关键词: 静脉血栓栓塞症, 斑马鱼, 二氢杨梅素, 中性粒细胞胞外诱捕网, 髓过氧化物酶, 炎症反应

Abstract:

This study aims to elucidate the effect and mechanism of dihydromyricetin (DHM) against venous thrombosis in zebrafish. Zebrafish at 3 days post-fertilization were randomly divided into six groups: a blank control, model, DHM low, medium, and high dose groups (50, 100, and 200 μg/mL), and an aspirin group (25 μg/mL). After six hours of treatment with DHM or aspirin, all groups except the blank control were exposed to 30 mg/mL arachidonic acid for 1 hour to induce caudal vein thrombosis. The caudal vein and cardiac erythrocytes of zebrafish were stained to evaluate the antivenous thrombosis effect of dihydromyricetin (DHM), live fluorescence imaging was employed to observe neutrophil activation and recruitment, RT-qPCR was performed to determine the mRNA expression levels of myeloperoxidase (MPO), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), reactive oxygen species (ROS) levels were measured using a fluorescent probe assay, intestinal hemorrhage was examined under a microscope to assess the safety of DHM, molecular docking was conducted to verify its binding activity with key targets. Results showed that compared to the model group, DHM significantly reduced thrombus formation in the caudal vein (P < 0.01), increased cardiac red blood cell staining area (P < 0.01), decreased neutrophil fluorescence intensity (P < 0.01), and suppressed mRNA expression of MPO (P < 0.001), TNF-α (P < 0.001), and IL-6 (P < 0.01). DHM also markedly inhibited ROS generation (P < 0.001). DHM did not cause intestinal bleeding at any tested concentrations. Molecular docking indicated that DHM exhibited favorable binding affinities with MPO, TNF-α, IL-6, and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2). These findings suggest that DHM may reduce caudal vein thrombosis in zebrafish by inhibiting the TNF-α/IL-6–ROS–MPO signaling axis, without inducing hemorrhagic effects.

Key words: venous thromboembolism, Zebrafish; Dihydromyricetin, neutrophil extracellular traps, myeloperoxidase, inflammation