注射用灯盏花素对脂多糖致巨噬细胞炎症损伤的抑制作用  

Protective effect of breviscapine injection on inflammatory injury in lipopolysaccharide-activated macrophages

作　　者：齐睿娟 孙桂波[1] 侯睿[1] 高源[1] 费巧玲[1] 韩宜芯 周鸿 齐云[1] QI Rui-juan;SUN Gui-bo;HOU Rui;GAO Yuan;FEI Qiao-ling;HAN Yi-xin;ZHOU Hong;QI Yun(Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences &Peking Union Medical College,Beijing 100193,China;Gerontology Center,Xiyuan Hospital of China Academy of Traditional Chinese Medicine,Beijing 100091,China)

机构地区：[1]中国医学科学院北京协和医学院药用植物研究所,北京100193 [2]中国中医科学研究院西苑医院老年病中心,北京100091

出　　处：《中国药理学与毒理学杂志》2018年第10期782-789,共8页Chinese Journal of Pharmacology and Toxicology

基　　金：国家中药标准化项目(ZYBZH-C-YN-59);国家科技重大专项(2015ZX09501004-001-003)~~

摘　　要：目的研究注射用灯盏花素(BSI)对脂多糖(LPS)致巨噬细胞炎症损伤的抑制作用及其机制。方法采用巨噬细胞集落刺激因子刺激小鼠骨髓细胞获得小鼠髓源巨噬细胞。BSI 6.25～400 mg·L^(-1)(终浓度)与小鼠巨噬细胞RAW264.7孵育24 h,MTT法测定细胞存活率。将小鼠巨噬细胞RAW264.7和髓源巨噬细胞与BSI 1.5625～50 mg·L^(-1)和LPS 40μg·L^(-1)共孵育24 h,Griess法测定上清炎症因子一氧化氮(NO)水平,ELISA法测定肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)水平。ICR雄性小鼠一次性ip给予BSI 2.5,5和10 mg·kg^(-1),0.5 h后尾静脉注射LPS 5 mg·kg^(-1)制备内毒素血症模型,给予LPS 3 h后,测定血清NO,TNF-α和IL-6水平。将小鼠巨噬细胞RAW264.7与BSI 1.5625～50 mg·L^(-1)和LPS 40μg·L^(-1)共孵育24 h,L-012荧光染色法测定胞内活性氧簇(ROS)浓度,JC-1荧光染色法检测胞内线粒体膜电位(MMP)水平,荧光素酶催化底物荧光素发光反应检测胞内ATP水平,光泽精化学发光法检测胞内NADPH氧化酶活性。还原型辅酶Ⅰ(NADHⅠ)-吩嗪硫酸甲酯(PMS)体系还原氮蓝四唑(NBT)法测定BSI清除超氧阴离子的作用。结果 BSI<100 mg·L^(-1)时对RAW264.7细胞存活率无影响。BSI 25和50 mg·L^(-1)可降低巨噬细胞RAW264.7上清NO含量(P<0.01),BSI 1.5625～50 mg·L^(-1)对小鼠巨噬细胞RAW264.7和髓源巨噬细胞上清中TNF-α和IL-6均无明显抑制作用;BSI 1.5625～50 mg·L^(-1)对小鼠髓源巨噬细胞上清NO,TNF-α和IL-6水平均无明显抑制作用。在LPS致内毒素血症小鼠模型上,BSI 2.5,5和10 mg·kg^(-1)对LPS诱导的血清NO,TNF-α和IL-6升高也无明显抑制作用。BSI可显著抑制LPS所致RAW264.7细胞胞内ROS上升,并对抗MMP与ATP的下降(P<0.01)。进一步机制研究表明,BSI 1.5626～50 mg·L^(-1)可显著抑制LPS所致RAW264.7细胞NADPH氧化酶活性升高(P<0.01);另外,BSI 3.125～50 mg·L^(-1)具有清除超氧阴离子的作用(P<0.01)。结论虽然BSI对LPS诱导体内外�OBJECTIVE To explore the protective effect of breviscapine injection(BSI) on inflammatory injury in lipopolysaccharide(LPS)-activated macrophages and its underlying mechanisms. METHODS Cell viability was detected by MTT assay. RAW264.7 macrophages were grown in DMEM supplemented with 10% fetal bovine serum. Bone marrow cells were differentiated into bone marrow-derived macrophages(BMDMs) in the presence of recombinant mouse macrophage colony-stimulating factor. RAW264.7cells and BMDMs were treated with BSI(1.5625-50 mg·L^-1) and LPS(40 μg·L^-1) for 24 h. The supernatant NO, TNF-α and IL-6 levels were detected by Griess reagent and ELISA kit, respectively. A mouse endotoxemia model was induced by LPS(5 mg·kg^-1) intravenously. The serum NO, TNF-α and IL-6 levels were detected 3 h after LPS application. Intracellular reactive oxygen species(ROS) was determined by L-012. The mitochondrial membrane potential(MMP) and the intracellular ATP level were detected by JC-1 and firefly luciferase reaction, respectively. The level of superoxide anion was measured by the method of NADH-PMS-NBT. NADPH oxidase activity was assayed by lucigenin chemiluminescence. RESULTS All experiments on the LPS-activated macrophages were carried out at the safe dosages of BSI that did not affect cell viability. BSI could slightly decrease supernatant NO at 25 and 50 mg·L^-1in LPS-activated RAW264.7(P<0.01), but did not affect NO in LPS-activated BMDMs.BSI(1.5625-50 mg·L^-1) did not reduce LPS-induced TNF-α or IL-6 in either RAW264.7 cells or BMDMs.Unexpectedly, BSI even increased the IL-6 level in LPS-activated RAW264.7 cells(P<0.05). In LPSinduced endotoxemia mice, BSI 2.5, 5.0 and 10 mg·kg^-1could not reduce serum NO, TNF-α and IL-6levels. However, BSI could potently lower the concentration of intracellular ROS, and withstand the decline of MMP and intracellular ATP at 6.25-50 mg·L^-1and 3.125-50 mg·L^-1, respectively(P<0.05,P<0.01) in LPS-activated RAW264.7 macrophages. Furthermore, BSI 3.125-50 mg·L^-1could directly scavenge

关 键 词：灯盏花素 脂多糖 巨噬细胞 抗氧化 

分 类 号：R285.5[医药卫生—中药学]