目的 探究補(bǔ)腎活血方（BSHX）在抗磷脂綜合征（APS）模型中的作用及其對(duì)中性粒細(xì)胞胞外誘捕網(wǎng)（NETs）的調(diào)控機(jī)制。方法 將小鼠隨機(jī)分為5組：對(duì)照組、模型組、阿司匹林（陽(yáng)性藥，0.585 mg·kg^-1）組和BSHX低、高劑量（5.8、11.60 g·kg^-1）組。除對(duì)照組外，采用β2糖蛋白I（β2GP-I）構(gòu)建APS小鼠模型，連續(xù)ig給藥15 d，每天2次；通過(guò)ELISA法測(cè)定血清中腫瘤壞死因子-α（TNF-α）、白細(xì)胞介素（IL）-6表達(dá)水平；采用全自動(dòng)凝血測(cè)試儀檢測(cè)血漿中活化部分凝血活酶時(shí)間（APTT）、凝血酶時(shí)間（TT）和凝血酶原時(shí)間（PT）。體外培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞（HUVECs），60 μmol·L^-1 β2GP-I刺激制備APS模型，同時(shí)給予不同濃度BSHX孵育24 h； MTT法檢測(cè)BSHX（40、80、120、160、200、240、280、320 μg·mL^-1）對(duì)細(xì)胞存活率的影響；熒光成像結(jié)合流式細(xì)胞術(shù)檢測(cè)BSHX（80、120 μg·mL^-1）對(duì)活性氧（ROS）水平的影響； Transwell實(shí)驗(yàn)檢測(cè)BSHX（120 μg·mL^-1）對(duì)細(xì)胞遷移能力的影響；免疫熒光及試劑盒法檢測(cè)NETs的形成； Western blotting檢測(cè)BSHX（120 μg·mL^-1）對(duì)炎癥相關(guān)（IL-6、TNF-α）、細(xì)胞黏附相關(guān)[細(xì)胞間黏附分子-1（ICAM-1）、血管細(xì)胞黏附分子-1（VCAM-1）]、凝血功能相關(guān)[組織因子（TF）、纖溶酶原激活抑制劑-1（PAI-1）]、NADPH氧化酶2（NOX2）、p38、細(xì)胞外調(diào)節(jié)蛋白激酶（ERK）、c-Jun氨基末端激酶（JNK）蛋白表達(dá)水平；將HUVECs細(xì)胞分為對(duì)照組、模型組、BSHX（120 μg·mL^-1）組和MK-2206組（Akt抑制劑，100 nmol·L^-1），Western blotting檢測(cè)ERK、p47、JNK、p38及磷酸化蛋白表達(dá)。結(jié)果 與模型組比較，給予APS小鼠BSHX后，APTT、TT、PT均顯著升高，TNF-α、IL-6表達(dá)水平被抑制（P＜0.05、0.01、0.001），說(shuō)明BSHX可緩解血液高凝狀態(tài)、抗炎。與模型組比較，BSHX可顯著改善APS模型HUVECs的細(xì)胞存活率，降低ROS水平和細(xì)胞遷移能力，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蛋白表達(dá)（P＜0.01、0.001）；給予Akt通路抑制劑后p-ERK、p-p47、p-JNK、p-p38和NOX2蛋白表達(dá)顯著降低（P＜0.01、0.001）。結(jié)論 補(bǔ)腎活血方可能通過(guò)調(diào)節(jié)Akt/ERK信號(hào)通路調(diào)控NETs的生成而發(fā)揮治療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

江西省中醫(yī)藥管理局科技計(jì)劃項(xiàng)目（2023A0020）；江西省衛(wèi)生健康委科技計(jì)劃基金資助項(xiàng)目（202410072）