[1]刘亚坤,吕风华,司澳洋,等.骨形成蛋白4诱导大鼠H9c2心肌细胞肥大的作用机制[J].新乡医学院学报,2018,35(4):260-265.[doi:10.7683/xxyxyxb.2018.04.002]
　LIU Ya-kun,LYU Feng-hua,SI Ao-yang,et al.Mechanisms of the bone morphogenetic protein 4 induced cardiomyocytes hypertrophy of rats[J].Journal of Xinxiang Medical University,2018,35(4):260-265.[doi:10.7683/xxyxyxb.2018.04.002]
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骨形成蛋白4诱导大鼠H9c2心肌细胞肥大的作用机制
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
35
期数:
2018年4
页码:
260-265
栏目:
基础研究
出版日期:
2018-04-05

文章信息/Info

Title:
Mechanisms of the bone morphogenetic protein 4 induced cardiomyocytes hypertrophy of rats
作者:
刘亚坤1; 吕风华1; 司澳洋1; 王　卓1; 吴　林2
（1.新乡医学院第一附属医院心内科，河南　卫辉　453100；2.河南大学淮河医院心内科，河南　开封　475000）
Author(s):
LIU Ya-kun1; LYU Feng-hua1; SI Ao-yang1; WANG Zhuo1; WU Lin2
（1.Department of Cardiology，the First Affiliated Hospital of Xinxiang Medical University，Weihui 453100，Henan Province，China；2.Department of Cardiology，Huaihe Hospital of Henan University，Kaifeng 475000，Henan Province，China）
关键词:
骨形成蛋白4; 心肌肥大; 磷脂酰肌醇3激酶-蛋白激酶B信号通路; 自噬
Keywords:
bone morphogenetic protein-4; cardiac hypertrophy; phosphatidylinositol-3-kinase/protein kinase B signal pathway; autophagy
分类号:
R542.2
DOI:
10.7683/xxyxyxb.2018.04.002
文献标志码:
A
摘要:
目的　探讨骨形成蛋白4（BMP4）诱导大鼠H9c2心肌细胞肥大的作用机制。方法　培养大鼠心肌细胞株H9c2，使细胞同步化。实验分2部分，第1部分取同步化的心肌细胞，加50 μg·L-1BMP4 60 μL进行干预，分别在干预后0、1、4、8、12、24 h检测磷酸化蛋白激酶B（P-Akt）蛋白的表达；第2部分将同步化的心肌细胞随机分为对照组、BMP4组、BMP4+LY294002组及BMP4+3MA组。对照组细胞应用体积分数1%胎牛血清培养基培养；BMP4组细胞应用含50 μg·L-1 BMP4的体积分数1%胎牛血清培养基培养；BMP4+LY294002组细胞先应用25 μmol·L-1的LY294002预处理120 min，再以含50 μg·L-1BMP4的体积分数1%胎牛血清培养基培养；BMP4+3MA组细胞先应用10 mmol·L-1的自噬抑制剂3MA预处理120 min，再以含50 μg·L-1BMP4的体积分数1%胎牛血清培养基培养。各组细胞给予BMP4 48 h后采用Image J软件测量细胞表面积。二喹啉甲酸法检测各组心肌细胞内蛋白含量。采用Western blot检测各组细胞给予BMP4 1 h后磷酸化磷脂酰肌醇3（P-PI3K）、P-Akt及自噬微管相关蛋白1轻链3（LC3）蛋白的表达，给予BMP4 48 h后检测脑钠肽（BNP）、α-平滑肌肌动蛋白（α-SMA）的表达。结果　BMP4作用心肌细胞0、1、4、8、12、24 h后P-Akt蛋白相对表达量分别为0.56±0.02、0.92±0.12、0.42±0.10、0.63±0.11、0.64±0.23、0.29±0.08，BMP4作用大鼠心肌细胞1 h后P-Akt蛋白相对表达量均高于其他时间点（P<0.05）。BMP4组大鼠心肌细胞表面积及总蛋白含量高于对照组（P<0.01）；BMP4+LY294002组与对照组大鼠心肌细胞表面积及总蛋白含量比较差异无统计学意义（P>0.05）；BMP4+3MA组大鼠心肌细胞表面积大于对照组（P<0.01），BMP4+3MA组与对照组大鼠心肌细胞总蛋白含量比较差异无统计学意义（P>0.05）；BMP4+LY294002、BMP4+3MA组H9c2大鼠心肌细胞表面积及总蛋白含量均低于BMP4组（P<0.05）；BMP4+LY294002组与BMP4+3MA组大鼠心肌细胞表面积及总蛋白含量比较差异无统计学意义（P>0.05）。BMP4组、BMP4+3MA组大鼠心肌细胞内P-PI3K蛋白表达水平高于对照组，BMP4+LY294002组心肌细胞内P-PI3K蛋白表达水平低于对照组（P<0.01）；BMP4、BMP4+3MA、BMP4+LY294002组大鼠心肌细胞内P-PI3K蛋白表达水平呈逐渐降低趋势，3组大鼠心肌细胞内P-PI3K蛋白表达水平两两比较差异均有统计学意义（P<0.05）。BMP4、BMP4+3MA组大鼠心肌细胞内P-Akt、LC3蛋白表达水平高于对照组（P<0.01），BMP4+LY294002组与对照组大鼠心肌细胞内P-Akt、LC3蛋白表达水平比较差异无统计学意义（P>0.05）；BMP4+3MA、BMP4+LY294002组大鼠心肌细胞内P-Akt、LC3蛋白表达水平低于BMP4组（P<0.05）；BMP4+3MA组与BMP4+LY294002组大鼠心肌细胞内P-Akt、LC3蛋白表达水平比较差异无统计学意义（P>0.05）。BMP4组大鼠心肌细胞内α-SMA蛋白表达水平高于对照组（P<0.01），BMP4+3MA、BMP4+LY294002组与对照组大鼠心肌细胞内α-SMA蛋白表达水平比较差异无统计学意义（P>0.05）；BMP4+3MA、BMP4+LY294002组大鼠心肌细胞内α-SMA蛋白表达水平低于BMP4组（P<0.05）；BMP4+3MA组大鼠心肌细胞内α-SMA蛋白表达水平高于BMP4+LY294002组（P<0.05）。BMP4、BMP4+3MA组大鼠心肌细胞内BNP蛋白表达水平高于对照组（P<0.01）；BMP4+LY294002组与对照组大鼠心肌细胞内BNP蛋白表达水平比较差异无统计学意义（P>0.05）；BMP4+3MA、BMP4+LY294002组大鼠心肌细胞内BNP蛋白表达水平低于BMP4组（P>0.05）。结论　BMP4可能通过激活PI3K-Akt通路增加自噬活性，从而诱导大鼠H9c2心肌细胞肥大。
Abstract:
Objective　To investigate the mechanisms of the bone morphogenetic protein 4 （BMP4）induced cardiomyocytes hypertrophy of rats.Methods　Rat myocardial cell line H9c2 was cultured to synchronize the cells.This experiment was divided into two parts.Part Ⅰ：the synchronized myocardial cells were taken and then the BMP4（50 μg·L-1） 60 μL was added to the cells for intervention.The expression of phosphorylated protein kinase B（P-Akt） protein was detected at 0，1，4，8，12，24 h after intervention.Part Ⅱ：the synchronized myocardial cells were randomly divided into control group，BMP4 group，BMP4+LY294002 group and BMP4+3MA group.The cells in the control group were cultured with volume fraction 1% fetal bovine serum medium；the cells in the BMP4 group were cultured with volume fraction 1% fetal bovine serum medium which contained 50 μg·L-1 BMP4；the cells in the BMP4+LY294002 group were given LY294002（25 μmol·L-1） pretreatment for 120 min，then the cells were cultured with volume fraction 1% fetal bovine serum medium which contained 50 μg·L-1 BMP4；the cells in the BMP4+3MA group were given autophagy inhibitor 3MA pretreatment（10 mmol·L-1） for 120 min，then the cells were cultured with volume fraction 1% fetal bovine serum medium which contained 50 μg·L-1 BMP4.The cell surface area was measured after giving BMP4 for 48 h by Image J software in all groups.The content of protein in myocardial cells in all groups was detected by bicinchoninic acid method.The protein expression of phosphorylated phosphatidylinositol-3-kinase（P-PI3K），P-Akt and microtubule associated protein 1 light chain 3 （LC3） in myocardial cells in all groups were detected at 1h after giving BMP4 by Western blot.The protein expression of brain natriuretic peptide（BNP）and α-smooth muscle actin（α-SMA） in myocardial cells in all groups were detected at 48 h after giving BMP4 by western blot.Results　The expression of P-Akt protein in myocardial cells at 0，1，4，8，12，24 h after giving BMP4 was 0.56±0.02，0.92±0.12，0.42±0.10，0.63±0.11，0.64±0.23，0.29±0.08 respectively；the expression of P-Akt protein in myocardial cells at 1 h after giving BMP4 was significantly higher than that at the other time point（P<0.05）.The surface area and protein content of myocardial cells in BMP4 group were significantly higher than those in the control group（P<0.01）；there was no statistic difference in the surface area and protein content of myocardial cells between the BMP4+LY294002 group and the control group（P>0.05）；the surface area of myocardial cells in the BMP4+3MA group was significantly higher than that in the control group（P<0.01）；there was no statistic difference in the protein content of myocardial cells between the BMP4+3MA group and the control group（P>0.05）.The expression of P-PI3K protein in myocardial cells in the BMP4 group，the BMP4+3MA group was significantly higher than that in the control group（P<0.01）；the expression of P-PI3K protein in myocardial cells in the BMP4+LY294002 group was significantly lower than that in the control group（P<0.01）.The expression of P-PI3K protein in myocardial cells in the BMP4，BMP4+3MA，BMP4+LY294002 group decreased gradually，there was statistic difference in the expression of P-PI3K protein among the BMP4，BMP4+3MA，BMP4+LY294002 groups（P<0.05）.The expression of P-Akt and LC3 protein in myocardial cells in the BMP4 group and the BMP4+3MA group was significantly higher than that in the control group（P<0.01）；there was no statistic difference in the expression of P-Akt and LC3 protein in myocardial cells between the BMP4+LY294002 group and the control group（P>0.05）.The expression of P-Akt and LC3 protein in myocardial cells in the BMP4+3MA group and the BMP4+LY294002 group was significantly lower than that in the BMP4 group（P<0.05）；there was no statistic difference in the expression of P-Akt and LC3 protein in myocardial cells between the BMP4+3MA group and the BMP4+LY294002 group（P>0.05）.The expression of α-SMA protein in myocardial cells in the BMP4 group was significantly higher than that in the control group（P<0.01）；there was no statistic difference in the expression of α-SMA protein in myocardial cells between the BMP4+3MA group，the BMP4+LY294002 group and the control group（P>0.05）；the expression of α-SMA protein in myocardial cells in the BMP4+3MA group and the BMP4+LY294002 group was significantly lower than that in the BMP4 group（P<0.05）；the expression of α-SMA protein in myocardial cells in the BMP4+3MA group was significantly higher than that in the BMP4+LY294002 group（P<0.01）.The expression of BNP protein in myocardial cells in the BMP4 group and the BMP4+3MA group was significantly higher than that in the control group（P<0.01）；there was no statistic difference in the BNP protein expression between the BMP4+LY294002 group and the control group（P>0.05）；the expression of BNP protein in myocardial cells in the BMP4+3MA group and the BMP4+LY294002 group was significantly lower than that in the BMP4 group（P<0.05）；there was no statistic difference in the BNP protein expression between the BMP4+3MA group and the BMP4+LY294002 group（P>0.05）.Conclusion　BMP4 may induce H9c2 cardiomyocyte hypertrophy by activating PI3K-Akt pathway and increasing autophagy activity.

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更新日期/Last Update: 2018-04-05