葛根素对凝血酶诱导的血管平滑肌细胞增殖的影响

[Effects of puerarin on proliferation of vascular smooth muscle cells induced by thrombin].

作者信息

Xu Yi-zhou, Wang Ning-fu, Li Pei-zhang, Wu Xin, Ling Feng, Zhang Xing-wei

机构信息

Department of Cardiology, First People's Hospital of Hangzhou, Hangzhou 310006, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2006 Feb 21;86(7):476-80.

PMID:16677576

Abstract

OBJECTIVE

To investigate the effects of puerarin on the proliferation of vascular smooth muscle cells (VSMC) induced by thrombin and the mechanism thereof.

METHODS

VSMCs were isolated from the thoracic aorta of a SD rat and cultured, then co-cultured with thrombin of the concentration 0.1, 0.3, 1.0, 3.0, and 10 U/L for 24 h, thrombin of the concentration of 1 U/L for 0, 6, 12, 24, 36, and 48 h respectively, or thrombin of the concentration of 1 U/L combined with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), or 1.5 x 10(-3) mol/L for 24 h. Flow cytometry was used to detect the cell number and cell cycle. Western blotting was used to indicate the protein expression of the oncogenes c-fos and bcl-2 RT-PCR was used to evaluate the thrombin receptor (TR) mRNA expression.

RESULTS

he numbers of the groups of VSMCs stimulated by 0.1, 0.3, 1.0, 3.0, and 10 U/L thrombin for 24 hours were 4.82 x 10(4)/ml +/- 0.11 x 10(4)/ml, 6.37 x 10(4)/ml +/- 0.09 x 10(4)/ml, 8.78 x 10(4)/ml +/- 0.08 x 10(4)/ml, 7.37 x 10(4)/ml +/- 0.07 x 10(4)/ml, and 5.28 x 10(4)/ml +/- 0.12 x 10(4)/ml respectively, all significantly higher than that of the control group (4.08 +/- 0.054 x 10(4)/ml, all P < 0.05). The effect of thrombin was in a dose-dependent manner within a concentration range of 0.1 - 1.0 U/L. The suppression rates of VSMC proliferation in the combination groups with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L were 10.9% +/- 1.6%, 32.1% +/- 3.3%, and 42.6% +/- 5.2% respectively in comparison with the thrombin group (all P < 0.05). The c-fos protein expression of the VSMCs after thrombin stimulation for 24 h increased by 156.0% +/- 11.3% (P < 0.05), and the bcl-2 protein expression of the VSMCs pretreated with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L, and then stimulated by thrombin was significantly lower than that of the VSMCs only stimulated by thrombin with the suppression rates of 20.7% +/- 2.1%, 31.6% +/- 5.2%, and 44.5% +/- 7.5% respectively (all P < 0.05). The bcl-2 protein expression of the VSMCs after thrombin stimulation for 24 h increased by 96.7% +/- 8.3% (P < 0.05), and the bcl-2 protein expression of the VSMCs pretreated with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L, and then stimulated by thrombin was significantly lower than that of the VSMCs only stimulated by thrombin with the suppression rates of 7.1% +/- 0.8%, 18.8% +/- 1.2%, and 39.6% +/- 6.4% respectively (all P < 0.05). The stimulation of thrombin increased the TR mRNA expression by 183.9% +/- 9.4%. The puerarin of the concentrations of 1.5 x 10(-5) mol/L and 1.5 x 10(-4) mol/L decreased the increase of TR mRNA expression induced by thrombin, however, without significant differences (both P > 0.05), and puerarin of the concentration of 1.5 x 10(-3) mol/L significantly suppressed the increase of TR mRNA expression induced by thrombin by 17.6% +/- 1.7% (P < 0.05).

CONCLUSION

Puerarin suppresses the proliferation and DNA synthesis of VSMC induced by thrombin. The inhibitory effect of puerarin is closely related with the suppression of the protein expression of c-fos and bcl-2n, and partly related with the suppression of the TR mRNA expression.

摘要

目的

探讨葛根素对凝血酶诱导的血管平滑肌细胞（VSMC）增殖的影响及其机制。

方法

从SD大鼠胸主动脉分离培养VSMC，然后分别与浓度为0.1、0.3、1.0、3.0和10 U/L的凝血酶共培养24 h，与浓度为1 U/L的凝血酶分别共培养0、6、12、24、36和48 h，或与浓度为1 U/L的凝血酶及浓度为1.5×10⁻⁵、1.5×10⁻⁴或1.5×10⁻³ mol/L的葛根素共培养24 h。采用流式细胞术检测细胞数量和细胞周期。采用蛋白质免疫印迹法检测癌基因c-fos和bcl-2的蛋白表达。采用逆转录聚合酶链反应（RT-PCR）评估凝血酶受体（TR）mRNA表达。

结果

浓度为0.1、0.3、1.0、3.0和10 U/L的凝血酶刺激VSMC 24 h后，细胞数量分别为4.82×10⁴/ml±0.11×10⁴/ml、6.37×10⁴/ml±0.09×10⁴/ml、8.78×10⁴/ml±0.08×10⁴/ml、7.37×10⁴/ml±0.07×10⁴/ml和5.28×10⁴/ml±0.12×10⁴/ml，均显著高于对照组（4.08±0.054×10⁴/ml，均P<0.05）。在0.1~1.0 U/L浓度范围内，凝血酶的作用呈剂量依赖性。浓度为1.5×10⁻⁵、1.5×10⁻⁴和1.5×10⁻³ mol/L的葛根素联合组VSMC增殖抑制率分别为10.9%±1.6%、32.1%±3.3%和42.6%±5.2%，与凝血酶组相比差异均有统计学意义（均P<0.05）。凝血酶刺激VSMC 24 h后，c-fos蛋白表达增加156.0%±11.3%（P<0.05）；浓度为1.5×10⁻⁵、1.5×10⁻⁴和1.5×10⁻³ mol/L的葛根素预处理VSMC后再经凝血酶刺激，bcl-2蛋白表达显著低于单纯凝血酶刺激组，抑制率分别为20.7%±2.1%、31.6%±5.2%和44.5%±7.5%（均P<0.05）。凝血酶刺激VSMC 24 h后，bcl-2蛋白表达增加96.7%±8.3%（P<0.05）；浓度为1.5×10⁻⁵、1.5×10⁻⁴和1.5×10⁻³ mol/L的葛根素预处理VSMC后再经凝血酶刺激，bcl-2蛋白表达显著低于单纯凝血酶刺激组，抑制率分别为7.1%±0.8%、18.8%±1.2%和39.6%±6.4%（均P<0.05）。凝血酶刺激使TR mRNA表达增加183.9%±9.4%。浓度为1.5×10⁻⁵ mol/L和1.5×10⁻⁴ mol/L的葛根素可降低凝血酶诱导的TR mRNA表达增加，但差异无统计学意义（均P>0.05）；浓度为1.5×10⁻³ mol/L的葛根素可显著抑制凝血酶诱导的TR mRNA表达增加，抑制率为17.6%±1.7%（P<0.05）。