Effects of Euonymine on Proliferation，Migration，and Cell Cycle of HUVECs and VSMCs
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摘要:
目的 探讨卫茅碱（Euonymine）对人脐静脉内皮细胞（HUVECs）及人血管平滑肌细胞（VSMCs）的影响，以明确其防治冠脉支架内再狭窄（in-stent restenosis，ISR）的可能机制。 方法 台盼蓝检测Euonymine对HUVECs成活率的影响；MTT法检测Euonymine对HUVECs及VSMCs增殖的影响；划痕法检测Euonymine对VSMCs迁移的影响。 结果 MTT结果显示，卫茅碱干预后, HUVECs和VSMCs的吸光度降低。另外，Euonymine分别作用HUVECs 24 h，VSMCs 48 h后，测的半数有效抑制浓度（IC50）分别为28.6 µg/mL和33.92 µg/mL。流式细胞结果显示，Euonymine（25 μg/mL）使HUVECs和VSMCs的细胞周期阻滞于G0/G1期，而Euonymine（100 µg/mL）使VSMCs有丝分裂被阻断于G0/G1期及G2/M期。 结论 Euonymine主要通过抑制VSMCs的增殖和迁移，影响细胞周期，对于抑制经皮冠状动脉介入治疗术（percutaneous coronary intervention，PCI）后新生内膜形成有潜在的临床应用价值。 Abstract:Objective To investigate the effects of Euonymine on isolated cultured human umbilical vein endothelial cells （HUVECs） and human vascular smooth muscle cells （VSMCs）, so as to elucidate the possible mechanism of Euonymine in prevention and treatment of ISR. Methods The effect of trypan blue assay on the viability of HUVECs. MTT assay was used to observe the effect of Euonymine on the proliferation of normal HUVECs and VSMCs. A wound healing assay was performed to detect the effect of Euonymine on the migration of VSMCs. Results The MTT results showed that the absorbance of HUVECs and VSMCs decreased after the Euonymine intervention. In addition, the IC50 of Euonymine on HUVECs after 24 hours and on VSMCs after 48 hours were 28.6 µg/mL and 33.92 µg/mL, respectively. Flow cytometry results showed that Euonymine （25 μg/mL） arrested the cell cycle of both HUVECs and VSMCs at the G0/G1 phase, while Euonymine （100 μg/mL） blocked the mitosis of VSMCs at both the G0/G1 and G2/M phases. Conclusion Euonymine affects the cell cycle mainly by inhibiting the proliferation and migration of VSMCs and has potential value of clinical applications in inhibiting neointima hyperplasia after percutaneous coronary intervention （PCI）. -
Key words:
- Euonymine /
- HUVECs /
- VSMCs /
- Anti-proliferative /
- Anti-migratory /
- Cell cycle
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图 2 正常HUVECs和VSMCs形态（×100）
A: 正常对照组HUVECs的形态；B: 正常对照组VSMCs的形态。
Figure 2. Typical appearance of HUVECs and VSMCs （×100）
图 4 Euonymine作用HUVECs 24 h的生长抑制率曲线
Figure 4. Curve inhibition rate of Euonymine treated HUVECs for 24 h
图 5 Euonymine作用VSMCs 48 h的生长抑制率曲线
Figure 5. Curve inhibition rate of Euonymine treated VSMCs for 48 h
图 6 卫茅碱抑制VSMCs迁移
A：划痕实验24 h后各实验组 VSMCs迁移的图像；B：Euonymine抑制VSMCs迁移距离的统计分析。与正常对照组比较，**P < 0.01；与模型组比较，#P < 0.05（$ \bar x \pm s $，n = 6）。
Figure 6. Euonymine inhibits the migration of VSMCs
图 7 卫茅碱对血清诱导的HUVECs细胞周期影响（
$ \bar x \pm s$ ，n = 6）A～C：不同浓度的Euonymine作用HUVECs后24 h的DNA直方图； D： Euonymine处理24 h的HUVECs的细胞周期的统计学分析。
Figure 7. Effect of Euonymine on the cell cycle of HUVECs induced by serum （
$ \bar x \pm s$ ，n = 6）
图 8 Euonymine对血清诱导的VSMCs细胞周期影响（
$\bar x \pm s $ ，n = 6）A～C：不同浓度的Euonymine作用VSMC后24 h的DNA直方图；D～E： Euonyming处理24 h和48 h的VSMCs的细胞周期分布。
Figure 8. Effect of Euonymine on the cell cycle of VSMCs induced by serum （
$\bar x \pm s $ ，n = 6）表 1 不同浓度Euonymine对HUVECs增殖的影响（
$ \bar x \pm s $ ，n = 6）Table 1. Effect of different concentrations of Euonymine on the proliferation of HUVECs （
$\bar x \pm s $ ，n = 6）分组 OD （570 nm） IR（%） F P 正常对照组 DMSO 0.768 ± 0.06 Euonymine（µg/mL） 12.5 0.654 ± 0.08△ 16.1 25 0.591 ± 0.19△ 25.0 5.97 0.030* 50 0.460 ± 0.07△ 43.5 100 0.152 ± 0.12△ 87.0 *P < 0.05；与正常对照组相比较，△P < 0.05。 
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表 2 不同浓度Euonymine对VSMCs增殖的影响（
$\bar x \pm s$ ，n = 6）Table 2. Effect of different concentrations of Euonymine on the proliferation of VSMCs （
$\bar x \pm s $ ，n = 6）分组 OD （570 nm） 24 h 48 h 72 h 96 h 正常对照组 DMSO 0.788 ± 0.16 0.841 ± 0.14 0.885 ± 0.08 0.925 ± 0.20 Euonymine
（µg/mL）12.5 0.688 ± 0.10 0.680 ± 0.13 0.676 ± 0.12△ 0.637 ± 0.04 25 0.626 ± 0.08△ 0.628 ± 0.19△ 0.607 ± 0.10△ 0.569 ± 0.12△ 50 0.524 ± 0.11△ 0.425 ± 0.15△ 0.419 ± 0.07△ 0.402 ± 0.18△ 100 0.312 ± 0.07△ 0.259 ± 0.13△ 0.219 ± 0.17△ 0.188 ± 0.08△ 200 0.259 ± 0.04△ 0.185 ± 0.16△ 0.156 ± 0.06△ 0.137 ± 0.18△ F 6.21 6.49 6.61 6.76 P 0.041* 0.041* 0 .031* 0.028* *P < 0.05；与正常对照组比较，△P < 0.05。
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