三七皂苷R1上调氧化低密度脂蛋白干预人脐静脉内皮细胞miR-421表达
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摘要
目的:研究三七皂苷R1(Notoginsenoside R1,NR1)减轻氧化低密度脂蛋白(ox-LDL)诱导人脐静脉内皮细胞(HUVEC)损伤作用机制。方法:预先向体外培养的HUVEC中加入不同浓度的三七皂苷R1 (25、50、100μmol/L)孵育2 h,再加入oxLDL(100μmol/L)干预24 h诱导细胞损伤,以阿伐他汀为阳性对照组; CCK8法检测各组细胞活力,流式细胞术检测细胞凋亡;ELISA检测细胞培养液中肿瘤坏死因子(TNF-α)、白细胞介素6(IL-6)、细胞间黏附分子1(ICAM-1)和血管细胞粘附分子-1(VCAM-1)的含量。qRT-PCR检测细胞内ICAM-1、VCAM-1、ACE2及miR-421的mRNA水平; Western blotting测定细胞ACE2的表达量。结果:与对照组相比,ox-LDL组细胞活力明显下降,细胞上清中TNF-α、IL-6、ICAM-1及VCAM-1的浓度明显升高,细胞凋亡率增高。三七皂苷R1 50、100μmol/L浓度较ox-LDL组能显著上调细胞存活率,显著降低其凋亡率,并下调细胞内TNF-α、IL-6、ICAM-1及VCAM-1的浓度,上调细胞内ACE2及miR-421表达。结论:三七皂苷R1能减轻ox-LDL诱导HUVEC损伤,其机制可能与增加细胞内miR-421表达,从而调节ICAM-1、VCAM-1、ACE2等表达有关。
Objective : To study the molecular mechanism of Notoginsenoside R1( NR1) on HUVEC injury induced by ox-LDL. Methods: After pre-treatment with the different concentrations( 25,50 and 100 μmol/L) of NR1 for 2 h,the cells were exposed to 100 μmol/L ox-LDL.Meanwhile,we used atorvastatin as positive control. The cell viability was measured by CCK8 assay. The contents of inflammatory cytokines,like TNF-α,IL-6,ICAM-1 and VCAM-1 were assayed by ELISA. The mRNA levels of ICAM-1,VCAM-1,ACE2 and miR-181 b were determined by qRT-PCR and the protein level of ACE2 was determined by western blotting. Results: ox-LDL induced the apoptosis and the inflammatory cytokines secretion in HUVEC. NR1 increased the cell livability and decreased apoptosis index,meanwhile down-regulated the expressions of TNF-α,IL-6,ICAM-1 and VCAM-1,especially at the concentrations of 50 and 100 μmol/L. Furthermore,NR1 regulated miR-421/ACE2 signaling in HUVEC. Conclusions: NR1 could reduce the HUVEC injury induced by ox-LDL,which is partly related to upregulation of miR-421 so as to reduce the inflammatory cytokines induced by ox-LDL.
Objective : To study the molecular mechanism of Notoginsenoside R1( NR1) on HUVEC injury induced by ox-LDL. Methods: After pre-treatment with the different concentrations( 25,50 and 100 μmol/L) of NR1 for 2 h,the cells were exposed to 100 μmol/L ox-LDL.Meanwhile,we used atorvastatin as positive control. The cell viability was measured by CCK8 assay. The contents of inflammatory cytokines,like TNF-α,IL-6,ICAM-1 and VCAM-1 were assayed by ELISA. The mRNA levels of ICAM-1,VCAM-1,ACE2 and miR-181 b were determined by qRT-PCR and the protein level of ACE2 was determined by western blotting. Results: ox-LDL induced the apoptosis and the inflammatory cytokines secretion in HUVEC. NR1 increased the cell livability and decreased apoptosis index,meanwhile down-regulated the expressions of TNF-α,IL-6,ICAM-1 and VCAM-1,especially at the concentrations of 50 and 100 μmol/L. Furthermore,NR1 regulated miR-421/ACE2 signaling in HUVEC. Conclusions: NR1 could reduce the HUVEC injury induced by ox-LDL,which is partly related to upregulation of miR-421 so as to reduce the inflammatory cytokines induced by ox-LDL.
引文
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[2]刘俊田.动脉粥样硬化发病的炎症机制的研究进展.西安交通大学学报(医学版),2015,(2)∶141~152.
[3]吉国辉,江斌,朱怡洁,等.不同前处理方法对川芎药材中三七皂苷R1含量的影响.食品与药品,2014,16(1)∶15~17.
[4]Jia C,Xiong M,Wang P,et al. Notoginsenoside R1? attenuates atherosclerotic lesions in ApoE deficient mouse model. PLo S One,2014,9(6)∶e99849.
[5]Chen W,Dang Y,Zhu C. Simultaneous determination of three major bioactive saponins of Panax notoginseng using liquid chromatography-tandem mass spectrometry and a pharmacokinetic study. Chin Med,2010,(5)∶12.
[6]赖小华,雷燕,杨静,等.基于microRNA-34a/SIRT1/p53通路探讨三七皂苷R1对血管内皮细胞衰老的影响.中国中药杂志,2018,(3)∶167~174.
[7]Kim E N,Kim M Y,Lim J H,et al. The protective effect of resveratrol on vascular aging by modulation of the renin-angiotensin system. Atherosclerosis,2018,270∶123~131.
[8]Anguiano L,Riera M,Pascual J,et al. investigators from the NEFRONA study. Circulating angiotensin converting enzyme 2 activity as a biomarker of silentatherosclerosis? in patients with chronic kidney disease. Atherosclerosis,2016,253∶135~143.
[9]吴颖,孙冰,肖静,等.三七皂苷R1对LPS诱导的小鼠心肌损伤的保护作用.中国药理学通报,2013,29(2)∶179~184.
[10]郭钰琪,齐冬梅,郝钰,等.三七皂苷R1调节炎性T细胞亚群保护血管内皮的实验研究.中国药理学通报,2012,28(2)∶240~244.
[11]Zhai Y,Meng X,Luo Y,et al. Notoginsenoside R1? ameliorates diabetic encephalopathy by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation. Onco target,2018,9(10)∶9344~9363.
[12]Su P,Du S,Li H,et al. Notoginsenoside R1? inhibits oxidized low-density lipoprotein induced inflammatory cytokines production in human endothelial EA. hy926 cells. Eur J Pharmacol,2016,770∶9~15.
[13]Lambert D W,Lambert L A,Clarke N E,et al. Angiotensin converting enzyme 2 is subject to post-transcriptional regulation by miR-421. Clin Sci,2014,127(4)∶243~249.
[14]程赛博.栀子苷调控miR-101/MKP-1/p38减轻动脉粥样硬化炎症损伤的机制研究.南方医科大学博士学位论文. 2018.