal B, Tan IIA, and Tan I. SME treatment reduced the frequency of necrotic toes, increased muscle mass, and alleviated hypoxia in the gastrocnemius muscle. SME significantly improved tumor vascular perfusion and notably enhanced pericyte coverage and basement membrane integrity. Pharmacokinetic analysis identified Tan I and Sal A as the key bioactive components that promote vascular normalization. Tan I inhibited FoxO1, preventing endothelial cell activation induced by angiopoietin 2 (Ang2), while Sal A bound to Ang2, facilitating Tie2 activation mediated by Ang1. Both in vitro and in vivo results demonstrated that the combination of Tan I and Sal A exerted a synergistic therapeutic effect on correcting abnormal blood vessels in ischemic hindlimbs and tumors. CONCLUSION: Our study innovatively revealed a reliable mouse model wherein the Ang2/Tie2 signaling cascade disrupted the endothelial homeostasis to aggravate the progression of hindlimb ischemia and tumor angiogenesis. This balance can be rescued by the combination therapy of Tan I and Sal A that were both from SME, leading to the occurrence of vascular normalization.