J Ethnopharmacol. 2026 Aug 29:122354. doi: 10.1016/j.jep.2026.122354. Online ahead of print.
ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Danshen-Chuanxiong (DS-CX), a classical herb pair composed of Salviae Miltiorrhizae Radix et Rhizoma (the dried roots and rhizomes of Salvia miltiorrhiza Bunge) and Chuanxiong Rhizoma (the dried rhizomes of Ligusticum chuanxiong Hort.), has been widely used in traditional Chinese medicine (TCM) for promoting blood circulation and resolving blood stasis. DS-CX has been developed into Guanxinning preparations, which are widely used to treat cardiovascular disorders. However, the potential role of DS-CX in deep vein thrombosis (DVT) and its underlying mechanisms remain poorly understood.
OBJECTIVE: This study aimed to investigate the protective effects of DS-CX against DVT and elucidate the molecular mechanisms, with a focus on the regulation of neutrophil extracellular traps (NETs).
METHODS: A mouse model of DVT induced by inferior vena cava stenosis was established to evaluate the effects of DS-CX pretreatment in vivo. Antithrombotic outcomes were assessed via ultrasonography (thrombus area and blood flow velocity), morphological observation (thrombus weight and length), coagulation-related parameters (PT, APTT, FIB, TT, D-Dimer, and TAT), histopathological examination, and the tail bleeding test. To determine the role of NETs, mice were co-treated with recombinant DNase I (rDNase I) or the PAD4 inhibitor Cl-amidine. NET formation was analyzed using ELISA detection, PicoGreen assays, and immunofluorescence staining. In vitro, mouse bone marrow-derived neutrophils (BMDNs) were stimulated with PMA to establish a reproducible NETosis model for mechanistic investigation. BMDNs were pre-incubated with DS-CX, rDNase I, or ROS/MAPK modulators, followed by PMA stimulation. Subsequently, NET-related markers and signaling pathway were determined through immunofluorescence, flow cytometry, SYTOX Green staining, and Western blotting. Furthermore, UPLC-MS/MS profiling combined with functional validation was performed to identify candidate active constituents responsible for the anti-NET effects of DS-CX.
RESULTS: DS-CX significantly reduced thrombus area, improved venous blood flow velocity, reduced thrombus weight and length, and ameliorated thrombus-associated pathological changes. Concurrently, coagulation function was modulated, with decreased D-Dimer, prolonged APTT, and reduced TAT. Importantly, DS-CX treatment did not significantly prolong tail bleeding time or alter hemoglobin levels. Both in vivo and in vitro results demonstrated that DS-CX suppressed NET formation, as evidenced by marked reductions in NETosis markers, including citrullinated histone H3 (citH3), myeloperoxidase (MPO), MPO-DNA complexes, neutrophil elastase (NE), and extracellular DNA. Additionally, the anti-NET efficacy of DS-CX was comparable to that of rDNase I, and co-administration with Cl-amidine yielded no additive benefits, suggesting a shared inhibitory pathway. Mechanistically, DS-CX suppressed NET formation by inhibiting ROS generation and subsequent MAPK signaling activation, as confirmed by pathway-specific inhibitors and activators. Furthermore, UPLC-MS/MS analysis and functional validation indicated that salvianolic acid B and tetramethylpyrazine are potential contributors to the anti-NET effects of DS-CX, with the combination showing enhanced inhibitory effects.
CONCLUSION: DS-CX has a distinctive antithrombotic effect against DVT without increasing the risk of bleeding, and its mechanism of action may be closely related to ROS-MAPK-mediated NET formation. Salvianolic acid B and tetramethylpyrazine may represent important contributors to these protective effects. These findings provide mechanistic insights into the traditional use of DS-CX and support its potential as a safer immunothrombosis-targeting approach for thrombotic disorders.
PMID:42667972 | DOI:10.1016/j.jep.2026.122354