益母草碱对血管紧张素Ⅱ诱导的心肌细胞肥大p38 MAPK信号通路和miRNA-1表达的影响
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摘要
目的:研究益母草碱对血管紧张素Ⅱ(angiotensin,AngⅡ)诱导心肌细胞肥大的作用,并分析其对p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)信号通路和微小RNA-1(microRNA-1,miRNA-1)表达的影响。方法:以AngⅡ(0. 1μmol·L~(-1))诱导肥大心肌细胞。实验分为空白组,模型组,p38 MAPK抑制剂组(SB203580,10μmol·L~(-1))以及益母草碱低、中、高浓度组(5,10,20μmol·L~(-1))。以图像分析软件测量心肌细胞表面积; Folin-酚试剂法(Lowry)检测心肌细胞蛋白含量;酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测细胞上清心房钠尿肽(atrial natriuretic peptide,ANP)含量;实时荧光定量聚合酶链式反应(Real-time PCR)检测心肌细胞miRNA-1 mRNA的表达水平;蛋白免疫印迹法(Western blot)检测心肌细胞内皮素-1(endothelin-1,ET-1),p38 MAPK,磷酸化p38 MAPK(p-p38 MAPK),肌细胞增强因子2(myocyte enhancer factor 2,MEF2),β-肌球蛋白重链(β-myosin heavy chain,β-MHC)和α-肌球蛋白重链(α-myosin heavy chain,α-MHC)蛋白的表达水平。结果:与空白组比较,模型组心肌细胞表面积、蛋白含量及细胞上清ANP含量明显升高(P <0. 05),ET-1,p38 MAPK,p-p38 MAPK,MEF2和β-MHC蛋白表达水平明显上调(P <0. 05),α-MHC蛋白及miRNA-1 mRNA的表达水平明显下调(P <0. 05)。与模型组比较,益母草碱高浓度组明显减少肥大心肌细胞表面积、蛋白含量及细胞上清ANP含量(P <0. 05);下调ET-1,p38 MAPK,p-p38 MAPK,MEF2和β-MHC蛋白表达水平(P <0. 05);上调α-MHC蛋白及miRNA-1的表达水平(P <0. 05)。结论:益母草碱高浓度组(20μmol·L~(-1))可抑制AngⅡ诱导的心肌细胞肥大,其机制可能与抑制p38MAPK信号通路活化和上调miRNA-1的表达有关。
Objective: To investigate the inhibitory effect of leonurine on cardiomyocyte hypertrophy induced by angiotensin Ⅱ( Ang Ⅱ) and its effect on p38 mitogen-activated protein kinase( p38 MAPK) signaling pathway and miRNA-1. Method: Cardiomyocyte hypertrophy was induced by Ang Ⅱ( 0. 1 μmol·L~(-1)) in primary neonatal cardiomyocytes. Experiments were designed in 6 groups as following: normal group,model group,p38 MAPK inhibitor group( SB203580,10 μmol·L~(-1)),low-dose( 5 μmol·L~(-1)),middle-dose( 10 μmol·L~(-1)) and high-dose( 20 μmol·L~(-1)) group. The cardiomyocyte surface area was measured by image software,and the protein contents were detected by Lowry. The concentrations of ANP in the culture supernatant were measured by enzymelinked immunosorbent assay( ELISA). The expression level of miRNA-1 was detected by Real-time fluorescence quantitative polymerase chain reaction( Real-time PCR),and the protein expression levels of endothelin-1( ET-1),p38 MAPK,p-p38 MAPK,myocyte enhancer factor 2( MEF2),β-myosin heavy chain( β-MHC),α-myosin heavy chain( α-MHC) were detected by Western blot. Result: Compared with normal group,the surface area of cardiomyocyte,the protein contents,the concentrations of ANP,and the protein expression levels of ET-1,p38 MAPK,p-p38 MAPK,MEF2,β-MHC in model group were higher( P < 0. 05),but the protein expression levels ofα-MHC and miRNA-1 were lower than those in normal group( P < 0. 05). Compared with model group,the surface area of cardiomyocyte,the protein contents,the concentrations of ANP,and the protein expression levels of ET-1,p38 MAPK,p-p38 MAPK,MEF2,β-MHC in high-dose group were lower( P < 0. 05),but the protein expression levels of α-MHC and miRNA-1 were higher than those in model group( P < 0. 05). Conclusion: Leonurine( 20 μmol·L~(-1)) could inhibit cardiomyocyte hypertrophy induced by AngⅡ,and the mechanism is related to the inhibition of activation of p38 MAPK signaling pathway and up-regulation the expression of miRNA-1.
Objective: To investigate the inhibitory effect of leonurine on cardiomyocyte hypertrophy induced by angiotensin Ⅱ( Ang Ⅱ) and its effect on p38 mitogen-activated protein kinase( p38 MAPK) signaling pathway and miRNA-1. Method: Cardiomyocyte hypertrophy was induced by Ang Ⅱ( 0. 1 μmol·L~(-1)) in primary neonatal cardiomyocytes. Experiments were designed in 6 groups as following: normal group,model group,p38 MAPK inhibitor group( SB203580,10 μmol·L~(-1)),low-dose( 5 μmol·L~(-1)),middle-dose( 10 μmol·L~(-1)) and high-dose( 20 μmol·L~(-1)) group. The cardiomyocyte surface area was measured by image software,and the protein contents were detected by Lowry. The concentrations of ANP in the culture supernatant were measured by enzymelinked immunosorbent assay( ELISA). The expression level of miRNA-1 was detected by Real-time fluorescence quantitative polymerase chain reaction( Real-time PCR),and the protein expression levels of endothelin-1( ET-1),p38 MAPK,p-p38 MAPK,myocyte enhancer factor 2( MEF2),β-myosin heavy chain( β-MHC),α-myosin heavy chain( α-MHC) were detected by Western blot. Result: Compared with normal group,the surface area of cardiomyocyte,the protein contents,the concentrations of ANP,and the protein expression levels of ET-1,p38 MAPK,p-p38 MAPK,MEF2,β-MHC in model group were higher( P < 0. 05),but the protein expression levels ofα-MHC and miRNA-1 were lower than those in normal group( P < 0. 05). Compared with model group,the surface area of cardiomyocyte,the protein contents,the concentrations of ANP,and the protein expression levels of ET-1,p38 MAPK,p-p38 MAPK,MEF2,β-MHC in high-dose group were lower( P < 0. 05),but the protein expression levels of α-MHC and miRNA-1 were higher than those in model group( P < 0. 05). Conclusion: Leonurine( 20 μmol·L~(-1)) could inhibit cardiomyocyte hypertrophy induced by AngⅡ,and the mechanism is related to the inhibition of activation of p38 MAPK signaling pathway and up-regulation the expression of miRNA-1.
引文
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[18]杨晋静,王敬萍,张月安,等. miRNA在心室重塑中的调控作用[J].中华医学杂志,2015,95(29):2408-2410.
[19] DUAN L, XIONG X, LIU Y, et al. miRNA-1:functional roles and dysregulation in heart disease[J].Mol Biosyst,2014,10(11):2775-2782.
[20] Karakikes I,Chaanine A H,Kang S,et al. Therapeutic cardiac-targeted delivery of miR-1 reverses pressure overload-induced cardiac hypertrophy and attenuates pathological remodeling[J]. J Am Heart Assoc,2013,2(2):e000078.
[21] Sucharov C C,Kao D P,Port J D,et al. Myocardial microRNAs associated with reverse remodeling in human heart failure[J]. JCI Insight,2017,2(2):e89169.
[22]魏运荣,卢清显,卢清君. MicroRNA与细胞信号转导通路研究进展[J].生物技术通报,2010,2:28-32.
[2]朱广旋,左建丽,许林,等.中药有效成分逆转左室心肌肥厚的研究进展[J].中国实验方剂学杂志,2017,23(17):228-234.
[3]梁博志,罗建华,杨冬花,等.益母草碱作用及机制研究进展[J].贵阳中医学院学报,2017,39(4):93-96.
[4]徐玉平,钱海兵,朱依谆.益母草碱对急性心肌梗死大鼠的影响[J].中国实验方剂学杂志,2016,22(17):113-116.
[5] GAO H, YANG X, GU X,et al. Synthesis and biological evaluation of the codrug of Leonurine and Aspirin as cardioprotective agents[J]. Bioorg Med Chem Lett,2016,26(19):4650-4654.
[6]梁赵文,罗建华,杨冬花,等.益母草碱对慢性心力衰竭心肌重构大鼠心房利钠肽、血管紧张素Ⅱ影响的研究[J].贵州医药,2016,40(12):1235-1238.
[7]刁爱芹,潘爱萍.机械刺激诱导心肌细胞肥大的分子机制[J].医学综述,2013,19(22):4074-4077.
[8] WU Q Q,NI J,ZHANG N,LIAO H H,et al.Andrographolide protects against aortic banding-induced experimental cardiac hypertrophy by inhibiting MAPKs signaling[J]. Front Pharmacol,2017,8:808.
[9] Busch A,Eken S M,Maegdefessel L. Prospective and therapeutic screening value of non-coding RNA as biomarkers in cardiovascular disease[J]. Ann Transl Med,2016,4(12):236.
[10]周颖,陈游洲,乔树宾. Apelin对血管紧张素Ⅱ诱导的心肌细胞肥大的作用及其机制[J].中国循环杂志,2014,29(9):733-737.
[11]刘新华.益母草碱的合成及其对心血管保护作用的研究[D].上海:复旦大学,2009.
[12]高欢.益母草碱衍生物的合成及心肌保护作用研究[D].长春:吉林大学,2015.
[13]吕林懋.心肌成纤维细胞来源的exosome在血管紧张素-2诱导的心肌肥大中的作用及机制研究[D].济南:山东大学,2015.
[14]周恒,唐其柱,邓伟,等.血管紧张素Ⅱ诱导的新生小鼠心肌细胞肥大模型[J].医学研究杂志,2015,44(11):26-30.
[15] Archer C R,Robinson E L,Drawnel F M,et al.Endothelin-1 promotes hypertrophic remodelling of cardiac myocytes by activating sustained signalling and transcription downstream of endothelin type A receptors[J]. Cell Signal,2017,36:240-254.
[16] Javadov S, Jang S, Agostini B. Crosstalk between mitogen-activated protein kinases and mitochondria in cardiac diseases:therapeutic perspectives[J].Pharmacol Ther,2014,144(2):202-225.
[17] WANG S,DING L,JI H,et al. The role of p38 MAPK in the development of diabetic cardiomyopathy[J]. Int J Mol Sci,2016,17(7):1037.
[18]杨晋静,王敬萍,张月安,等. miRNA在心室重塑中的调控作用[J].中华医学杂志,2015,95(29):2408-2410.
[19] DUAN L, XIONG X, LIU Y, et al. miRNA-1:functional roles and dysregulation in heart disease[J].Mol Biosyst,2014,10(11):2775-2782.
[20] Karakikes I,Chaanine A H,Kang S,et al. Therapeutic cardiac-targeted delivery of miR-1 reverses pressure overload-induced cardiac hypertrophy and attenuates pathological remodeling[J]. J Am Heart Assoc,2013,2(2):e000078.
[21] Sucharov C C,Kao D P,Port J D,et al. Myocardial microRNAs associated with reverse remodeling in human heart failure[J]. JCI Insight,2017,2(2):e89169.
[22]魏运荣,卢清显,卢清君. MicroRNA与细胞信号转导通路研究进展[J].生物技术通报,2010,2:28-32.