冠心宁片减轻ox-LDL损伤内皮细胞的药效物质研究  

Guanxinning Tablet and their active substances alleviate ox-LDL-induced endothelial cell injury

作　　者：余敏 王木兰 赵璐 王毅[1,3] YU Min;WANG Mu-lan;ZHAO Lu;WANG Yi(College of Pharmaceutical Science,Zhejiang University,Hangzhou 310058,China;Chiatai Qingchunbao Pharmaceutical Co.,Ltd.,Hangzhou 310023,China;Innovative Institute for Artificial Intelligence in Medicine of Zhejiang University,Hangzhou 310018,China)

机构地区：[1]浙江大学药学院,浙江杭州310058 [2]正大青春宝药业有限公司,浙江杭州310023 [3]浙江大学智能创新药物研究院,浙江杭州310018

出　　处：《药学学报》2023年第1期139-148,共10页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(82173941)。

摘　　要：本文研究冠心宁片对氧化低密度脂蛋白(oxidized low density lipoprotein, ox-LDL)诱导人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)损伤的干预作用,为冠心宁片治疗动脉粥样硬化提供实验依据。在ox-LDL对HUVECs细胞的损伤下,采用CCK-8 (cell counting kit-8)法检测细胞的活力;采用相应的试剂盒检测细胞培养上清中的乳酸脱氢酶(lactate dehydrogenase, LDH)含量;用普通相差显微镜观察不同组别的细胞形态;采用DCFH-DA和DAF-FM DA探针分别检测细胞中的活性氧(reactive oxygen species, ROS)和NO水平;单核细胞黏附实验检测HUVECs对THP-1的招募情况;用TMRM染料检测线粒体膜电位水平;ELISA法检测细胞的白细胞介素-6 (interleukin-6, IL-6)、细胞间黏附因子-1 (intercellular adhesion molecule-1, ICAM-1)和单核细胞趋化蛋白-1(monocyte chemoattractant protein-1, MCP-1)分泌情况。结果显示,冠心宁片对HUVECs具有浓度依赖性促增殖作用,在100μg·mL-1 ox-LDL刺激下,内皮细胞形态发生明显改变,此时NO水平显著下降, ROS水平显著上升并伴随着线粒体膜电位下降,内皮细胞对THP-1细胞的招募增加, IL-6、ICAM-1和MCP-1也显著升高,产生了氧化应激和炎性损伤。而冠心宁片及其组成的丹参、川芎提取物能够明显改善细胞形态、提高NO水平、降低ROS的产生,并且还能减少炎症相关蛋白IL-6和MCP-1的分泌。在NO这个内皮基础功能的指标上,丹参和川芎具有显著的协同作用。其中丹酚酸B和丹参素发挥主要的药效作用,丹参素和阿魏酸联合药效优于单给药药效。以上结果表明,冠心宁片及其药效物质具有改善内皮基础功能、抵抗氧化应激、缓解炎性损伤的作用,并且丹参和川芎协同增效,可能与其调控氧化应激和炎症有关,在动脉粥样硬化的防治中具有应用前景。This study investigated the intervention effect of Guanxinning Tablet on human umbilical vein endothelial cells(HUVECs) injury induced by oxidized low density lipoprotein(ox-LDL),providing experimental basis for Guanxinning Tablet in the treatment of atherosclerosis-related diseases.Under the damage of HUVECs by ox-LDL,the cell viability was detected by CCK-8(cell counting kit-8) assay;lactate dehydrogenase(LDH) in the cell culture supernatant was detected by the corresponding kit;the cell morphology of different groups was observed by common phase contrast microscope;reactive oxygen species(ROS) and NO levels in the cells were detected by DCFH-DA and DAF-FM DA probes,respectively;monocyte adhesion assay was used to detect the recruitment of THP-1 in HUVECs,and TMRM dye was used to detect the level of mitochondrial membrane potential;interleukin-6(IL-6),intercellular adhesion molecule-1(ICAM-1) and monocyte chemoattractant protein-1(MCP-1) secretion in the cells was detected by ELISA assay.The results showed that Guanxinning Tablet had a concentration-dependent proliferative effect on HUVECs.Under the stimulation of 100 μg·mL-1 ox-LDL,the morphology of endothelial cells was significantly changed.At this time,NO level was significantly decreased,ROS level was significantly increased and accompanied by a decrease in mitochondrial membrane potential.The recruitment of THP-1 cells by endothelial cells and IL-6,ICAM-1 and MCP-1 were also significantly increased,resulting in oxidative stress and inflammatory injury.Guanxinning Tablet and its composed extracts could significantly improve cell morphology,increase NO level,decrease ROS production,and also reduce the secretion of inflammation-related proteins IL-6 and MCP-1.Salvia miltiorrhiza and Ligusticum striatum DC.have significant synergistic effects on NO.Among them,salvianolic acid B and salvianic acid A exerted the main effects,and the combined efficacy of salvianic acid A and ferulic acid was superior to that of single administration.The above results showed th

关 键 词：冠心宁片 丹参 川芎 动脉粥样硬化 协同作用 氧化低密度脂蛋白 炎症 氧化应激 

分 类 号：R966[医药卫生—药理学]