Xu Yi-zhou, Wang Ning-fu, Li Pei-zhang, Wu Xin, Ling Feng, Zhang Xing-wei
Department of Cardiology, First People's Hospital of Hangzhou, Hangzhou 310006, China.
Zhonghua Yi Xue Za Zhi. 2006 Feb 21;86(7):476-80.
To investigate the effects of puerarin on the proliferation of vascular smooth muscle cells (VSMC) induced by thrombin and the mechanism thereof.
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.
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).
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.
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。
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。
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）。
葛根素可抑制凝血酶诱导的VSMC增殖和DNA合成。其抑制作用与抑制c-fos和bcl-2蛋白表达密切相关，部分与抑制TR mRNA表达有关。
