黄芪水提物3种效应成分对高糖诱导的内皮细胞损伤的保护作用及机制研究  

Protective effects and mechanism of three active components of water extract of milkvetch root on high glucose-induced endothelial cell damage

作　　者：马盼 刘汉滢 彭美中 牛艺婷 杨美美 徐艺宸 刘卓容 韩静 MA Pan;LIU Hanying;PENG Meizhong;NIU Yiting;YANG Meimei;XU Yichen;LIU Zhuorong;HAN Jing(School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 102488,China;Beijing Key Laboratory of TCM Syndrome and Formula,Beijing 102488,China;Key Laboratory of TCM Syndrome and Formula of the Ministry of Education,Beijing 102488,China;Institute of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 102488,China)

机构地区：[1]北京中医药大学中医学院,北京102488 [2]证候与方剂基础研究北京市重点实验室 [3]证候与方剂基础研究教育部重点实验室(北京中医药大学) [4]北京中医药大学中医药研究院

出　　处：《北京中医药大学学报》2024年第2期188-198,共11页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(No.81873165,No.82074238)。

摘　　要：目的考察黄芪水提物效应成分对高糖(25 mmol/L)诱导的人脐静脉内皮细胞(HUVEC)损伤的保护作用,并探讨其作用机制。方法给雄性SD大鼠连续灌胃黄芪水提物[20 g/(kg·d)]7 d,分别于末次灌胃后1、2、4 h取血,合并上清,行超高效液相色谱质谱(UPLC⁃MS)分析,筛选黄芪水提物在大鼠体内代谢后的效应成分。将细胞分为正常组、模型组、不同浓度效应成分组及效应成分配伍组。用CCK-8试剂盒检测细胞活力,探讨黄芪水提物效应成分的最佳浓度。通过活性氧(ROS)实验检测细胞氧化损伤情况。酶联免疫吸附测定法检测各组细胞超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)含量;蛋白质印迹法检测核因子E2相关因子(Nrf2)、血红素氧合酶1(HO⁃1)蛋白表达水平。结果芒柄花素、大豆苷元、毛蕊异黄酮是黄芪水提物的效应成分。与正常组比较,模型组细胞活力增加(P<0.05);与模型组比较,芒柄花素各浓度组(26、13、6.5μmol/L)细胞活力降低(P<0.05),大豆苷元高浓度组(0.11μmol/L)细胞活力降低(P<0.05)。与正常组比较,模型组细胞ROS水平升高(P<0.05);与模型组比较,芒柄花素高、中浓度组(26、13μmol/L),大豆苷元不同浓度组(0.11、0.06、0.03μmol/L),毛蕊异黄酮高浓度组(7.00μmol/L)及各配伍组细胞ROS水平均降低(P<0.05)。效应成分配伍对细胞的抑制率高于单个效应成分,且高浓度配伍组抑制率最高。与正常组比较,模型组细胞Nrf2、HO⁃1蛋白表达量降低(P<0.05);与模型组比较,效应成分高浓度配伍组Nrf2、HO⁃1蛋白表达量升高(P<0.05)。与正常组相比,模型组细胞SOD、CAT含量降低(P<0.05)、MDA含量升高(P<0.05);与模型组相比,效应成分高浓度配伍组SOD、CAT含量均升高(P<0.05)、MDA含量降低(P<0.05)。结论黄芪水提物效应成分芒柄花素、大豆苷元及毛蕊异黄酮有保护高糖诱导的HUVEC损伤的作用,其机制可能与调节Nrf2、HO⁃1�Objective We aimed to(i)investigate the protective effect of active components of water extract of huangqi(milkvetch root)on high glucose(25 mmol/L)induced injury of human umbilical vein endothelial cells(HUVEC)and(ii)elucidate the underlying mechanism.Methods Male SD rats were continuously fed with water extract[20 g/(kg·d)]for 7 d,and blood samples were colleeted 1,2 and 4 h after the last gavage,combined with supernatant,and performed ultra⁃performance liquid chromatography(UPLC⁃MS)analysis to screen the active components of the extract in rats.Cells were divided into normal group,model group,different active components groups treated with different concentrations,and the concerted application of active components group.Cell viability was measured by Cell Counting Kit⁃8 to explore the optimal concentration of the effective components of milkvetch root.Oxidative damage was measured by reactive oxygen species(ROS).The enzyme⁃linked immunosorbent assay measured the contents of superoxide dismutase(SOD),catalase(CAT)and malondialdehyde(MDA);the protein expression level of nuclear factor E2 related factor(Nrf 2)and heme oxygenase 1(HO⁃1)by Western blotting.Results Formononetin,daidzein,and calycosin are the active components of milkvetch root water extract.Compared with the normal group,the cell viability of the model group was increased(P<0.05).Compared with the model group,cell viability was decreased in each concentration(26,13,6.5μmol/L)of formononetin group(P<0.05),a high dose of daidzein(0.11μmol/L)could reduce cell viability(P<0.05).Compared with the normal group,the ROS levels in the model group were increased(P<0.05);compared with the model group,the ROS levels in the formononetin high and medium concentrations(26,13μmol/L)groups,different concentrations of daidzein(0.11,0.06,0.03μmol/L),the calycosin high concentration group(7.00μmol/L)were decreased(P<0.05).The combined active components inhibitive rate was higher than the single active components,and highest in the high concentration gro

关 键 词：芒柄花素 大豆苷元 毛蕊异黄酮 配伍 黄芪 核因子E2相关因子2/血红素氧合酶1信号通路 人脐静脉内皮细胞 糖尿病视网膜病变 

分 类 号：R285.5[医药卫生—中药学]