Mechanism investigation of Shi-Xiao-San in treating blood stasis syndrome based on network pharmacology, molecular docking and in vitro/vivo pharmacological validation

Ethnopharmacological relevance: Shixiao San (SXS) is a traditional Chinese formula that has been widely used in clinical practice to treat blood stasis syndromes, such as hyperlipidemia, atherosclerotic, thrombosis and coro-nary heart disease. However, the effectiveness and mechanism of SXS have not been studied in detail yet.Aim of the study: Current study aimed to identify the compounds in SXS, evaluate the formula efficacies using network pharmacology, molecular docking, and verify the pharmacological effects by in vivo and in vitro experiments.Materials and methods: The compounds in SXS were analyzed using UPLC-QTOF-MS. Potential target genes for identified compounds were obtained from three databases. DAVID database was used to perform GO and KEGG pathway enrichment analyses. PPI network was constructed to screen core targets. Molecular docking was used to examine interactions between active compounds and potential targets. The mechanism was also verified by model of acute blood stasis rats and human umbilical vein cells.Results: In total, 45 compounds were identified from SXS. Among the detected phytochemicals, quercetin, iso-rhamnetin, kaempferol, D-catechin, naringenin and amentoflavone were identified as the active constituents. SXS is primarily involved in the modulation of hypoxic state, vascular regulation, and inflammation response, ac-cording to GO and KGG pathway enrichment analysis. A network of protein-protein interactions (PPIs) was constructed and five core targets were identified as VEGFA, AKT1, EGFR, PTGS2, and MMP9. Molecular docking simulation revealed good binding affinity of the five putative targets with the corresponding compounds. SXS reduced HIF-1 alpha and COX-2 levels and increased the eNOS expression levels in hypoxic HUVECs. SXS can reduce the whole blood viscosity in adrenaline induced acute blood stasis rats and relieve blood stasis.Conclusions: SXS removes blood stasis might through VEGFA/AKT/eNOS/COX-2 pathway and flavonoids are the main active components in the formula.