Leonurus japonicus Alleviates Thrombosis by Modulating Lipid Metabolism and Inhibiting TLR4/STAT3/TNF‐α/IL‐17A Pathways in both Rat and Zebrafish

Leonurus japonicus Houtt. (LJ) is a pivotal traditional Chinese medicine remedy for thrombosis; however, its potential mechanisms and active constituents remain unclear. In this study, LJ exhibits a significant antithrombotic effect on an arteriovenous bypass thrombosis rat, characterized by reductions in thrombus wet and dry weights, decreased maximum aggregation rate, increased thrombus formation inhibition rate, aggregation inhibition rate, alterations in fibrinogen and antithrombin III levels, and shortened coagulation indicators. Moreover, LJ significantly enhanced the erythrocyte staining area and intensity in an arachidonic acid-induced zebrafish heart. Network pharmacology indicated that LJ alleviates thrombosis by regulating TNF and IL-17 pathways. Metabolomics revealed that LJ significantly modulated 29 endogenous differential metabolites, linking it to the regulation of primary bile acid biosynthesis, IL-17, and TNF pathways. Zebrafish experiments and molecular docking suggested that kaempferol and apigenin exhibit antithrombotic effects by modulating TNF and IL-17 proteins. Finally, molecular dynamics simulation validated that kaempferol and apigenin have strong binding capabilities with TLR4, STAT3, TNF-α, and IL-17A, and their binding free energies are -20 to -27 and -13 to -15 kcal/mol, respectively. In conclusion, LJ exerts its antithrombotic effect by regulating lipid metabolism and inhibiting TLR4/STAT3/TNF-α/IL-17A pathways, with kaempferol and apigenin identified as bio-constituents. This plant holds significant potential for development and utilization in antithrombotic therapies.