[1]陈德秀,李家富,冯健.激活TGR5通过抑制CaN/NAFT3减轻 ET-1诱导的心肌细胞肥大[J].第三军医大学学报,2019,41(10):939-946.
 CHEN Dexiu,LI Jiafu,FENG Jian.G protein-coupled bile acid receptor 1 activation attenuates endothelin-1-induced rat cardiomyocyte hypertrophy by inhibiting CaN/NAFT3 pathway[J].J Third Mil Med Univ,2019,41(10):939-946.
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激活TGR5通过抑制CaN/NAFT3减轻 ET-1诱导的心肌细胞肥大(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第10期
页码:
939-946
栏目:
基础医学
出版日期:
2019-05-30

文章信息/Info

Title:
G protein-coupled bile acid receptor 1 activation attenuates endothelin-1-induced rat cardiomyocyte hypertrophy by inhibiting CaN/NAFT3 pathway
作者:
陈德秀李家富冯健
西南医科大学附属医院心血管内科,医学电生理学教育部重点实验室
Author(s):
CHEN Dexiu LI Jiafu FENG Jian

Department of Cardiology, Key Laboratory of Medical Electrophysiology of Ministry of Education, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan Province, 646000, China

关键词:
G蛋白偶联胆汁酸受体1 内皮素-1 心肌细胞肥大 钙调神经磷酸酶 活化T细胞核因子3
Keywords:
G protein-coupled bile acid receptor 1 endothelin-1 cardiomyocyte hypertrophy calcineurin activated T cell nuclear factor 3
分类号:
R322.1;R392.3;R542.2
文献标志码:
A
摘要:

目的 观察G蛋白偶联胆汁酸受体1(G protein-coupled bile acid receptor 1,TGR5)受体激活后对内皮素-1(endothelin-1,ET-1)诱导心肌细胞肥大的作用及其机制的探讨。方法 原代培养乳鼠心肌细胞,分为空白对照组和ET-1组,ET-1组的浓度分别为10-6、10-7、10-8 mmol/L,分别培养12、24、36、48 h,分别检测各组心肌细胞表面积和总蛋白浓度,建立心肌肥大细胞模型。将心肌细胞分为对照组、ET-1组、ET-1+INT-777(TGR5受体激动剂)组、ET-1+ INT-777+TGR5 siRNA(干扰TGR5表达)组、ET-1+ INT-777+TGR5 siRNA-NC(空病毒)组。采用图像分析系统测定心肌细胞表面积,BCA法测定细胞总蛋白量,RT-PCR检测TGR5、钙调神经磷酸酶(calcineurin,CaN)的mRNA,Western blot方法检测心房尿钠因子(atrial natriuretic factor,ANF)、β-肌球蛋白重链(β-myosin heavy chain,β-MHC)、TGR5、CaN、活化T细胞核因子3(activated T cell nuclear factor 3,NFAT3)的蛋白表达变化。结果 48 h内,ET-1诱导心肌细胞肥大在10-8 mmol/L~10-6 mmol/L浓度范围内成明显浓度依赖性和时间依赖性(P<0.05),其中ET-1 10-6 mmol/L培养48 h心肌细胞表面积为(3 624.7 ±71.60)um2,总蛋白量(51.810±1.47)μg,显著高于对照组表面积(1 560.8±3 188.94)um2和总蛋白(37.827±0.47)μg(P<0.05)。与对照组相比,ET-1组心肌细胞表面积、总蛋白、ANF及β-MHC表达增加(P<0.05),CaN及NFAT3的表达增加(P<0.05)。与ET-1组心肌细胞表面积(4 167.59±271.11)um2、总蛋白(57.765±0.553)μg、ANF/GAPDH(0.587±0.012)、β-MHC/GAPDH(0.422±0.016)、CaN/GAPDH(0.529±0.006)及NFAT3/Histone3(0.811±0.014)相比,给予TGR5受体激动剂组心肌细胞表面积(2 421.69±123.61)um2、总蛋白(42.714±0.542)μg、ANF/GAPDH(0.229±0.011)、β-MHC/GAPDH(0.230±0.018)、CaN/GAPDH(0.247±0.008)及NFAT3/Histone3(0.407±0.008)的表达表达增加受到抑制(P<0.05)。予以siTGR5转染细胞后,部分消除了上述的抑制作用(P<0.05)。结论 激活TGR5可改善ET-1诱导心肌细胞肥大,其机制可能部分与抑制CaN/NFAT3信号通路有关。

Abstract:

Objective To investigate the inhibitory effect of G protein-coupled bile acid receptor 1 (TGR5) on endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy and explore the mechanism. Methods Primary cultured neonatal rat cardiomyocytes were exposed to ET-1 at 10-6, 10-7, or 10-8 mmol/L for 12, 24, 36, or 48 h, and the surface area and total protein concentration of the cells were measured to verify the establishment of cardiac mast cell models. In the subsequent experiment, rat cardiomyocytes were treated with ET-1, ET-1+INT-777 (a TGR5 receptor agonist), ET-1+INT-777+TGR5 siRNA, or ET-1+ INT-777+TGR5 siRNA-NC (an empty viral vector). The surface area of the cardiomyocytes was measured using an image analysis system, and the total protein content in the cells was determined with BCA method; The mRNA levels of TGR5 and calcineurin (CaN) were detected using RT-PCR, and the protein levels of atrial natriuretic factor (ANF), β-myosin heavy chain (β-MHC), TGR5, CaN and activated T cell nuclear factor 3 (NFAT3) were determined using Western blotting. Results Within 48 h, ET-1 concentration- and time-dependently induced cardiomyocyte hypertrophy in the concentration range of 1×10-8  to 1×10-6 mmol/L (P<0.05). ET-1 treatment resulted in significantly increased surface area (3 624.7±71.60 μm2) and total protein content (51.810±1.47 μg) in the cardiomyocytes at 48 h as compared with the those in the control cells (1 560.8±3 188.94 μm2 and 37.827±0.47 μg, respectively; P<0.05). ET-1 exposure of the cells caused also significantly increased expression of ANF, β-MHC, CaN and NFAT3 (P<0.05). Compared with the cells treated with ET-1 alone, the cells with both ET-1 and INT-777 treatment showed significantly reduced total cell surface area, total protein content, and protein expressions of ANF, β-MHC, CaN and NFAT3 (P<0.05). Transfection with siTGR5 partially eliminated the inhibitory effects of INT-777 on ET-1-induced cardiomyocyte hypertrophy (P<0.05). Conclusion Activation of TGR5 may ameliorate ET-1-induced cardiomyocyte hypertrophy, the mechanism of which is related to the inhibition of CaN/NFAT3 signaling pathway.
 

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更新日期/Last Update: 2019-05-24