基于microRNA-34a/SIRT1/p53通路探讨三七皂苷R_1对血管内皮细胞衰老的影响
|
摘要
该研究旨在探讨三七皂苷R_1能否通过调控miRNA-34a/SIRT1/p53通路发挥延缓H_2O_2诱导的血管内皮细胞衰老的作用。以人脐静脉内皮细胞(HUVEC)作为研究对象,建立H_2O_2诱导的衰老模型,以白藜芦醇作为阳性对照药,实验分为青年组、H_2O_2模型组、三七皂苷R1组、白藜芦醇组。药物组提前加入三七皂苷R1(30μmo L·L~(-1))或白藜芦醇(10μmo L·L~(-1))干预24 h,然后用H_2O_2(100μmo L·L~(-1))造模4 h,最后换成完全培养基培养24 h。采用衰老特异性β-半乳糖苷酶(SA-β-gal)染色法鉴定细胞衰老程度,CCK-8法检测细胞增殖能力,流式细胞仪分析细胞周期,WST-1法检测细胞内超氧化物歧化酶(SOD)活力,QRT-PCR检测miRNA-34a,SIRT1和p53的核酸表达,Western-blot技术检测SIRT1,p53和衰老相关蛋白p21,p16的蛋白表达。结果发现,与模型组相比,三七皂苷R1组和白藜芦醇组可有效减低衰老内皮细胞的β-半乳糖苷酶染色阳性率,增强细胞增殖能力和SOD活力,减少G0/G1期细胞比例,增加S期细胞比例,抑制了miRNA-34a和p53的核酸表达,促进了SIRT1的核酸表达,此外抑制了p53,p21和p16的蛋白表达,促进了SIRT1的蛋白表达,差异均有统计意义(P<0.05)。该研究表明,H_2O_2诱导的衰老的内皮细胞可以作为衰老研究的模型,三七皂苷R1延缓血管内皮细胞衰老的效果明显,其机制可能是通过调控miRNA-34a/SIRT1/p53通路延缓了血管内皮细胞衰老的进程。
This study aimed to investigate the effect of notoginsenoside R_1 in delaying H_2O_2-induced vascular endothelial cell senescence through microRNA-34 a/SIRT1/p53 signal pathway. In this study,human umbilical vein endothelial cells( HUVECs) were selected as the study object; the aging model induced by hydrogen peroxide( H_2O_2) was established,with resveratrol as the positive drug. HUVECs were randomly divided into four groups,youth group,senescence model group,notoginsenoside R_1 group and resveratrol group. Notoginsenoside R_1 group and resveratrol group were modeled with 100 μmo L·L~(-1) H_2O_2 for 4 h after 24 h treatment with notoginsenoside R_1( 30 μmo L·L~(-1)) and resveratrol( 10 μmo L·L~(-1)) respectively. At the end,each group was cultured with complete medium for 24 h. The degree of cellular senescence was detected by senescence-associated β-galactosidase( SA-β-Gal) staining kit,the cell viability was detected by cell counting kit-8,the cell cycle distribution was analyzed by flow cytometry,and the cellular SOD activity was detected by WST-1 method in each group. The expressions of SIRT1,p53,p21 and p16 proteins in HUVECs were detected by Western blot. In addition,the mRNA expressions of miRNA-34 a,SIRT1 and p53 in HUVECs were assayed by Real-time PCR.These results indicated that notoginsenoside R_1 significantly reduced the positive staining rate of senescent cells,enhanced the cell proliferation capacity and intracellular SOD activity,decreased the proportion of cells in G0/G1 phase,and increased the percentage of cells in S phase simultaneously compared with the senescence model group. Moreover,notoginsenoside R_1 decreased the mRNA expressions of miRNA-34 a and p53 and the protein expression of p53,p21 and p16. At the same time,notoginsenoside R_1 increased the protein and mRNA expressions of SIRT1. The differences in these results between the senescence model group and the notoginsenoside R_1 group were statistically significant( P < 0. 05). However,there was not statistically significant difference in these results between the notoginsenoside R_1 group and the resveratrol group. In conclusion,the senescence of endothelial cells induced by H_2O_2 can be used as a model for studying aging. Notoginsenoside R_1 has an obvious anti-aging effect on vascular endothelial cells in this study. The possible mechanism is that notoginsenoside R_1 can delay the senescence process of vascular endothelial cells induced by H_2O_2 by regulating microRNA-34 a/SIRT1/p53 signal pathway.
This study aimed to investigate the effect of notoginsenoside R_1 in delaying H_2O_2-induced vascular endothelial cell senescence through microRNA-34 a/SIRT1/p53 signal pathway. In this study,human umbilical vein endothelial cells( HUVECs) were selected as the study object; the aging model induced by hydrogen peroxide( H_2O_2) was established,with resveratrol as the positive drug. HUVECs were randomly divided into four groups,youth group,senescence model group,notoginsenoside R_1 group and resveratrol group. Notoginsenoside R_1 group and resveratrol group were modeled with 100 μmo L·L~(-1) H_2O_2 for 4 h after 24 h treatment with notoginsenoside R_1( 30 μmo L·L~(-1)) and resveratrol( 10 μmo L·L~(-1)) respectively. At the end,each group was cultured with complete medium for 24 h. The degree of cellular senescence was detected by senescence-associated β-galactosidase( SA-β-Gal) staining kit,the cell viability was detected by cell counting kit-8,the cell cycle distribution was analyzed by flow cytometry,and the cellular SOD activity was detected by WST-1 method in each group. The expressions of SIRT1,p53,p21 and p16 proteins in HUVECs were detected by Western blot. In addition,the mRNA expressions of miRNA-34 a,SIRT1 and p53 in HUVECs were assayed by Real-time PCR.These results indicated that notoginsenoside R_1 significantly reduced the positive staining rate of senescent cells,enhanced the cell proliferation capacity and intracellular SOD activity,decreased the proportion of cells in G0/G1 phase,and increased the percentage of cells in S phase simultaneously compared with the senescence model group. Moreover,notoginsenoside R_1 decreased the mRNA expressions of miRNA-34 a and p53 and the protein expression of p53,p21 and p16. At the same time,notoginsenoside R_1 increased the protein and mRNA expressions of SIRT1. The differences in these results between the senescence model group and the notoginsenoside R_1 group were statistically significant( P < 0. 05). However,there was not statistically significant difference in these results between the notoginsenoside R_1 group and the resveratrol group. In conclusion,the senescence of endothelial cells induced by H_2O_2 can be used as a model for studying aging. Notoginsenoside R_1 has an obvious anti-aging effect on vascular endothelial cells in this study. The possible mechanism is that notoginsenoside R_1 can delay the senescence process of vascular endothelial cells induced by H_2O_2 by regulating microRNA-34 a/SIRT1/p53 signal pathway.
引文
[1]Puca A A,Carrizzo A,Villa F,et al.Vascular ageing:the role of oxidative stress[J].Int J Biochem Cell Biol,2013,45(3):556.
[2]El Assar M,Angulo J,Rodríguez-Ma1as L.Oxidative stress and vascular inflammation in aging[J].Free Radic Biol Med,2013,10(65C):380.
[3]Boon R A,Iekushi K,Lechner S,et al.MicroRNA-34a regulates cardiac ageing and function[J].Nature,2013,495(7439):107.
[4]Ito T,Yagi S,Yamakuchi M.MicroRNA-34a regulation of endothelial senescence[J].Biochem Biophys Res Commun,2010,398(4):735.
[5]米雪楠,惠汝太,张伟丽.MicroRNAs参与血管内皮细胞衰老机制的研究进展[J].中国医学前沿杂志:电子版,2013,5(4):52.
[6]Guo Y,Li P,Gao L,et al.Kallistatin reduces vascular senescence and aging by regulating microRNA-34a-SIRT1 pathway[J].Aging Cell,2017,16(4):837.
[7]Li N,Wang K,Li P F.MicroRNA-34 family and its role in cardiovascular disease[J].Crit Rev Eukaryot Gene Expr,2015,25(4):293.
[8]Badi I,Burba I,Ruggeri C,et al.MicroRNA-34a induces vascular smooth muscle cells senescence by SIRT1 downregulation and promotes the expression of age-associated pro-inflammatory secretory factors[J].J Gerontol A Biol Sci Med Sci,2015,70(11):1304.
[9]杨晶晶,刘英,王承潇,等.三七皂苷R1的现代研究进展[J].中国医院药学杂志,2015,35(5):463.
[10]万强,潘芸芸,贾钰华,等.三七皂苷R1抑制JNK通路减轻H2O2诱导心肌细胞损伤的作用[J].时珍国医国药,2015,26(9):2061.
[11]Wang X B,Zhu L,Huang J,et al.Resveratrol-induced augmentation of telomerase activity delays senescence of endothelial progenitor cell[J].Chin Med J,2011,124(24):4310.
[12]Barodka V M,Joshi B L,Berkowitz D E,et al.Review article:implications of vascular aging[J].Anesth Analg,2011,112(5):1048.
[13]姜平,黎健.血管衰老及其机制[J].生物化学与生物物理进展,2014,41(3):295.
[14]Hayashi T,Matsui-Hirai H,Miyazaki-Akita A,et al.Endothelial cellular senescence is inhibited by nitric oxide:implications in atherosclerosis associated with menopause and diabetes[J].Proc Natl Acad Sci USA,2006,103(45):17018.
[15]Dimiri G P,Lee X,Basile G,et al.A biomarker that identifies senescent human cells in culture and in aging skin in vivo[J].Proc Natl Acad Sci U S A,1995,92(1):93.
[16]Rodriguez M A,Garcia E C,Briones A,et al.Changes in plasma oxidative state with age and their influence on contractions elici2ted by noradrenaline in the rat tail artery[J].Life Sci,1999,65(9):915.
[17]石淙,万腊根.细胞增殖的检测方法[J].实验与检验医学,2012,30(2):153.
[18]Adelibieke Y,Shimizu H,Muteliefu G,et al.Indoxyl sulfate induces endothelial cell senescence by increasing reactive oxygen species production and p53 activity[J].J Ren Nutr,2012,22(1):86.
[19]王强,雷燕,杨静,等.益气活血中药抗衰老研究现状[J].中国中医药信息杂志,2015,22(2):119.
[20]修成奎,雷燕,王强,等.人参三七川芎提取物对复制性衰老血管平滑肌细胞骨架蛋白微丝的影响[J].中国中药杂志,2016,41(3):484.
[21]Wang Y,Ren Y,Xing L,et al.Endothelium-dependent vasodilation effects of Panax notoginseng and its main components are mediated by nitric oxide and cyclooxygenase pathways[J].Exp Ther Med,2016,12(6):3998.
[22]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[J].中国药理学通报,2012,28(2):240.
[23]Zhang H S,Wang S Q.Notoginsenoside R1inhibits TNF-alphainduced fibronectin production in smooth muscle cells via the ROS/ERK pathway[J].Free Radic Biol Med,2006,40(9):1664.
[24]Zhang H S,Wang S Q.Notoginsenoside R1from Panax notoginseng inhibits TNF-alpha-induced PAI-1 production in human aortic smooth muscle cells[J].Vascul Pharmacol,2006,44(4):224.
[25]金吉春,金兴林.microRNA的概述及其研究[J].医学研究生学报,2013,26(10):1109.
[26]于瑞杰,汪俊军.microRNA在动脉粥样硬化发病机制中的作用[J].医学研究生学报,2013,26(9):970.
[27]Dimmeler S,Nicotera P.MicroRNAs in age-related disease[J].EMBO Mol Med,2013,5(2):180.
[28]刘婷,洪骏,汪俊军.microRNA与心血管衰老的研究进展[J].医学研究生学报,2015,28(5):539.
[29]Feng J,Li S,Chen H.TanshinoneⅡAinhibits myocardial remodeling induced by pressure overload via suppressing oxidative stress and inflammation:possible role of silent information regulator 1[J].Eur J Pharmacol,2016,791(deficiency):632.
[30]Kitada M,Ogura Y,Koya D.The protective role of Sirt1 in vascular tissue:its relationship to vascular aging and atherosclerosis[J].Aging,2016,8(10):2290.
[31]赵思达,常春康.p53与细胞衰老关系的研究进展[J].诊断学理论与实践,2014,13(6):636.
[32]Ito T,Yagi S,Yamakuchi M.MicroRNA-34a regulation of endothelial senescence[J].Biochem Biophys Res Commun,2010,398(4):735.
[2]El Assar M,Angulo J,Rodríguez-Ma1as L.Oxidative stress and vascular inflammation in aging[J].Free Radic Biol Med,2013,10(65C):380.
[3]Boon R A,Iekushi K,Lechner S,et al.MicroRNA-34a regulates cardiac ageing and function[J].Nature,2013,495(7439):107.
[4]Ito T,Yagi S,Yamakuchi M.MicroRNA-34a regulation of endothelial senescence[J].Biochem Biophys Res Commun,2010,398(4):735.
[5]米雪楠,惠汝太,张伟丽.MicroRNAs参与血管内皮细胞衰老机制的研究进展[J].中国医学前沿杂志:电子版,2013,5(4):52.
[6]Guo Y,Li P,Gao L,et al.Kallistatin reduces vascular senescence and aging by regulating microRNA-34a-SIRT1 pathway[J].Aging Cell,2017,16(4):837.
[7]Li N,Wang K,Li P F.MicroRNA-34 family and its role in cardiovascular disease[J].Crit Rev Eukaryot Gene Expr,2015,25(4):293.
[8]Badi I,Burba I,Ruggeri C,et al.MicroRNA-34a induces vascular smooth muscle cells senescence by SIRT1 downregulation and promotes the expression of age-associated pro-inflammatory secretory factors[J].J Gerontol A Biol Sci Med Sci,2015,70(11):1304.
[9]杨晶晶,刘英,王承潇,等.三七皂苷R1的现代研究进展[J].中国医院药学杂志,2015,35(5):463.
[10]万强,潘芸芸,贾钰华,等.三七皂苷R1抑制JNK通路减轻H2O2诱导心肌细胞损伤的作用[J].时珍国医国药,2015,26(9):2061.
[11]Wang X B,Zhu L,Huang J,et al.Resveratrol-induced augmentation of telomerase activity delays senescence of endothelial progenitor cell[J].Chin Med J,2011,124(24):4310.
[12]Barodka V M,Joshi B L,Berkowitz D E,et al.Review article:implications of vascular aging[J].Anesth Analg,2011,112(5):1048.
[13]姜平,黎健.血管衰老及其机制[J].生物化学与生物物理进展,2014,41(3):295.
[14]Hayashi T,Matsui-Hirai H,Miyazaki-Akita A,et al.Endothelial cellular senescence is inhibited by nitric oxide:implications in atherosclerosis associated with menopause and diabetes[J].Proc Natl Acad Sci USA,2006,103(45):17018.
[15]Dimiri G P,Lee X,Basile G,et al.A biomarker that identifies senescent human cells in culture and in aging skin in vivo[J].Proc Natl Acad Sci U S A,1995,92(1):93.
[16]Rodriguez M A,Garcia E C,Briones A,et al.Changes in plasma oxidative state with age and their influence on contractions elici2ted by noradrenaline in the rat tail artery[J].Life Sci,1999,65(9):915.
[17]石淙,万腊根.细胞增殖的检测方法[J].实验与检验医学,2012,30(2):153.
[18]Adelibieke Y,Shimizu H,Muteliefu G,et al.Indoxyl sulfate induces endothelial cell senescence by increasing reactive oxygen species production and p53 activity[J].J Ren Nutr,2012,22(1):86.
[19]王强,雷燕,杨静,等.益气活血中药抗衰老研究现状[J].中国中医药信息杂志,2015,22(2):119.
[20]修成奎,雷燕,王强,等.人参三七川芎提取物对复制性衰老血管平滑肌细胞骨架蛋白微丝的影响[J].中国中药杂志,2016,41(3):484.
[21]Wang Y,Ren Y,Xing L,et al.Endothelium-dependent vasodilation effects of Panax notoginseng and its main components are mediated by nitric oxide and cyclooxygenase pathways[J].Exp Ther Med,2016,12(6):3998.
[22]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[J].中国药理学通报,2012,28(2):240.
[23]Zhang H S,Wang S Q.Notoginsenoside R1inhibits TNF-alphainduced fibronectin production in smooth muscle cells via the ROS/ERK pathway[J].Free Radic Biol Med,2006,40(9):1664.
[24]Zhang H S,Wang S Q.Notoginsenoside R1from Panax notoginseng inhibits TNF-alpha-induced PAI-1 production in human aortic smooth muscle cells[J].Vascul Pharmacol,2006,44(4):224.
[25]金吉春,金兴林.microRNA的概述及其研究[J].医学研究生学报,2013,26(10):1109.
[26]于瑞杰,汪俊军.microRNA在动脉粥样硬化发病机制中的作用[J].医学研究生学报,2013,26(9):970.
[27]Dimmeler S,Nicotera P.MicroRNAs in age-related disease[J].EMBO Mol Med,2013,5(2):180.
[28]刘婷,洪骏,汪俊军.microRNA与心血管衰老的研究进展[J].医学研究生学报,2015,28(5):539.
[29]Feng J,Li S,Chen H.TanshinoneⅡAinhibits myocardial remodeling induced by pressure overload via suppressing oxidative stress and inflammation:possible role of silent information regulator 1[J].Eur J Pharmacol,2016,791(deficiency):632.
[30]Kitada M,Ogura Y,Koya D.The protective role of Sirt1 in vascular tissue:its relationship to vascular aging and atherosclerosis[J].Aging,2016,8(10):2290.
[31]赵思达,常春康.p53与细胞衰老关系的研究进展[J].诊断学理论与实践,2014,13(6):636.
[32]Ito T,Yagi S,Yamakuchi M.MicroRNA-34a regulation of endothelial senescence[J].Biochem Biophys Res Commun,2010,398(4):735.