目的 研究三棱含药血清对脂多糖（lipopolysaccharides，LPS）诱导人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVEC）损伤的影响。方法 SPF级雄性SD大鼠随机分为空白组和三棱组，制备空白血清及含药血清。建立LPS诱导的HUVEC损伤模型，设置对照组、模型组、5%空白血清组和三棱含药血清（1%、3%、5%）组，采用MTT法检测三棱含药血清对细胞活力的影响；采用荧光显微镜分析各组细胞线粒体膜电位（mitochondrial membrane potential，MMP）和活性氧（reactive oxygen species，ROS）水平；采用ELISA法检测各组细胞上清液乳酸脱氢酶（lactate dehydrogenase，LDH）、半胱氨酸天冬氨酸蛋白酶-1（cystein-asparate protease-1，Caspase-1）活性和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）、IL-6、IL-17、组织因子（tissue factor，TF）、血管性血友病因子（von Willebrand factor，vWF）、凝血酶调节蛋白（thrombomodulin，TM）水平；采用Western blotting检测各组细胞微管相关蛋白1轻链3 II（microtubule-associated protein l light chain 3 II），LC3 II）、Beclin-1、Parkin和PINK蛋白表达情况；采用qRT-PCR法测定各组细胞IL-1β、IL-18和NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3，NLRP3）mRNA表达情况。结果 与对照组比较，模型组细胞活力、MPP显著降低（P＜0.01），细胞内ROS水平显著升高（P＜0.01），上清液LDH、Caspase-1活性和TNF-α、IL-6、IL-17、TF、vWF、TM水平均显著升高（P＜0.01），LC3 II、Beclin-1、Parkin和PINK蛋白表达水平均显著升高（P＜0.01），IL-1β、IL-18和NLRP3 mRNA表达水平显著升高（P＜0.01）。与模型组比较，三棱含药血清组细胞活力、MPP显著升高（P＜0.01），细胞内ROS水平显著降低（P＜0.05、0.01），上清液LDH、Caspase-1活性和TNF-α、IL-6、IL-17、TF、vWF、TM水平均显著降低（P＜0.01），LC3 II、Beclin-1、Parkin和PINK蛋白表达水平均显著降低（P＜0.01），IL-1β、IL-18和NLRP3 mRNA表达水平显著降低（P＜0.01）。结论 三棱含药血清具有内皮细胞保护作用，可以降低LPS诱导的细胞氧化损伤和炎症，降低凝血级联反应。

Objective To study the effect of Sanleng (Sparganii Rhizoma, SR) medicated serum on lipopolysaccharides (LPS)-induced injury of human umbilical vein endothelial cells (HUVEC). Methods SPF male SD rats were randomly divided into blank group and SR group, blank serum and drug-containing serum were prepared. LPS-induced HUVEC injury model was established, and were divided into control group, model group, 5% blank serum group and SR medicated serum (1%, 3%, 5%) group, MTT method was used to detect the effect of SR medicated serum on cell viability. Fluorescence microscopy was used to analyze the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels of cells in each group; ELISA was used to detect lactate dehydrogenase (LDH), cystein-asparate protease-1 (Caspase-1) activities and tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-17, tissue factor (TF), von Willebrand factor (vWF), thrombomodulin (TM) in supernatant of cells in each group. Western blotting was used to detect the protein expressions of microtubule-associated protein l light chain 3 II (LC3 II), Beclin-1, Parkin and PINK of cells in each group; qRT-PCR was used to detect the mRNA expressions of IL-1β, IL-18 and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) of cells in each group. Results Compared with control group, cell viability and MPP in model group were significantly decreased (P < 0.01), level of intracellular ROS was significantly increased (P < 0.01), activities of LDH and Caspase-1 in the supernatant and TNF-α, IL-6, IL-17, TF, vWF, TM levels were significantly increased (P < 0.01), LC3 II, Beclin-1, Parkin and PINK protein expression levels were significantly increased (P < 0.01), IL-1β, IL-18 and NLRP3 mRNA expression levels were significantly increased (P < 0.01). Compared with model group, cell viability and MPP in SR drug-containing serum group were significantly increased (P < 0.01), level of intracellular ROS was significantly decreased (P < 0.05, 0.01), activities of LDH and Caspase-1 in the supernatant and TNF-α, IL-6, IL-17, TF, vWF, TM levels were significantly decreased (P < 0.01), protein expressions of LC3 II, Beclin-1, Parkin and PINK were significantly decreased (P < 0.01), IL-1β, IL-18 and NLRP3 mRNA expression levels were significantly decreased (P < 0.01). Conclusion SR medicated serum has the protective effect of endothelial cells, which can reduce LPS-induced oxidative damage and inflammation, and reduce the coagulation cascade.

R285.5

基金项目:山东省自然科学基金资助项目(ZR2015HL117);山东省中医药科技发展计划项目(2017-165);山东省重点研发计划项目(2017CXGC1308);山东省自然科学基金重点项目(ZR2020KH017);山东省高校中药质量控制与全产业链建设协同创新中心项目(CYLXTCX2020-03,CYLXTCX2021-02)