乌头碱抑制血管紧张素Ⅱ诱导的心肌细胞肥大
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摘要
研究乌头碱(aconitine,AC)对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导H9c2细胞肥大的抑制作用,并探讨其作用机制。通过AngⅡ诱导H9c2细胞肥大建立模型,并分别与不同浓度的乌头碱共同处理,Western blot法检测atrial natriuretic peptide(ANP)、brain natriuretic peptide(BNP)、β-myosin heavy chain(β-MHC)、α-smooth muscle actin(α-SMA)在蛋白水平的表达;实时荧光定量PCR技术(qRT-PCR)检测肥大基因ANP,BNP,β-MHC mRNA的表达。采用激光扫描共聚焦显微镜检测肌原纤维中的重要组成成分F-actin标记的荧光强度。乌头碱可明显逆转AngⅡ所诱导的H9c2细胞总蛋白含量的升高; qRTPCR检测结果显示乌头碱可明显抑制AngⅡ所诱导的ANP,BNP,β-MHC mRNA的上调; Western blot法检测提示乌头碱可明显抑制AngⅡ所诱导的ANP,BNP,β-MHC蛋白的上调;荧光标记检测F-actin的表达水平,乌头碱可以抑制AngⅡ所诱导的F-actin的表达。乌头碱明显下调AngⅡ所诱导的心肌细胞肥大的多项指标,揭示乌头碱可能通过抑制肥大因子的上调来缓解AngⅡ所引起的心肌肥大,这可能是乌头碱发挥治疗作用的机制之一。
This paper was aimed to investigate the inhibitory effect of aconitine(AC) on angiotensin Ⅱ(Ang Ⅱ)-induced H9 c2 cell hypertrophy and explore its mechanism of action. The model of hypertrophy was induced by Ang Ⅱ(1×10-6 mol·L-1),and cardiomyocytes were incubated with different concentrations of AC. Western blot was used to quantify the protein expression levels of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),β-myosin heavy chain(β-MHC),and α-smooth muscle actin(α-SMA). Real-time quantitative PCR(qRT-PCR) was used to quantify the mRNA expression levels of cardiac hypertrophic markers ANP,BNP and β-MHC. In addition,the fluorescence intensity of the F-actin marker,an important component of myofibrils,was detected by using laser confocal microscope. AC could significantly reverse the increase of total protein content in H9 c2 cells induced by Ang Ⅱ; qRT-PCR results showed that AC could significantly inhibit the ANP,BNP and β-MHC mRNA up-regulation induced by AngⅡ. Western blot results showed that AC could significantly inhibit the ANP,BNP and β-MHC protein up-regulation induced by AngⅡ. In addition,F-actin expression induced by Ang Ⅱ could be inhibited by AC,and multiple indicators of cardiomyocyte hypertrophy induced by Ang Ⅱ could be down-regulated,indicating that AC may inhibit cardiac hypertrophy by inhibiting the expression of hypertrophic factors,providing new clues for exploring the cardiovascular protection of AC.
This paper was aimed to investigate the inhibitory effect of aconitine(AC) on angiotensin Ⅱ(Ang Ⅱ)-induced H9 c2 cell hypertrophy and explore its mechanism of action. The model of hypertrophy was induced by Ang Ⅱ(1×10-6 mol·L-1),and cardiomyocytes were incubated with different concentrations of AC. Western blot was used to quantify the protein expression levels of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),β-myosin heavy chain(β-MHC),and α-smooth muscle actin(α-SMA). Real-time quantitative PCR(qRT-PCR) was used to quantify the mRNA expression levels of cardiac hypertrophic markers ANP,BNP and β-MHC. In addition,the fluorescence intensity of the F-actin marker,an important component of myofibrils,was detected by using laser confocal microscope. AC could significantly reverse the increase of total protein content in H9 c2 cells induced by Ang Ⅱ; qRT-PCR results showed that AC could significantly inhibit the ANP,BNP and β-MHC mRNA up-regulation induced by AngⅡ. Western blot results showed that AC could significantly inhibit the ANP,BNP and β-MHC protein up-regulation induced by AngⅡ. In addition,F-actin expression induced by Ang Ⅱ could be inhibited by AC,and multiple indicators of cardiomyocyte hypertrophy induced by Ang Ⅱ could be down-regulated,indicating that AC may inhibit cardiac hypertrophy by inhibiting the expression of hypertrophic factors,providing new clues for exploring the cardiovascular protection of AC.
引文
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[2] Zhao D D,Wang J,Cui Y J,et al. Pharmacological effects of Chinese herb aconite(Fuzi)on cardiovascular system[J]. J Tradit Chin Med,2012,32(3):1.
[3]熊亮,彭成,缪璐琳,等.基于“毒-效整合分析思路”探讨有毒中药附子的物质基础[J].世界中医药,2017,12(11):2568.
[4]陈荣昌,孙桂波,张强,等.附子及其复方中药的药理作用研究进展[J].中草药,2014,45(6):883.
[5]王胜林,董耀荣.附子水煎液对心梗后心力衰竭大鼠心室重构的影响[J].辽宁中医杂志,2010(S1):243.
[6]卢志强,张艳军,庄朋伟,等.附子对急性心力衰竭大鼠血流动力学影响及其机制研究[J].中草药,2015,46(21):3223.
[7] Ayanga B A,Badal S S,Wang Y,et al. Dynamin-related protein 1 deficiency improves mitochondrial fitness and protects against progression of diabetic nephropathy[J]. J Am Soc Nephrol,2016,27(9):2733.
[8]王楚盈,李玉梅,刘畅,等.附子人参有效组分配伍对阿霉素致慢性心衰大鼠血流动力学的影响及其机制研究[J].中药新药与临床药理,2015,26(1):39.
[9]王晓丽,李丽静,李玉梅,等.附子与人参不同配伍对心肌细胞的减毒作用[J].中国实验方剂学杂志,2015,22(11):153.
[10]李芸霞,彭成.基于毒效整合分析的附子药动学、药效学研究[J].世界中医药,2017(11):2579.
[11]孙蓉,严敏,王懿,等.附子水提组分含药血清对大鼠离体心功能影响的“毒-效”关联研究[J].中国药物警戒,2014(10):587.
[12]王丹,贾德贤,李真真,等.草乌的安全性评价与风险控制措施的探讨[J].中国中药杂志,2018,43(15):3093.
[13]谢晓芳,彭成.附子心脏毒效的多维评价和整合分析研究进展[J].世界中医药,2017,12(11):2555.
[14] Liu S,Chen S,Li M,et al. Autophagy activation attenuates angiotensinⅡ-induced cardiac fibrosis[J]. Arch Biochem Biophys,2016,590:37.
[15] Zhang B,Jiang J,Yue Z,et al. Store-operated Ca2+entry(SOCE)contributes to angiotensinⅡ-induced cardiac fibrosis in cardiac fibroblasts[J]. J Pharmacol Sci,2016,132(3):171.
[16] Francis G S,Felker G M,Tang W H W. A test in context:critical evaluation of natriuretic peptide testing in heart failure[J].J Am Coll Cardiol,2016,67(3):330.
[17] Kim H N,Januzzi J L. Natriuretic peptide testing in heart failure[J]. Circulation,2011,123(18):2015.
[18] Sedaghat-Hamedani F,Kayvanpour E,Frankenstein L,et al. Biomarker changes after strenuous exercise can mimic pulmonary embolism and cardiac injury--a Meta analysis of 45 studies[J].Clin Chem,2015,61(10):1246.
[19] Kim K,Chini N,Fairchild D G,et al. Evaluation of cardiac toxicity biomarkers in rats from different laboratories[J]. Toxicol Pathol,2016,44(8):1072.