番石榴叶总黄酮对AngⅡ诱导的乳鼠心肌细胞肥大的抑制作用及机制
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摘要
目的观察番石榴叶总黄酮对血管紧张素Ⅱ(AngⅡ)诱导的乳鼠心肌细胞肥大的抑制作用,并探讨其作用机制。方法体外分离培养乳鼠心肌细胞,将细胞分为模型组和番石榴叶总黄酮低、中、高剂量组。模型组加入AngⅡ0. 1μmol/L诱导心肌细胞肥大,番石榴叶总黄酮低、中、高剂量组先分别加入番石榴叶总黄酮50、100、150μg/m L预处理24 h,然后加入AngⅡ0. 1μmol/L培养24 h。另设正常对照组,加入含10%FBS的DMEM高糖培养基培养24 h,后改以含0. 1%FBS的DMEM进行无血清培养48 h。采用免疫荧光染色法测算细胞面积,Bradford法检测心肌细胞总蛋白含量,Western blotting法检测心肌细胞中血管紧张素Ⅱ1型受体(AT1R)、蛋白激酶C(PKC)蛋白表达。结果与正常对照组比较,模型组和番石榴叶总黄酮低、中、高剂量组心肌细胞面积均增加;与模型组比较,番石榴叶总黄酮低、中、高剂量组心肌细胞面积缩小,其中高剂量组小于低、中剂量组(P均<0. 01)。与对照组比较,模型组心肌细胞内总蛋白含量升高(P <0. 05);与模型组比较,番石榴叶总黄酮各剂量组总蛋白含量均减少,其中高剂量组少于中、低剂量组(P均<0. 05)。与对照组比较,模型组心肌细胞中AT1R、PKC蛋白表达升高(P均<0. 01)。结论 AngⅡ可诱导乳鼠心肌细胞肥大,番石榴叶总黄酮预处理可抑制心肌细胞体积增大、总蛋白合成,从而抑制心肌肥厚;该机制与调控AT1R-PKC通路有关。
Objective To investigate the inhibitory effect and mechanism of total flavones from Psidium guajava leaves on cardiomyocyte hypertrophy induced by angiotensin Ⅱ( AngⅡ) in vivo of neonatal mice. Methods The cardiomyocytes isolated from neonatal Wistar rats were cultured in vitro and divided into the model group and low-,medium-and high-dose groups of total flavonoids from Psidium guajava leaves,meanwhile,we set up the normal control group. AngⅡ 0. 1 μmol/L was added to the model group to induce cardiomyocyte hypertrophy. The cells in the low-,medium-and high-dose groups of total flavonoids from Psidium guava leaves were first pretreated with 50,100,and 150 μg/m L total flavonoids from Psidium guajava leaves for 24 h,and then cultured with AngⅡ 0. 1 μmol/L for 24 h. The cells in the normal control group were first cultured in DMEM high sugar medium containing 10% FBS for 24 h,then received the serum-free culture with DMEM containing 0. 1% FBS for 48 h. Cell area was measured by immunofluorescence staining. The total protein content of myocardial cells of each group was detected with Bradford method. The expression of angiotensin Ⅱ type 1 receptor( AT1 R)and protein kinase C( PKC) was detected by Western blotting. Results Compared with the normal control group,the area of myocardial cells in the model group and low-,medium-and high-dose groups increased; compared with the model group,the area of myocardial cells in the low-,medium-and high-dose groups decreased; and the area of myocardial cells in the high-dose group was smaller than that in the low-and medium-dose group( all P < 0. 01). Compared with the control group,the total protein content in the myocardial cells of the model group increased( P < 0. 01). Compared with the model group,the total protein content of guava leaf flavonoids in each dose group decreased,and the high-dose group was less than the medium-and low-dose groups( all P < 0. 05). Compared with the normal control group,the expression of AT1 R and PKC of the model group increased( both P < 0. 01). Compared with the model group,the expression of AT1 R and PKC in different dosage groups decreased( all P < 0. 01). Conclusion AngⅡ can induce the cardiomyocyte hypertrophy in the neonatal mice,and pretreatment with total flavonoids from Psidium guajava leaves can inhibit the increase of cell size and protein synthesis,and thus inhibit cardiomyocyte hypertrophy,which is related to the regulation of AT1 R-PKC pathway.
Objective To investigate the inhibitory effect and mechanism of total flavones from Psidium guajava leaves on cardiomyocyte hypertrophy induced by angiotensin Ⅱ( AngⅡ) in vivo of neonatal mice. Methods The cardiomyocytes isolated from neonatal Wistar rats were cultured in vitro and divided into the model group and low-,medium-and high-dose groups of total flavonoids from Psidium guajava leaves,meanwhile,we set up the normal control group. AngⅡ 0. 1 μmol/L was added to the model group to induce cardiomyocyte hypertrophy. The cells in the low-,medium-and high-dose groups of total flavonoids from Psidium guava leaves were first pretreated with 50,100,and 150 μg/m L total flavonoids from Psidium guajava leaves for 24 h,and then cultured with AngⅡ 0. 1 μmol/L for 24 h. The cells in the normal control group were first cultured in DMEM high sugar medium containing 10% FBS for 24 h,then received the serum-free culture with DMEM containing 0. 1% FBS for 48 h. Cell area was measured by immunofluorescence staining. The total protein content of myocardial cells of each group was detected with Bradford method. The expression of angiotensin Ⅱ type 1 receptor( AT1 R)and protein kinase C( PKC) was detected by Western blotting. Results Compared with the normal control group,the area of myocardial cells in the model group and low-,medium-and high-dose groups increased; compared with the model group,the area of myocardial cells in the low-,medium-and high-dose groups decreased; and the area of myocardial cells in the high-dose group was smaller than that in the low-and medium-dose group( all P < 0. 01). Compared with the control group,the total protein content in the myocardial cells of the model group increased( P < 0. 01). Compared with the model group,the total protein content of guava leaf flavonoids in each dose group decreased,and the high-dose group was less than the medium-and low-dose groups( all P < 0. 05). Compared with the normal control group,the expression of AT1 R and PKC of the model group increased( both P < 0. 01). Compared with the model group,the expression of AT1 R and PKC in different dosage groups decreased( all P < 0. 01). Conclusion AngⅡ can induce the cardiomyocyte hypertrophy in the neonatal mice,and pretreatment with total flavonoids from Psidium guajava leaves can inhibit the increase of cell size and protein synthesis,and thus inhibit cardiomyocyte hypertrophy,which is related to the regulation of AT1 R-PKC pathway.
引文
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[17] Liu X,Wang Y,Zhang H,et al. Different protein kinase C isoenzymes mediate inhibition of cardiac rapidly actvating delayed rectifier K+current by different G-protein coupled receptors[J].Br J Pharmacol,2017,174(23):4464-4477.
[18]Pakhomov N,Pustovit K,Potekhina V,et al. Negative inotropic effects of diadenosine tetraphosphate are mediated by protein kinase C and phosphodiesterases stimulation in the rat heart[J]. Eur J Pharmacol,2018,820:97-105.
[2]Tham YK,Berndrdo BC,Ooi JY,et al. Pathophysiology of cardiac hypertrophy and heart failure:signaling pathways and novel therapeutic targets[J]. Arch Toxicol,2015,89(9):1401-1438.
[3]Billet S,Aguilar F,Baudry C,et al. Role of the angiotensinⅡAT1 receptor activation in cardiovascular diseases[J]. Kidney Int,2008,74(11):1379-1384.
[4]Sionis A,Sionis Green A,Manito Lorite N,et al. Comments on the 2016 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure[J]. Rev Esp Cardiol(Engl Ed),2016,69(12):1119-1125.
[5]李甜甜,王东明.血管紧张素Ⅱ致心肌细胞肥大作用研究进展[J].汕头大学医学院学报,2011,24(3):185-188.
[6]郑海燕,王晓杰.黄酮类化合物药理作用的探讨[J].中国生化药物杂志,2017,37(5):441-443.
[7]周文婷,王雪飞,邬利娅·伊明,等.榅桲总黄酮对自发性高血压大鼠心肌肥厚的抑制作用及机制研究[J].中国药理学通报,2015,31(11):1540-1546.
[8]赵永青,田伟,彭海平,等.水杉总黄酮对胰岛素样生长因子-Ⅰ诱导的乳鼠心肌细胞肥大的抑制作用[J].中国实验方剂学杂志,2006,12(9):38-40.
[9]陈金灶,郭兰,韩淑英,等.荞麦花叶总黄酮对压力超负荷大鼠心肌肥厚的影响[J].中药药理与临床,2013,29(5):67-69.
[10]吴剑,邹云增. ACE/AngⅡ/AT1轴与ACE2/Ang(1-7)/Mas轴的平衡失调在心血管病中的作用[J].中国医学前沿杂志(电子版),2013,5(6):38-42.
[11]蔡荣林,胡玲,申国明,等.电针预处理对急性心肌缺血再灌注损伤大鼠心肌组织水通道蛋白1表达及蛋白激酶C活性的影响[J].中国针灸,2017,37(2):157-161.
[12] Ferreira JC,Brum PC,Mochly-Rosen D.βⅡPKC andεPKC isozymes as potential pharmacological targets in cardiac hypertrophy and heartfailure[J]. J Mol Cell Cardiol,2011,51(4):479-484.
[13]Ringvold HC,Khalil RA. Protein kinase C as regulator of vascular smooth muscle function and potential target in vascular disorders[J]. Adv Pharmacol,2017,78:203-301.
[14]Passer D,Vrugt AV,Atmanli A,et al. Atypical protein kinase C dependent polarized cell division is required for myocardial trabeculation[J]. Cell Rep,2016,14(7):1662-1672.
[15]邓强,龙鼎新.蛋白激酶C研究进展[J].生命的化学,2018,38(3):433-437.
[16]姜勇,罗深秋.细胞信号转导的分子基础与功能调控[M].北京:科学出版社,2005:59.
[17] Liu X,Wang Y,Zhang H,et al. Different protein kinase C isoenzymes mediate inhibition of cardiac rapidly actvating delayed rectifier K+current by different G-protein coupled receptors[J].Br J Pharmacol,2017,174(23):4464-4477.
[18]Pakhomov N,Pustovit K,Potekhina V,et al. Negative inotropic effects of diadenosine tetraphosphate are mediated by protein kinase C and phosphodiesterases stimulation in the rat heart[J]. Eur J Pharmacol,2018,820:97-105.