丹参酮ⅡA对大鼠心肌成纤维细胞增殖的影响
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摘要
目的探讨丹参酮ⅡA对大鼠心肌成纤维细胞增殖的影响。方法培养Wistar大鼠原代心肌成纤维细胞,分为对照组、血管紧张素Ⅱ(AngⅡ)组、丹参酮A组(0.01 mmol/L丹参酮ⅡA)、丹参酮B组(0.1 mmol/L丹参酮ⅡA)、丹参酮C组(1 mmol/L丹参酮ⅡA)。采用四甲基偶氮唑盐比色法测定细胞增殖率;采用酶联免疫吸附试验检测羟脯氨酸含量;采用实时定量聚合酶链式反应(PCR)法检测大鼠心肌成纤维细胞胶原蛋白Ⅰ型、Ⅲ型、基质金属蛋白酶2(MMP-2)及金属蛋白酶特异性组织抑制2(TIMP-2)mRNA的表达水平;采用Western-blotting测定半乳糖凝集素3蛋白表达水平。结果 5组心肌成纤维细胞间细胞增殖率、羟脯氨酸表达水平、胶原蛋白Ⅰ型、Ⅲ型、MMP-2、TIMP-2 mRNA表达水平、半乳糖凝集素3蛋白表达水平的比较,差异均有统计学意义(F=14.440、13.510、15.860、42.950、3.727、7.357、127.600,P均<0.001)。且与对照组相比,AngⅡ组及丹参酮ⅡA各浓度组细胞增殖率[(100.0±2.8)%、(171.2±11.4)%、(162.3±15.9)%、(154.6±17.5)%、(128.7±13.2)%]、羟脯氨酸表达水平[(1.13±0.14)、(2.07±0.23)、(1.89±0.17)、(1.74±0.20)、(1.35±0.16)μg/mg]、胶原蛋白Ⅰ型、Ⅲ型、MMP-2、TIMP-2 mRNA表达水平、半乳糖凝集素3蛋白表达水平均明显上升(P均<0.05)。与AngⅡ组比较,丹参酮ⅡA各浓度组细胞增殖率、羟脯氨酸表达水平、胶原蛋白Ⅰ型、Ⅲ型、TIMP-2 mRNA表达水平、半乳糖凝集素3蛋白表达水平均显著下降,且丹参酮C组最低(P均<0.05)。而MMP-2 mRNA表达水平在AngⅡ组与丹参酮ⅡA各浓度组间比较,差异均无统计学意义(P均> 0.05),且丹参酮ⅡA各浓度组间MMP-2 mRNA表达水平比较,差异亦均无统计学意义(P均> 0.05)。结论丹参酮ⅡA可有效抑制心肌成纤维细胞的增殖,其机制可能与抑制半乳糖凝集素3蛋白表达,降低TIMP-2表达相关。
Objective To investigate the effect of tanshinone ⅡA on proliferation of my ocardial fibroblasts in rats. Methods Cultured primary myocardial fibroblasts of Wistar rats were randomly divided into the control group, AngiotensinⅡ(AngⅡ) group, tanshinone A group(0.01 mmol/L of tanshinone ⅡA), tanshinone B group(0.1 mmol/L of tanshinone ⅡA) and tanshinone C group(1 mmol/L of tanshinone ⅡA). The cell proliferation rate was measured by methye thiazolye telrazlium. The level of hydroxyproline was detected by enzyme linked immunosorbent assay. The expressions of fibroblast collagen Ⅰ, collagen Ⅲ, matrix metalloproteinase-2(MMP-2)and tissue inhibitor of metalloproteinase-2(TIMP-2) mRNA were tested by real-time quantita tive polymerase chain reaction(PCR). The expression of galectin-3 protein was detected by Westernblotting. Results The cell proliferation rates, hydroxyproline levels, and expressions of fibroblast collagen Ⅰ, collagen Ⅲ, MMP-2, TIMP-2 mRNA and galectin-3 protein all showed significant differences among five groups(F = 14.440, 13.510, 15.860, 42.950, 3.727, 7.357, 127.600; all P <0.001). The cell proliferation rates [(100.0 ± 2.8)%,(171.2 ± 11.4)%,(162.3 ± 15.9)%,(154.6 ±17.5)%,(128.7 ± 13.2)%], hydroxyproline levels [(1.13 ± 0.14),(2.07 ± 0.23),(1.89 ± 0.17),(1.74 ±0.20),(1.35 ± 0.16) μg/mg], and expressions of fibroblast collagen Ⅰ, collagen Ⅲ, MMP-2,TIMP-2 mRNA and galectin-3 protein in the Ang Ⅱ group and tanshinone groups with different concentrations were all much higher than those in the control group(all P < 0.05). The cell proliferation rates, hydroxyproline levels, expressions of fibroblast collagen Ⅰ, collagen Ⅲ, TIMP-2 mRNA and galectin-3 protein in tanshinone groups with different concentrations were much lower than those in the Ang Ⅱ group, and the lowest in the tanshinone C group(all P < 0.05).However, the expression of MMP-2 mRNA showed no significant differences between the Ang Ⅱgroup and tanshinone groups with different concentrations, and among different tanshinone groups(all P > 0.05). Conclusion Tanshinone ⅡA can effectively inhibit the proliferation of myocardial fibroblasts, and its mechanism may be related to inhibition of galectin-3 protein and decrease of TIMP-2 expression.
Objective To investigate the effect of tanshinone ⅡA on proliferation of my ocardial fibroblasts in rats. Methods Cultured primary myocardial fibroblasts of Wistar rats were randomly divided into the control group, AngiotensinⅡ(AngⅡ) group, tanshinone A group(0.01 mmol/L of tanshinone ⅡA), tanshinone B group(0.1 mmol/L of tanshinone ⅡA) and tanshinone C group(1 mmol/L of tanshinone ⅡA). The cell proliferation rate was measured by methye thiazolye telrazlium. The level of hydroxyproline was detected by enzyme linked immunosorbent assay. The expressions of fibroblast collagen Ⅰ, collagen Ⅲ, matrix metalloproteinase-2(MMP-2)and tissue inhibitor of metalloproteinase-2(TIMP-2) mRNA were tested by real-time quantita tive polymerase chain reaction(PCR). The expression of galectin-3 protein was detected by Westernblotting. Results The cell proliferation rates, hydroxyproline levels, and expressions of fibroblast collagen Ⅰ, collagen Ⅲ, MMP-2, TIMP-2 mRNA and galectin-3 protein all showed significant differences among five groups(F = 14.440, 13.510, 15.860, 42.950, 3.727, 7.357, 127.600; all P <0.001). The cell proliferation rates [(100.0 ± 2.8)%,(171.2 ± 11.4)%,(162.3 ± 15.9)%,(154.6 ±17.5)%,(128.7 ± 13.2)%], hydroxyproline levels [(1.13 ± 0.14),(2.07 ± 0.23),(1.89 ± 0.17),(1.74 ±0.20),(1.35 ± 0.16) μg/mg], and expressions of fibroblast collagen Ⅰ, collagen Ⅲ, MMP-2,TIMP-2 mRNA and galectin-3 protein in the Ang Ⅱ group and tanshinone groups with different concentrations were all much higher than those in the control group(all P < 0.05). The cell proliferation rates, hydroxyproline levels, expressions of fibroblast collagen Ⅰ, collagen Ⅲ, TIMP-2 mRNA and galectin-3 protein in tanshinone groups with different concentrations were much lower than those in the Ang Ⅱ group, and the lowest in the tanshinone C group(all P < 0.05).However, the expression of MMP-2 mRNA showed no significant differences between the Ang Ⅱgroup and tanshinone groups with different concentrations, and among different tanshinone groups(all P > 0.05). Conclusion Tanshinone ⅡA can effectively inhibit the proliferation of myocardial fibroblasts, and its mechanism may be related to inhibition of galectin-3 protein and decrease of TIMP-2 expression.
引文
1 Nishimura RA,Otto CM,Bonow RO,et al.2014 AH-A/ACC guideline for the management of patients with valvular heart disease:executive summary:a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines[J].JAm Coll Cardiol,2014,63(22):2438-2488.
2 Song M,Li A,Gong J,et al.Effects of combined prednisone+fluvastatin on cholesterol and bilirubin in pediatric patients with minimal change nephropathy[J].Clin Ther,2013,35(3):286-293.
3常文静,赵智明,赵凌杰,等.丹参酮ⅡA对压力负荷增加大鼠心肌纤维化的改善作用[J].微循环学杂志,2013,23(2):34-36,39.
4闫俊,冯娟,杨雪,等.丹参酮ⅡA的药理作用及疾病治疗的最新进展[J].实用药物与临床,2015,18(8):972-977.
5 Kaur H,Takefuji M,Ngai CY,et al.Targeted ablation of periostin-expressing activated fibroblasts prevents adverse cardiac remodeling in mice[J].Circ Res,2016,118(12):1906-1917.
6崔琳,王幼平,谢世阳,等.血管紧张素Ⅱ对原代培养大鼠心肌成纤维细胞增殖及转分化的影响[J].中西医结合心脑血管病杂志,2017,15(21):2680-2684.
7邓文浩,李树仁,张跃华.半乳糖凝聚素-3在心肌梗死后心力衰竭家兔心肌纤维化中的作用及其对心室重构的影响[J].医学研究杂志,2017,46(9):120-125.
8蔡皇娥,阮琴韵,傅丽云,等.自发性高血压大鼠心肌半乳糖凝集素3与胶原纤维及左心功能的关系[J].中华高血压杂志,2017,25(1):31-37.
9 Yu L,Ruifrok WP,Meissner M,et al.Genetic and pharmacological inhibition of galectin-3 prevents cardiac remodeling by interfering with myocardial fibrogenesis[J].Circ Heart Fail,2013,6(1):107-117.
10 de Boer RA,Lok DJ,Jaarsma T,et al.Predictive value of plasma galectin-3 levels in heart failure with reduced and preserved ejection fraction[J].Ann Med,2011,43(1):60-68.
11 Wu CK,Su MY,Lee JK,et al.Galectin-3 level and the severity of cardiac diastolic dysfunction using cellular and animal models and clinical indices[J].Sci Rep,2015(5):17007.
12 Dong R,Zhang M,Hu Q,et al.Galectin-3 as a novel biomarker for disease diagnosis and a target for therapy(Review)[J].Int J Mol Med,2018,41(2):599-614.
13 Seliger SL,Ginsberg E,Gottdiener J,et al.Soluble ST2 and galectin-3 are associated with subclinical diastolic dysfunction in older adults[J].Journal of the American College of Cardiology,2014,63(12):A769.
14唐关敏,钱钢,翟昌林,等.丹参酮ⅡA对缺血性心力衰竭大鼠心肌纤维化的影响[J].医学研究杂志,2018,47(9):105-108.
15王亚利,胡申江,王欢,等.不同剂量瑞舒伐他汀对急性冠脉综合征患者血清高敏C反应蛋白和基质金属蛋白酶-9的影响[J/CD].中华危重症医学杂志(电子版),2014,7(3):195-198.
16 Elnakish MT,Kuppusamy P,Khan M.Stem cell transplantation as a therapy for cardiac fibrosis[J].J Pathol,2013,229(2):347-354.
17 Tayebjee MH,Lip GY,MacFadyen RJ,et al.Matrix metalloproteinases in coronary artery disease:clinical and therapeutic implications and pathological significance[J].Curr Med Chem,2005,12(8):917-925.
18张蓓蓓,张静,常文静,等.丹参酮ⅡA对压力超负荷大鼠心肌纤维化的影响[J].国际心血管病杂志,2015,42(4):251-255.
19 Ren ZH,Tong YH,Xu W,et al.TanshinoneⅡAattenuates inflammatory responses of rats with myocardial infarction by reducing MCP-1 expression[J].Phytomedicine,2010,17(3-4):212-218.
20 Fang J,Xu SW,Wang P,et al.TanshinoneⅡ-Aattenuates cardiac fibrosis and modulates collagen metabolism in rats with renovascular hypertension[J].Phytomedicine,2010,18(1):58-64.
21吴逢选,张京臣,姜久昆,等.转化生长因子β1/Smads通路在百草枯中毒所致上皮-间充质转变和肺纤维化中的作用研究[J/CD].中华危重症医学杂志(电子版),2018,11(1):3-10.
2 Song M,Li A,Gong J,et al.Effects of combined prednisone+fluvastatin on cholesterol and bilirubin in pediatric patients with minimal change nephropathy[J].Clin Ther,2013,35(3):286-293.
3常文静,赵智明,赵凌杰,等.丹参酮ⅡA对压力负荷增加大鼠心肌纤维化的改善作用[J].微循环学杂志,2013,23(2):34-36,39.
4闫俊,冯娟,杨雪,等.丹参酮ⅡA的药理作用及疾病治疗的最新进展[J].实用药物与临床,2015,18(8):972-977.
5 Kaur H,Takefuji M,Ngai CY,et al.Targeted ablation of periostin-expressing activated fibroblasts prevents adverse cardiac remodeling in mice[J].Circ Res,2016,118(12):1906-1917.
6崔琳,王幼平,谢世阳,等.血管紧张素Ⅱ对原代培养大鼠心肌成纤维细胞增殖及转分化的影响[J].中西医结合心脑血管病杂志,2017,15(21):2680-2684.
7邓文浩,李树仁,张跃华.半乳糖凝聚素-3在心肌梗死后心力衰竭家兔心肌纤维化中的作用及其对心室重构的影响[J].医学研究杂志,2017,46(9):120-125.
8蔡皇娥,阮琴韵,傅丽云,等.自发性高血压大鼠心肌半乳糖凝集素3与胶原纤维及左心功能的关系[J].中华高血压杂志,2017,25(1):31-37.
9 Yu L,Ruifrok WP,Meissner M,et al.Genetic and pharmacological inhibition of galectin-3 prevents cardiac remodeling by interfering with myocardial fibrogenesis[J].Circ Heart Fail,2013,6(1):107-117.
10 de Boer RA,Lok DJ,Jaarsma T,et al.Predictive value of plasma galectin-3 levels in heart failure with reduced and preserved ejection fraction[J].Ann Med,2011,43(1):60-68.
11 Wu CK,Su MY,Lee JK,et al.Galectin-3 level and the severity of cardiac diastolic dysfunction using cellular and animal models and clinical indices[J].Sci Rep,2015(5):17007.
12 Dong R,Zhang M,Hu Q,et al.Galectin-3 as a novel biomarker for disease diagnosis and a target for therapy(Review)[J].Int J Mol Med,2018,41(2):599-614.
13 Seliger SL,Ginsberg E,Gottdiener J,et al.Soluble ST2 and galectin-3 are associated with subclinical diastolic dysfunction in older adults[J].Journal of the American College of Cardiology,2014,63(12):A769.
14唐关敏,钱钢,翟昌林,等.丹参酮ⅡA对缺血性心力衰竭大鼠心肌纤维化的影响[J].医学研究杂志,2018,47(9):105-108.
15王亚利,胡申江,王欢,等.不同剂量瑞舒伐他汀对急性冠脉综合征患者血清高敏C反应蛋白和基质金属蛋白酶-9的影响[J/CD].中华危重症医学杂志(电子版),2014,7(3):195-198.
16 Elnakish MT,Kuppusamy P,Khan M.Stem cell transplantation as a therapy for cardiac fibrosis[J].J Pathol,2013,229(2):347-354.
17 Tayebjee MH,Lip GY,MacFadyen RJ,et al.Matrix metalloproteinases in coronary artery disease:clinical and therapeutic implications and pathological significance[J].Curr Med Chem,2005,12(8):917-925.
18张蓓蓓,张静,常文静,等.丹参酮ⅡA对压力超负荷大鼠心肌纤维化的影响[J].国际心血管病杂志,2015,42(4):251-255.
19 Ren ZH,Tong YH,Xu W,et al.TanshinoneⅡAattenuates inflammatory responses of rats with myocardial infarction by reducing MCP-1 expression[J].Phytomedicine,2010,17(3-4):212-218.
20 Fang J,Xu SW,Wang P,et al.TanshinoneⅡ-Aattenuates cardiac fibrosis and modulates collagen metabolism in rats with renovascular hypertension[J].Phytomedicine,2010,18(1):58-64.
21吴逢选,张京臣,姜久昆,等.转化生长因子β1/Smads通路在百草枯中毒所致上皮-间充质转变和肺纤维化中的作用研究[J/CD].中华危重症医学杂志(电子版),2018,11(1):3-10.