Background
Shensong Yangxin Capsule (SSYX), traditional Chinese medicine, has been used to treat arrhythmias, angina, cardiac remodeling, cardiac fibrosis, and so on, but its effect on cardiac energy metabolism is still not clear. The
Conclusions
SSYX could increase myocardial energy metabolism in AngII-induced cardiac hypertrophy. Therefore, SSYX might be considered to be an alternative therapeutic remedy for myocardial hypertrophy. 参松养心胶囊在血管紧张度Ⅱ诱导的心肌肥大中对心肌细胞能量代谢的影响摘要背景: 作为中国的传统中药，参松养心胶囊（Shensong Yangxin Capsule, SSYX）可以用来治疗心律失常、心绞痛、心肌重构、心肌纤维化等等，但是它对心脏能量代谢的影响还尚不明确，本研究的目的主要是探究参松养心胶囊在血管紧张度 (Angiotensin）Ⅱ诱导的心肌肥大过程中对心肌细胞能量代谢的影响。 方法: 我们用2 μl （10-6mol/L） Ang(Angiotensin) II诱导Sprague-Dawley (SD)大鼠的乳鼠心肌细胞（Neonatal rat cardiomyocytes ，NRCMs）48小时，用心肌细胞的α-actinin染色观察细胞体积大小，用实时定量聚合酶链反应（Real-time Polymerase chain Reaction , PCR）检测心肌肥厚标记物BNP（Brain natriuretic peptide）mRNA表达以判断心肌细胞肥大模型构建是否成功。然后， 用1 μl浓度梯度为0.25, 0.5, and 1.0 μg/ml的参松养心胶囊溶液处理细胞24小时。同时，为了探究参松养心胶囊对心肌细胞能量代谢影响的时间效应，我们用0.5 μg/ml参松养心胶囊溶液分别作用细胞0，6，12，24和48小时。用线粒体Mito-Red Tracker探针标记心肌细胞中的线粒体并用共聚焦显微镜观察线粒体密度，用PCR检测线粒体生物起源相关因子-PGC-1α（peroxisome proliferator-activated receptor coactivator-1 alpha），能量平衡关键因子-AMPK (AMP-activated protein kinase)，脂肪酸代谢因子-CPT-1（carnitine acyltransferase enzyme-1），葡萄糖氧化关键因子-GLUT-4（glucose transporter protein 4）的mRNA表达。 结果: 随着参松养心胶囊溶液药物浓度的增加，SSYX处理组的线粒体密度逐渐增多(0.25 μg/ml, 18.3300 ± 0.8895 vs. 24.4900 ± 0.9041, t = 10.240, P < 0.0001; 0.5 μg/ml, 18.3300 ± 0.8895 vs. 25.9800 ± 0.8187, t = 12.710, P < 0.0001; 1.0 μg/ml, 18.3300 ± 0.8895 vs. 24.2900 ± 1.3120, t = 9.902, P < 0.0001; 每组 n均等于5)；而且，SSYX也可以促进AngII诱导心肌细胞肥大过程中能量代谢相关因子-PGC-1α (0.25 μg/ml, 0.8892 ± 0.0848 vs. 1.0970 ± 0.0994, t = 4.319, P = 0.0013; 0.5 μg/ml, 0.8892 ± 0.0848 vs. 1.2330 ± 0.0564, t = 7.150, P < 0.0001; 1.0 μg/ml, 0.8892 ± 0.0848 vs. 1.1640 ± 0.0755, t = 5.720, P < 0.0001; 每组 n均等于5), AMPK (0.25 μg/ml, 0.8872 ± 0.0779 vs. 1.1500 ± 0.0507, t = 7.239,P < 0.0001; 0.5 μg/ml, 0.8872 ± 0.0779 vs. 1.228 ± 0.0623, t = 9.379,P < 0.0001; 1.0 μg/ml, 0.8872 ± 0.0779 vs. 1.3020 ± 0.0450, t = 11.400; ,P < 0.0001; n均等于5), CPT-1 (1.0 μg/ml, 0.7348 ± 0.0594 vs. 0.9880 ± 0.0851, t = 4.994, P = 0.0007, n = 5) 和GLUT-4 (0.5 μg/ml, 1.5640 ± 0.0599vs. 1.7720 ± 0.0660, t = 3.783, P = 0.0117; 1.0 μg/ml, 1.5640 ± 0.0599 vs. 2.0490 ± 0.1280, t = 8.808, P < 0.0001; 每组 n均等于5) 的mRNA和蛋白质表达，并且随着SSYX药物浓度的增加，这种作用效果越发显著；当用0.5 μg/ml相同剂量的参松养心胶囊溶液分别处理细胞0，6，12，24和48小时后，AMPK (6 h, 14.6100 ± 0.6205 vs. 16.5200 ± 0.7450, t = 3.456, P = 0.0250; 12 h, 14.6100 ± 0.6205 vs. 18.3200 ± 0.9965, t = 6.720, P < 0.0001; 24 h, 14.6100 ± 0.6205 vs. 21.8800 ± 0.8208, t = 13.160, P < 0.0001; 48 h, 14.6100 ± 0.6205 vs. 23.7400 ± 1.0970, t = 16.530, P < 0.0001; 每组 n均等于5), PGC-1α (12 h, 11.4700 ± 0.7252 vs. 16.9000 ± 1.0150, t = 7.910; 24 h, 11.4700 ± 0.7252 vs. 20.8800 ± 1.234, t = 13.710, P < 0.0001; 48 h, 11.4700 ± 0.7252 vs. 22.0300 ± 1.4180, t = 15.390, P < 0.0001; n均等于5), CPT-1 (24 h, 15.1600 ± 1.0960 vs. 18.5800 ± 0.9049, t = 6.048, P < 0.0001, n = 5), GLUT-4 (6 h, 10.2100 ± 0.9485 vs. 12.9700 ± 0.8221, t = 4.763, P = 0.0012; 12 h, 10.2100 ± 0.9485 vs. 16.9100 ± 0.8481, t = 11.590, P < 0.0001; 24 h, 10.2100 ± 0.9485 vs. 19.0900 ± 0.9797, t = 15.360, P < 0.0001; 48 h, 10.2100 ± 0.9485 vs. 14.1900 ± 0.9611, t = 6.877, P < 0.0001; 每组 n均等于5)的mRNA和蛋白质表达均随着时间的延长而逐渐增多。 结论: 参松养心胶囊可以促进血管紧张度Ⅱ诱导的心肌肥大过程中心肌细胞的能量代谢，这可能成为治疗心肌肥大的新的治疗方法。.
Methods
We used 2 μl (10-6 mol/L) AngII to treat neonatal rat cardiomyocytes (NRCMs) for 48 h. Myocardial α-actinin staining showed that the myocardial cell volume increased. Expression of the cardiac hypertrophic marker-brain natriuretic peptide (BNP) messenger RNA (mRNA) also increased by real-time polymerase chain reaction (PCR). Therefore, it can be assumed that the model of hypertrophic cardiomyocytes was successfully constructed. Then, NRCMs were treated with 1 μl of different concentrations of SSYX (0.25, 0.5, and 1.0 μg/ml) for another 24 h. To explore the time-depend effect of SSYX on energy metabolism, 0.5 μg/ml SSYX was added into cells for 0, 6, 12, 24, and 48 h. Mitochondria was assessed by MitoTracker staining and confocal microscopy. mRNA and protein expression of mitochondrial biogenesis-related genes - Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), energy balance key factor - adenosine monophosphate-activated protein kinase (AMPK), fatty acids oxidation factor - carnitine palmitoyltransferase-1 (CPT-1), and glucose oxidation factor - glucose transporter- 4 (GLUT-4) were measured by PCR and Western blotting analysis.
Results
With the increase in the concentration of SSYX (from 0.25 to 1.0 μg/ml), an increased mitochondrial density in AngII-induced cardiomyocytes was found compared to that of those treated with AngII only (0.25 μg/ml, 18.3300 ± 0.8895 vs. 24.4900 ± 0.9041, t = 10.240, P < 0.0001; 0.5 μg/ml, 18.3300 ± 0.8895 vs. 25.9800 ± 0.8187, t = 12.710, P < 0.0001; and 1.0 μg/ml, 18.3300 ± 0.8895 vs. 24.2900 ± 1.3120, t = 9.902, P < 0.0001; n = 5 per dosage group). SSYX also increased the mRNA and protein expression of PGC-1α (0.25 μg/ml, 0.8892 ± 0.0848 vs. 1.0970 ± 0.0994, t = 4.319, P = 0.0013; 0.5 μg/ml, 0.8892 ± 0.0848 vs. 1.2330 ± 0.0564, t = 7.150, P < 0.0001; and 1.0 μg/ml, 0.8892 ± 0.0848 vs. 1.1640 ± 0.0755, t = 5.720, P < 0.0001; n = 5 per dosage group), AMPK (0.25 μg/ml, 0.8872 ± 0.0779 vs. 1.1500 ± 0.0507, t = 7.239, P < 0.0001; 0.5 μg/ml, 0.8872 ± 0.0779 vs. 1.2280 ± 0.0623, t = 9.379, P < 0.0001; and 1.0 μg/ml, 0.8872 ± 0.0779 vs. 1.3020 ± 0.0450, t = 11.400, P < 0.0001; n = 5 per dosage group), CPT-1 (1.0 μg/ml, 0.7348 ± 0.0594 vs. 0.9880 ± 0.0851, t = 4.994, P = 0.0007, n = 5), and GLUT-4 (0.5 μg/ml, 1.5640 ± 0.0599 vs. 1.7720 ± 0.0660, t = 3.783, P = 0.0117; 1.0 μg/ml, 1.5640 ± 0.0599 vs. 2.0490 ± 0.1280, t = 8.808, P < 0.0001; n = 5 per dosage group). The effect became more obvious with the increasing concentration of SSYX. When 0.5 μg/ml SSYX was added into cells for 0, 6, 12, 24, and 48 h, the expression of AMPK (6 h, 14.6100 ± 0.6205 vs. 16.5200 ± 0.7450, t = 3.456, P = 0.0250; 12 h, 14.6100 ± 0.6205 vs. 18.3200 ± 0.9965, t = 6.720, P < 0.0001; 24 h, 14.6100 ± 0.6205 vs. 21.8800 ± 0.8208, t = 13.160, P < 0.0001; and 48 h, 14.6100 ± 0.6205 vs. 23.7400 ± 1.0970, t = 16.530, P < 0.0001; n = 5 per dosage group), PGC-1α (12 h, 11.4700 ± 0.7252 vs. 16.9000 ± 1.0150, t = 7.910, P < 0.0001; 24 h, 11.4700 ± 0.7252 vs. 20.8800 ± 1.2340, t = 13.710, P < 0.0001; and 48 h, 11.4700 ± 0.7252 vs. 22.0300 ± 1.4180, t = 15.390; n = 5 per dosage group), CPT-1 (24 h, 15.1600 ± 1.0960 vs. 18.5800 ± 0.9049, t = 6.048, P < 0.0001, n = 5), and GLUT-4 (6 h, 10.2100 ± 0.9485 vs. 12.9700 ± 0.8221, t = 4.763, P = 0.0012; 12 h, 10.2100 ± 0.9485 vs. 16.9100 ± 0.8481, t = 11.590, P < 0.0001; 24 h, 10.2100 ± 0.9485 vs. 19.0900 ± 0.9797, t = 15.360, P < 0.0001; and 48 h, 10.2100 ± 0.9485 vs. 14.1900 ± 0.9611, t = 6.877, P < 0.0001; n = 5 per dosage group) mRNA and protein increased gradually with the prolongation of drug action time. Conclusions: SSYX could increase myocardial energy metabolism in AngII-induced cardiac hypertrophy. Therefore, SSYX might be considered to be an alternative therapeutic remedy for myocardial hypertrophy. 参松养心胶囊在血管紧张度Ⅱ诱导的心肌肥大中对心肌细胞能量代谢的影响摘要背景: 作为中国的传统中药，参松养心胶囊（Shensong Yangxin Capsule, SSYX）可以用来治疗心律失常、心绞痛、心肌重构、心肌纤维化等等，但是它对心脏能量代谢的影响还尚不明确，本研究的目的主要是探究参松养心胶囊在血管紧张度 (Angiotensin）Ⅱ诱导的心肌肥大过程中对心肌细胞能量代谢的影响。 方法: 我们用2 μl （10-6mol/L） Ang(Angiotensin) II诱导Sprague-Dawley (SD)大鼠的乳鼠心肌细胞（Neonatal rat cardiomyocytes ，NRCMs）48小时，用心肌细胞的α-actinin染色观察细胞体积大小，用实时定量聚合酶链反应（Real-time Polymerase chain Reaction , PCR）检测心肌肥厚标记物BNP（Brain natriuretic peptide）mRNA表达以判断心肌细胞肥大模型构建是否成功。然后， 用1 μl浓度梯度为0.25, 0.5, and 1.0 μg/ml的参松养心胶囊溶液处理细胞24小时。同时，为了探究参松养心胶囊对心肌细胞能量代谢影响的时间效应，我们用0.5 μg/ml参松养心胶囊溶液分别作用细胞0，6，12，24和48小时。用线粒体Mito-Red Tracker探针标记心肌细胞中的线粒体并用共聚焦显微镜观察线粒体密度，用PCR检测线粒体生物起源相关因子-PGC-1α（peroxisome proliferator-activated receptor coactivator-1 alpha），能量平衡关键因子-AMPK (AMP-activated protein kinase)，脂肪酸代谢因子-CPT-1（carnitine acyltransferase enzyme-1），葡萄糖氧化关键因子-GLUT-4（glucose transporter protein 4）的mRNA表达。 结果: 随着参松养心胶囊溶液药物浓度的增加，SSYX处理组的线粒体密度逐渐增多(0.25 μg/ml, 18.3300 ± 0.8895 vs. 24.4900 ± 0.9041, t = 10.240, P < 0.0001; 0.5 μg/ml, 18.3300 ± 0.8895 vs. 25.9800 ± 0.8187, t = 12.710, P < 0.0001; 1.0 μg/ml, 18.3300 ± 0.8895 vs. 24.2900 ± 1.3120, t = 9.902, P < 0.0001; 每组 n均等于5)；而且，SSYX也可以促进AngII诱导心肌细胞肥大过程中能量代谢相关因子-PGC-1α (0.25 μg/ml, 0.8892 ± 0.0848 vs. 1.0970 ± 0.0994, t = 4.319, P = 0.0013; 0.5 μg/ml, 0.8892 ± 0.0848 vs. 1.2330 ± 0.0564, t = 7.150, P < 0.0001; 1.0 μg/ml, 0.8892 ± 0.0848 vs. 1.1640 ± 0.0755, t = 5.720, P < 0.0001; 每组 n均等于5), AMPK (0.25 μg/ml, 0.8872 ± 0.0779 vs. 1.1500 ± 0.0507, t = 7.239,P < 0.0001; 0.5 μg/ml, 0.8872 ± 0.0779 vs. 1.228 ± 0.0623, t = 9.379,P < 0.0001; 1.0 μg/ml, 0.8872 ± 0.0779 vs. 1.3020 ± 0.0450, t = 11.400; ,P < 0.0001; n均等于5), CPT-1 (1.0 μg/ml, 0.7348 ± 0.0594 vs. 0.9880 ± 0.0851, t = 4.994, P = 0.0007, n = 5) 和GLUT-4 (0.5 μg/ml, 1.5640 ± 0.0599vs. 1.7720 ± 0.0660, t = 3.783, P = 0.0117; 1.0 μg/ml, 1.5640 ± 0.0599 vs. 2.0490 ± 0.1280, t = 8.808, P < 0.0001; 每组 n均等于5) 的mRNA和蛋白质表达，并且随着SSYX药物浓度的增加，这种作用效果越发显著；当用0.5 μg/ml相同剂量的参松养心胶囊溶液分别处理细胞0，6，12，24和48小时后，AMPK (6 h, 14.6100 ± 0.6205 vs. 16.5200 ± 0.7450, t = 3.456, P = 0.0250; 12 h, 14.6100 ± 0.6205 vs. 18.3200 ± 0.9965, t = 6.720, P < 0.0001; 24 h, 14.6100 ± 0.6205 vs. 21.8800 ± 0.8208, t = 13.160, P < 0.0001; 48 h, 14.6100 ± 0.6205 vs. 23.7400 ± 1.0970, t = 16.530, P < 0.0001; 每组 n均等于5), PGC-1α (12 h, 11.4700 ± 0.7252 vs. 16.9000 ± 1.0150, t = 7.910; 24 h, 11.4700 ± 0.7252 vs. 20.8800 ± 1.234, t = 13.710, P < 0.0001; 48 h, 11.4700 ± 0.7252 vs. 22.0300 ± 1.4180, t = 15.390, P < 0.0001; n均等于5), CPT-1 (24 h, 15.1600 ± 1.0960 vs. 18.5800 ± 0.9049, t = 6.048, P < 0.0001, n = 5), GLUT-4 (6 h, 10.2100 ± 0.9485 vs. 12.9700 ± 0.8221, t = 4.763, P = 0.0012; 12 h, 10.2100 ± 0.9485 vs. 16.9100 ± 0.8481, t = 11.590, P < 0.0001; 24 h, 10.2100 ± 0.9485 vs. 19.0900 ± 0.9797, t = 15.360, P < 0.0001; 48 h, 10.2100 ± 0.9485 vs. 14.1900 ± 0.9611, t = 6.877, P < 0.0001; 每组 n均等于5)的mRNA和蛋白质表达均随着时间的延长而逐渐增多。 结论: 参松养心胶囊可以促进血管紧张度Ⅱ诱导的心肌肥大过程中心肌细胞的能量代谢，这可能成为治疗心肌肥大的新的治疗方法。.