红车轴草总黄酮对H_2O_2诱导的血管内皮细胞损伤的保护作用  

Protective effects of red clover flavonoids against H_2O_2-induced injury in vascular endothelial cells

作　　者：高蒙蒙[1] 孙桂波[1] 斯建勇[1] 秦蒙[1] 罗云[1] 孟祥宝[1] 王敏[1] 孙晓波[1] 

机构地区：[1]中国医学科学院药用植物研究所,北京100193

出　　处：《中国药理学通报》2013年第2期201-207,共7页Chinese Pharmacological Bulletin

基　　金：国家重大新药创制专项(No 2012ZX09103201-004;2012ZX09501001-004;2012ZX09301002-001);国家科技支撑计划项目(No 2008BAI51B02);国家"973"计划资助课题(No 2009 CB522805)

摘　　要：目的观察红车轴草总黄酮对H2O2诱导的血管内皮细胞损伤的保护作用,并探讨其可能的作用机制。方法利用体外培养的人脐静脉血管内皮细胞ECV-304,采用H2O2诱导细胞损伤模型,观察红车轴草总黄酮对H2O2所致的血管内皮细胞损伤的保护作用。MTT法检测细胞存活率、试剂盒测定细胞中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性及细胞中丙二醛(MDA)的含量、乳酸脱氢酶(LDH)的漏出量;荧光法检测Caspase-3的活性;Hoechst33342及PI荧光染色法观察红车轴草总黄酮对H2O2诱导血管内皮细胞凋亡的影响;JC-1染色法检测红车轴草总黄酮对H2O2损伤血管内皮细胞线粒体膜电位的影响,检测各组细胞内ROS的表达水平。结果与空白对照组相比,H2O2损伤组细胞存活率降低、细胞内LDH,漏出量增加(P<0.001);抗氧化酶SOD、CAT、GSH-Px活性降低、MDA含量升高(P<0.01);Caspase-3活性升高(P<0.001)、ROS含量升高、线粒体膜电位降低;与模型组相比,红车轴草总黄酮各剂量组(12.5、25、50mg.L-1)及槲皮素组(30 mg.L-1)细胞存活率明显增加(P<0.01,P<0.05),红车轴草总黄酮各剂量组(12.5、25、50 mg.L-1)及槲皮素组(30 mg.L-1)均能明显降低LDH漏出量(P<0.01,P<0.05)、明显增强SOD、CAT及GSH-Px活力(P<0.01,P<0.05)、降低MDA、ROS含量及Caspase-3活性(P<0.05,P<0.01)、升高线粒体膜电位(P<0.01)。结论红车轴草总黄酮对H2O2诱导的内皮细胞损伤有明显的保护作用,其初步作用机制可能与抗自由基,减轻脂质过氧化、降低Caspase-3活性及稳定线粒体膜电位有关。Aim To investigate the protective effects of red clover flavonoids against H2O2-induced injury in vascular endothelial cells and to probe into its possible mechanisms.Methods All the experiments were carried out with human umbilical vein vascular endothelial cells ECV-304 in vitro,which were divided into 6 groups： control,model,quercetin（30 mg·L^-1） and red clover flavonoids（12.5,25,50 mg·L^-1） preincubation.Cell viability was determined colorimetrically by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium（MTT） assay;Hoechst33342 and PI staining were used for the qualitative and quantitative analyses of the apoptotic endothelial cells.JC-1 staining was used for the detection of mitochondrial membrane potential of endothelial cells injured by H2O2.Then ROS production of endothelial cells in different groups was detected.The supernates and the cells were collected respectively after different treatments for measuring LDH and MDA levels,as well as SOD,CAT and GSH-Px with the corresponding detection kit according to the manufacturers instructions.Fluorescence spectroscopy was used to detect the activity of caspase-3.Results Red clover flavonoids restrained the apoptotic of endothelial cells;the activity of SOD,CAT and GSH-Px increased and caspase-3 decreased;the contents of LDH,MDA and ROS decreased and mitochondrial membrane potential increased.Conclusion Red clover flavonoids exert protective effects against H2O2-induced injury in endothelial cells,which may act through its anti-oxidative effects and the decrease of caspase-3 activity and the stabilization of mitochondrial membrane potential.

关 键 词：红车轴草总黄酮 H2O2 内皮细胞 抗氧化 线粒体膜电位 CASPASE-3 

分 类 号：R284.1[医药卫生—中药学] R322.123[医药卫生—中医学]