葛根素对凝血酶诱导的血管平滑肌细胞增殖的影响

目的观察葛根素对凝血酶诱导的血管平滑肌细胞(VSMC)增殖的影响,对原癌基因c-fos和bc l-2蛋白质以及凝血酶受体(TR)mRNA表达的作用。方法以细胞计数法,流式细胞仪测定DNA含量,细胞周期分析法观察凝血酶及葛根素对VSMC增殖和DNA合成的影响。在凝血酶及葛根素作用24 h后,用W estern印迹法检测c-fos和bc l-2蛋白表达,以半定量逆转录聚合酶链式反应(RT-PCR)检测TR mRNA的表达。结果凝血酶对VSMC有明显促增殖作用,促增殖效应在24 h末达峰值(细胞计数凝血酶组8.64×104/m l±0.12×104/m l,对照组4.20×104/m l±0.11×104/m l,P<0.05),且凝血酶浓度在0.1~1.0 U/L之间呈剂量依赖关系;葛根素呈剂量依赖性地抑制凝血酶诱导的细胞增殖、DNA合成以及VSMC c-fos和bc l-2蛋白的表达(葛根素浓度为1.5×10-3mol/L时,抑制率分别为42.6%±5.2%、58.2%±7.9%、44.5%±7.5%和39.6%±6.4%,均P<0.05);高浓度(1.5×10-3mol/L)的葛根素可显著抑制凝血酶诱导的TR mRNA上调(抑制率为17.6%±1.7%,P<0.05)。结论葛根素能抑制凝血酶诱导的VSMC增殖,这可能与其抑制c-fos和bc l-2蛋白有关,并部分与其抑制TR mRNA表达有关。

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

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.