【摘要】 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；qRTPCR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（DDimer）、纤维蛋白降解产物（fibrinogen and fibrin degradation products,FDP）、TF、ET-1、s VCAM-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m RNA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。更多还原

【Abstract】 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 odianisidine 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.更多还原