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

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 that Guanxinning Tablet and their active substances had the effects of improving endothelial basal function,resisting oxidative stress,and alleviating inflammatory injury,and Salvia miltiorrhiza and Ligusticum striatum DC.synergized,which may be related to their regulation of oxidative stress and inflammation and have application prospects in the treatment of atherosclerosis-related diseases.