小檗碱活化AMPK-eNOS减轻油酸所致人主动脉内皮细胞损伤  

Protective Effect of Berberine on Oleic Acid-Induced Injury in Human Aortic Endothelial Cells via AMPK-eNOS Activation

作　　者：周慧 刘敬 侯文锋 王尧清 张文彦 许波[1] 马成俊[1] 李忌[1] 孟庆国[3] 孙喜灵[4] 王振华[1] ZHOU Hui;LIU Jing;HOU Wenfeng;WANG Yaoqing;ZHANG Wenyan;XU Bo;MA Chengjun;LI Ji;MENG Qingguo;SUN Xiling;WANG Zhenhua(Center for Mitochondria and Healthy Aging, College of Life Sciences, Yantai University, Yantai 264005, China;Key Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education, School of Pharmacy, Shihezi University,Shihezi 832002, China;Key Laboratory of Molecular Pharmacology and Drug Evaluation, Ministry of Education, Collaborative Yantai 264005, China;Shandong Province Key Laboratory of TCM Syndrome, Binzhou Medical University, Yantai 264003, China)

机构地区：[1]烟台大学生命科学学院,线粒体与健康衰老研究中心,山东烟台264005 [2]石河子大学药学院,新疆特种植物药资源教育部重点实验室,新疆石河子832002 [3]烟台大学新型制剂与生物技术药物研究山东省高校协同创新中心,分子药理和药物评价教育部重点实验室,山东烟台264005 [4]滨州医学院山东省中医证候研究重点实验室,山东烟台264003

出　　处：《食品科学》2018年第5期213-219,共7页Food Science

基　　金：山东省泰山学者建设工程专项(tshw201502046);国家自然科学基金面上项目(21372190;81273628;81473104);中科院战略性先导科技专项(A类)子课题(XDA12040320);烟台市科技计划项目(2014LGS004)

摘　　要：目的:考察小檗碱对油酸所致内皮细胞损伤的保护作用,并探究其作用机制。方法:以体外培养人主动脉内皮细胞系(human aorta endothelial cells,HAEC)为受试对象;分别以油酸、油酸联合小檗碱、油酸联合单磷酸腺苷活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)激活剂(AICAR)或抑制剂(Compound C)处理HAEC;采用油红O染色法观察细胞内脂滴合成;采用比色法检测细胞内甘油三酯、总胆固醇含量,硝酸还原酶法检测NO的水平,2’,7’-二氯荧光黄双乙酸盐探针检测细胞内总活性氧(reactive oxygen species,ROS)水平,Western Blotting检测细胞总AMPK、内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)及其磷酸化(p-AMPK、p-eNOS)水平。结果:油酸可浓度依赖性地抑制细胞增殖,小檗碱组各项指标较正常对照组无显著性差异,油酸联合小檗碱组的细胞活性较油酸组明显好转(P<0.01)。与正常对照组比较,油酸组的脂质浸润程度明显,油酸在不同浓度下使内皮细胞的脂质含量呈剂量依赖性增加,加入小檗碱可明显减少这种脂质堆积。油酸组的NO水平较正常对照组明显减少,小檗碱能显著抑制油酸所致的内皮NO水平的减少,并且减少由于油酸所致的ROS水平的升高;油酸抑制了AMPK的活化,使p-AMPK和p-eNOS的蛋白水平明显降低(P<0.01);小檗碱可明显逆转油酸所致AMPK和eNOS磷酸化水平下降,Compound C可拮抗其作用。结论:小檗碱可减轻油酸所致血管内皮细胞损伤,或与其活化AMPK/eNOS信号相关。Objective:To investigate the protective effect and underlying mechanism of berberine chloride on oleicacid(OA)-induced damage in human aortic endothelial cells(HAECs).Methods:HAECs were treated with oleic acid alone or in combination with berberine,adenosine monophosphate-activated protein kinase(AMPK)activator AICAR(5-aminoimidazole-4-carboxamide1-β-D-ribofuranoside),or the AMPK inhibitor compound C,respectively.Cell proliferation was measured by3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)assay.Intracellular lipid accumulation was observed after Oil Red O staining and determined by triglycerides(TG)and total cholesterol(TC)assay kits.The secretion of nitric oxide(NO)by HAECs was detected by nitrate reductase assay.The phosphorylation of adenosine monophosphate-activated protein kinase(AMPK)and endothelial nitric oxide synthase(eNOS)was measured by Western Blotting assay.Results:OA exposure inhibited cell proliferation and resulted in lipid accumulation in HAECs in a concentration-dependent manner.Co-treatment with berberine resulted in no significant differences in all tested parameters compared with the normal control group and showed significantly improved cell viability compared with treatment with OA alone(P<0.01).The degree of lipid infiltration was higher after OA treatment compared with the normal control group.OA concentration-dependently induced lipid accumulation in endothelial cells,while lipid accumulation was significantly reduced by added berberine.OA significantly decreased NO levels compared with the normal control group,but this decrease was significantly reversed by berberine;furthermore,berberine repressed the increase caused by OA in reactive oxygen species(ROS).OA inhibited AMPK activation and consequently significantly attenuated the levels of p-AMPK and p-eNOS(P<0.01).Accordingly,berberine could reverse the reduced phosphorylation of AMPK and eNOS induced by OA,but this effect was antagonized by Compound C.Conclusion:Berberine can alleviate endothelial cell injur

关 键 词：小檗碱 油酸 单磷酸腺苷活化蛋白激酶 内皮型一氧化氮合酶 人主动脉内皮细胞 

分 类 号：R966[医药卫生—药理学]