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

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bcl-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24h后，用Western印迹法检测c-fos和bcl-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24h末达峰值（细胞计数凝血酶组8．64×10^4／ml±0．12×10^4／ml，对照组4．20×10^4ml±0．11×10^4/ml，P〈0．05），且凝血酶浓度在0．1～1．0U／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VSMCc—fos和bcl02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bcl-2蛋白有关，并部分与其抑制TR mRNA表达有关。

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 × 10^-5, 1.5 × 10^-4, or 1.5 × 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-los 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 4.82×10^4/ml±0.11 × 10^4/nd, 6.37 ×10^4/ml ±0.09× 10^4/ml, 8.78 × 10^4/ml±0.08 × 10^4/ml, 7.37 × 10^4/ml ±0.07 × 10^4/ml, and 5.28 × 10^4/ml s0.12 × 10^4/ml respectively, all significantly higher than that of the control group （4.08 ± 0. 054 × 104/ml, all P 〈 0.05 ）. The effect of thrnmbin 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 × 10 -5, 1.5 × 10-4, and 1.5×10-3 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 × 10^-5, 1.5 × 10^-4, and 1.5 × 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 × 10^-5, 1.5 × 10^-4, and 1.5 × 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 by183 . 9% ±9.4%. The puerarin of the concentrations of 1.5 × 10^-5 mol/L and 1.5 × 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 × 10^-3 mol/L significantly suppressed the increase of TR mRNA expression induced by thrombin by 17.6% ± 1.7% （ P 〈 0. 05 ）. CondusionPuerarin 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-fes and bcl- 2n, and partly related with the suppression of the TR mRNA expression.