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

Effect of Leonurine on p38 MAPK Signaling Pathway and miRNA-1 in Cardiomyocyte Hypertrophy Induced by AngⅡ

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.