摘要
探讨灯盏花素对内皮细胞的保护作用及机制。采用用不同浓度的灯盏花素(10,20,40μmol/L)对人脐静脉内皮细胞(HUVEC细胞)预处理4 h,再采用氧化型低密度脂蛋白诱导细胞氧化损伤20 h,然后检测细胞的改变,包括采用MTT方法检测细胞增殖活力、流式细胞仪检测细胞凋亡及活性氧含量,以及蛋白免疫印迹及定量PCR方法检测细胞信号通路关键分子的改变。实验结果发现,灯盏花素可以逆转氧化型低密度脂蛋白对内皮细胞的损伤并呈剂量依赖效应,并减少内皮细胞的凋亡。为探索灯盏花素的作用机制,首先检测了细胞与多种浓度及时间(2,4,6 h)的灯盏花素预孵育后的活性氧含量改变,结果发现灯盏花素可剂量及时间依赖地减少活性氧的产生。进一步发现,细胞信号通路分析发现灯盏花素可促进BCL-2的表达,抑制BAX的表达及细胞色素C的释放及caspase-3的剪切。灯盏花素可减少Keap1及激活Nrf2的核内转运,促进下游抗氧化酶NAD(P)H:醌氧化还原酶1(NQO1)和谷胱甘肽-S-转移酶Mu1型(GSTM1)的基因转录及蛋白表达,增强NQO1酶活。此外,灯盏花素还可减少IKK及IKB及抑制NF-κB核内转运,而促进e NOS的表达。本研究表明灯盏花素对氧化型低密度脂蛋白诱导的内皮细胞损伤有保护作用,其作用可能与其抗氧化作用及抑制NF-κB活化有关。
This study was to investigate the protective effects and mechanisms of breviscapine on endothelial cells.ox-LDL was used to induce oxidative damage on human umbilical vein endothelial cells（HUVEC）for 20 h with or without 4 h pretreatment of various concentrations of breviscapine（10;20;40 μmol/L）;then observe the protective effect and mechanism of breviscapine on endothelial cells insulted by ox-LDL.MTT method was used to detect cell viability;flow cytometry was used to detect cell apoptosis and reactive oxygen species;and Western blot and RT-PCR was used to detect cell signaling pathways.The results showed that breviscapine recovered HUVEC cell viability in a dose-dependent manner which inhibited by ox-LDL;and it also protected cell from apoptosis induced by ox-LDL.To explore the mechanisms of breviscapine;reactive oxygen species （ROS）was determined after pretreatment of various concentrations of breviscapine or different durations（2;4;6 h）of brevis-capine.Results showed that breviscapine decreased ROS production in a dose-and time-dependent manner.Fur-thermore;cell signaling pathway analysis showed that breviscapine increased the expression of BCL-2;decreased the expression of BAX and the release of Cytochrome C and cleavage of caspase-3.Breviscapine decreased Keap1 and activated the nuclear import of Nrf2;and subsequently increased the mRNA and protein expression of down-stream antioxidant enzyme as NAD（P）H：quinone oxidoreductase 1（NQO1）and glutathione transferase-S-Mu 1（GSTM1）;and increasing the activity of NQO1.Besides;breviscapine decreased IKK and IKB;and inhibited nuclear translocation of NF-κB;while increasing the expression of eNOS.This study demonstrated that breviscap-ine has a protective role on ox-LDL-induced endothelial cell injury;which may be related to its antioxidant effects and inhibition of NF-κB activation.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2015年第5期610-616,共7页
Journal of China Pharmaceutical University
基金
天津滨海新区卫生局联合攻关项目(No.2012BWKL026)~~
关键词
灯盏花素
活性氧
内皮细胞
细胞凋亡
breviscapine
reactive oxygen species
endothelial cells
cell apoptosis
