Huo-Xue-Xiao-Zhong Decoction prevents thrombus formation of deep vein thrombosis by targeting SOAT2 to modulate the AKT and ERK pathways

ETHNOPHARMACOLOGICAL RELEVANCE: Huo-Xue-Xiao-Zhong Decoction (HXXZ), a traditional Chinese medicine formulation comprising 14 herbal components, has demonstrated significant efficacy and favorable safety profiles in clinical trials for the prevention of deep vein thrombosis (DVT). However, the underlying molecular mechanisms responsible for its therapeutic effects remain to be elucidated. AIM OF THE STUDY: This study aims to investigate the molecular mechanisms through which HXXZ prevents thrombus formation in DVT. MATERIALS AND METHODS: The chemical profile of HXXZ was characterized using HPLC-Q-TOF-MS. Therapeutic efficacy was evaluated in a mouse model of DVT (n=8 per group) and in a TNF-α-induced human umbilical vein endothelial cell (HUVEC) injury model. A multi-faceted approach integrating network pharmacology, molecular docking, and transcriptomic analysis was employed to identify potential targets of HXXZ. Following gain- and loss-of-function experiments, levels of inflammation-related, coagulation-related, and endothelial injury markers were assessed using ELISA, Western blotting, and immunofluorescence staining. RESULTS: HPLC-Q-TOF-MS analysis identified 38 major constituents in HXXZ. In vivo, treatment with moderate to high doses of HXXZ significantly reduced thrombus length and mass, while modulating levels of IL-1β, IL-6, NO, vWF, P-Selectin, FXI, FVIII, TM, PC, eNOS, EPCR, and ICAM-1 in plasma and thrombus tissue. In vitro, HXXZ attenuated TNF-α-induced apoptosis and endothelial dysfunction, and reduced total cholesterol, cholesteryl ester, and free cholesterol levels in HUVECs. Network pharmacology revealed 181 active compounds and 53 DVT-related targets, with enrichment in the PI3K-AKT and MAPK pathways. Molecular docking confirmed favorable binding potential interactions between HXXZ constituents and AKT1, ERK2, KLF11, and KLF15. Transcriptomic analysis identified CD14, SOAT2, and MCL1 as key genes modulated by HXXZ. Functional studies demonstrated that SOAT2 knockdown enhanced the protective effects of HXXZ on HUVECs, whereas SOAT2 overexpression produced opposing effects. CONCLUSIONS: HXXZ prevents thrombus formation in DVT through a multi-component, centered on the downregulation of SOAT2 and consequent suppression of the AKT-ERK pathway, revealing a previously unrecognized pharmacological target for DVT management.