目的 探究补肾活血方（BSHX）在抗磷脂综合征（APS）模型中的作用及其对中性粒细胞胞外诱捕网（NETs）的调控机制。方法 将小鼠随机分为5组：对照组、模型组、阿司匹林（阳性药，0.585 mg·kg^-1）组和BSHX低、高剂量（5.8、11.60 g·kg^-1）组。除对照组外，采用β2糖蛋白I（β2GP-I）构建APS小鼠模型，连续ig给药15 d，每天2次；通过ELISA法测定血清中肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6表达水平；采用全自动凝血测试仪检测血浆中活化部分凝血活酶时间（APTT）、凝血酶时间（TT）和凝血酶原时间（PT）。体外培养人脐静脉内皮细胞（HUVECs），60 μmol·L^-1 β2GP-I刺激制备APS模型，同时给予不同浓度BSHX孵育24 h； MTT法检测BSHX（40、80、120、160、200、240、280、320 μg·mL^-1）对细胞存活率的影响；荧光成像结合流式细胞术检测BSHX（80、120 μg·mL^-1）对活性氧（ROS）水平的影响； Transwell实验检测BSHX（120 μg·mL^-1）对细胞迁移能力的影响；免疫荧光及试剂盒法检测NETs的形成； Western blotting检测BSHX（120 μg·mL^-1）对炎症相关（IL-6、TNF-α）、细胞黏附相关[细胞间黏附分子-1（ICAM-1）、血管细胞黏附分子-1（VCAM-1）]、凝血功能相关[组织因子（TF）、纤溶酶原激活抑制剂-1（PAI-1）]、NADPH氧化酶2（NOX2）、p38、细胞外调节蛋白激酶（ERK）、c-Jun氨基末端激酶（JNK）蛋白表达水平；将HUVECs细胞分为对照组、模型组、BSHX（120 μg·mL^-1）组和MK-2206组（Akt抑制剂，100 nmol·L^-1），Western blotting检测ERK、p47、JNK、p38及磷酸化蛋白表达。结果 与模型组比较，给予APS小鼠BSHX后，APTT、TT、PT均显著升高，TNF-α、IL-6表达水平被抑制（P＜0.05、0.01、0.001），说明BSHX可缓解血液高凝状态、抗炎。与模型组比较，BSHX可显著改善APS模型HUVECs的细胞存活率，降低ROS水平和细胞迁移能力，抑制NETs的形成，抑制IL-6、TNF-α、ICAM-1、VCAM-1、TF、PAI-1、NOX2、p-JNK、p-ERK和p-p38蛋白表达（P＜0.01、0.001）；给予Akt通路抑制剂后p-ERK、p-p47、p-JNK、p-p38和NOX2蛋白表达显著降低（P＜0.01、0.001）。结论 补肾活血方可能通过调节Akt/ERK信号通路调控NETs的生成而发挥治疗APS作用。

Objective To explore the effect of Bushen Huoxue Decoction (BSHX) on antiphospholipid syndrome (APS) model and its regulatory mechanism on neutrophil extracellular traps (NETs). Methods Mice were randomly divided into five groups: control group, model group, aspirin (positive drug, 0.585 mg·kg^-1) group and BSHX low and high dose (5.8, 11.60 g·kg^-1) groups. Except for the control group, APS mouse models were established by β2 glycoprotein I (β2GP-I), and intragastric administration was given for 15 days, twice a day. The expression levels of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in serum were determined by ELISA. The activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) in plasma were detected by a fully automatic coagulation analyzer. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and APS models were prepared by stimulating with 60 μmol·L^-1 β2GP-I. Different concentrations of BSHX were incubated for 24 hours. The effect of BSHX (40, 80, 120, 160, 200, 240, 280, 320 μg·mL^-1) on cell survival rate was detected by MTT assay. The effect of BSHX (80, 120 μg·mL^-1) on reactive oxygen species (ROS) levels was detected by fluorescence imaging combined with flow cytometry. The effect of BSHX (120 μg·mL^-1) on cell migration ability was detected by Transwell assay. The formation of NETs was detected by immunofluorescence and kit method. The expression levels of inflammatory-related (IL-6, TNF-α), cell adhesion-related [intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1)], coagulation function-related [tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1)], NADPH oxidase 2 (NOX2), p38, extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) proteins were detected by Western blotting. HUVECs cells were divided into control group, model group, BSHX (120 μg·mL^-1) group and MK-2206 group (Akt inhibitor, 100 nmol·L^-1), and the expression of ERK, p47, JNK, p38 and phosphorylated proteins was detected by Western blotting. Results Compared with the model group, after administration of BSHX to APS mice, APTT, TT and PT were significantly increased, and the expression levels of TNF-α and IL-6 were inhibited (P < 0.05, 0.01, 0.001), indicating that BSHX can alleviate the hypercoagulable state of blood and anti-inflammation. Compared with the model group, BSHX could significantly improve the cell survival rate of HUVECs in APS models, reduce ROS levels and cell migration ability, inhibit the formation of NETs, and inhibit the expression of IL-6, TNF-α, ICAM-1, VCAM-1, TF, PAI-1, NOX2, p-JNK, p-ERK and p-p38 proteins (P < 0.01, 0.001); after administration of Akt pathway inhibitor, the expression of p-ERK, p-p47, p-JNK, p-p38 and NOX2 proteins was significantly reduced (P < 0.01, 0.001). Conclusion BSHX might exert therapeutic effects on APS by regulating the generation of NETs through the Akt/ERK signaling pathway.

R285.5

江西省中医药管理局科技计划项目（2023A0020）；江西省卫生健康委科技计划基金资助项目（202410072）