目的 探讨腰痹通胶囊（Yaobitong Capsules，YBT）对深静脉血栓形成（deep vein thrombosis，DVT）的干预作用及其分子机制。方法 采用大鼠下肢深静脉结扎模型、苯肼诱导的斑马鱼血瘀模型和氧糖剥夺（oxygen-glucose deprivation，OGD）诱导人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）损伤模型多尺度评价YBT的作用。大鼠下肢深静脉结扎模型中，采用血栓质量与长度比值、苏木素-伊红（hematoxylin-eosin，HE）染色评估血栓病理改变；检测凝血六项、血浆组织因子（tissue factor，TF）、内皮素-1（endothelin-1，ET-1）、可溶性血管细胞黏附分子-1（soluble vascular cell adhesion molecule-1，sVCAM-1）、白细胞介素-6（interleukin-6，IL-6）、IL-1β、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）、缺氧诱导因子-1α（hypoxia inducible factor-1α，HIF-1α）等指标。斑马鱼血瘀模型中，以邻联茴香胺染色和共聚焦成像观察红细胞瘀滞与血小板聚集情况；qRT-PCR检测凝血相关基因（fga、fgb、fgg、f2）表达。OGD诱导的HUVEC细胞损伤模型中，采用qRT-PCR、Western blotting和免疫荧光检测TF、VCAM-1、ET-1、HIF-1α的表达及核因子-κB（nuclear factor-κB，NF-κB）p65的磷酸化与核转位情况。结果 在大鼠下肢深静脉结扎模型中，YBT能够降低血栓质量与长度比值，改善组织病理变化（P＜0.05），延长活化部分凝血活酶时间（activated partial thromboplastin time，APTT）（P＜0.001），降低纤维蛋白原（fibrinogen，FIB）、D-二聚体（D-Dimer）、纤维蛋白降解产物（fibrinogen and fibrin degradation products，FDP）、TF、ET-1、sVCAM-1、IL-6、IL-1β、TNF-α、MDA和HIF-1α水平（P＜0.01、0.001），并提高SOD活性（P＜0.05、0.01）。在斑马鱼血瘀模型中，YBT能够显著增加斑马鱼心脏红细胞染色强度与循环血小板数目（P＜0.001），并下调凝血相关基因的表达（P＜0.01、0.001）。在OGD诱导的HUVEC细胞损伤模型中，YBT能够下调TF、VCAM-1、ET-1、HIF-1α的mRNA表达（P＜0.05、0.01、0.001），下调TF与HIF-1α的蛋白表达（P＜0.05、0.01、0.001），并抑制NF-κB p65的磷酸化及核转位（P＜0.05、0.01）。结论 YBT可能通过抑制HIF-1α/NF-κB p65/TF信号轴（下调HIF-1α表达，抑制NF-κB p65磷酸化及核转位，进而减少TF过度表达），减轻缺氧-炎症级联反应，保护血管内皮功能并改善高凝状态，从而有效抑制DVT。

Objective To investigate the effect and mechanism of Yaobitong Capsules (腰痹通胶囊, YBT) on deep vein thrombosis (DVT). Methods Multi-scale evaluation of the effect of YBT was conducted using a rat lower limb deep vein ligation model, a zebrafish blood stasis model induced by phenylhydrazine, and an oxygen glucose deprivation (OGD)-induced human umbilical vein endothelial cell (HUVEC) injury model. In the rat lower limb deep vein ligation model, the ratio of thrombus weight to length and hematoxylin-eosin (HE) staining were used to evaluate the pathological changes of thrombus. Six coagulation parameters, plasma tissue factor (TF), endothelin-1 (ET-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD), malondialdehyde (MDA), hypoxia inducible factor-1α (HIF-1α) and other indicators were detected. In the zebrafish blood stasis model, the stasis of red blood cells and platelet aggregation were assessed via o-dianisidine staining and confocal microscopy, respectively. qRT-PCR was employed to measure the expressions of coagulation-related genes (fga, fgb, fgg, f2). In the OGD induced HUVEC cell injury model, qRT-PCR, Western blotting and immunofluorescence were used to detect the expressions of TF, VCAM-1, ET-1, HIF-1α, as well as the phosphorylation and nuclear translocation of nuclear factor-κB (NF-κB) p65. Results In the rat model of deep vein ligation of the lower limbs, YBT could reduce the ratio of thrombus weight to length, improve histopathological changes (P < 0.05), prolong activated partial thromboplastin time (APTT) (P < 0.001), reduce fibrinogen (FIB), D-dimer, fibrinogen and fibrin degradation products (FDP), TF, ET-1, sVCAM-1, IL-6, IL-1β, TNF -α, MDA and HIF-1α levels (P < 0.01, 0.001), and increase SOD activity (P < 0.05, 0.01). In the zebrafish blood stasis model, YBT could significantly increase cardiac erythrocyte staining intensity and circulating platelet number (P < 0.001), and down-regulate the expressions of coagulation related genes (P < 0.01, 0.001). In the OGD induced HUVEC cell injury model, YBT could down-regulate the mRNA expressions of TF, VCAM-1, ET-1 and HIF-1α (P < 0.05, 0.01, 0.001), down-regulate the protein expressions of TF and HIF-1α (P < 0.05, 0.01, 0.001), inhibit the phosphorylation and nuclear translocation of NF-κB p65 (P < 0.05, 0.01). Conclusion YBT may effectively inhibit DVT by inhibiting HIF-1α/NF-κB p65/TF axis (down-regulating HIF-1α expression, inhibiting NF-κB p65 phosphorylation and nuclear translocation, thereby reducing TF overexpression), alleviating the hypoxia inflammation cascade, protecting endothelial function, and improving hypercoagulability.

R285.5

江苏省基础研究计划自然科学基金-前沿引领技术基础研究专项（BK20232014）