Zhang Zhuo-qi, Cao Xi-chuan, Zhang Ling, Zhu Wen-ling
Department of Cardiology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.
Zhonghua Yi Xue Za Zhi. 2005 Jun 8;85(21):1484-8.
To study the anti-proliferation, pro-apoptosis and cell cycle blocking effects of shikonin on rat vascular smooth muscle cell (VSMC) in vitro.
VSMCs were primarily cultured by explant method from the thoracic aorta of male SD rats. Shikonin of different concentration, 4, 2, 1, 0.5, 0.25, and 0 micromol/L was added. The cell viability was detected by MTT method. Cell growth curve was drawn by trypan blue exclusion method. (3)H-thymidine incorporation was used to calculate the inhibition rate of DNA synthesis. Flow cytometry was used to detect the cell cycle. Cell apoptosis was observed by fluorescence microscopy. Western blotting was performed to detect the expression of different cell apoptosis and cell cycle regulatory proteins, such as cyclin D(1) and E, proliferating cell nuclear antigen (PCNA), p21(waf1/cip1), p27(kip1), and p53.
Compared with control group, shikonin had no obvious cytotoxic effect on cell viability at the concentration of 0.25-1 micromol/L (P > 0.05). While it could inhibit, both time- and dose-dependently, the growth of VSMC, which was predominant of 1 micromol/L at 72 h (1.9 x 10(5)/well vs 5.8 x 10(5)/well, P < 0.05), and DNA synthesis was also significantly inhibited in a time- and dose-dependent manner with inhibition rate varied from 33 to 98% (P < 0.05 or P < 0.01). 1 micromol/L shikonin significantly blocked the cell cycle progression in proliferative VSMC, decreased S, G(2)/M phase (P < 0.05) and increased G(0)/G(1) phase (P < 0.05) to quiescent level with sub-G(1) apoptotic distribution at 48 h (10.9% +/- 0.3%). Shikohin at the concentration of 1-2 micromol/L significantly increased the percentage of apoptotic cells in a time- and dose-dependent manner compared with control group (2.8%-23.7% vs 0.2%-0.4%, P < 0.05), and typical apoptotic nuclear morphological changes were observed. 1 micromol/L shikonin significantly down-regulated cyclin D(1), E and PCNA expression, up-regulated p21(wif1/cip1) expression, and did not obviously influence the p27(kip1) and p53 expression.
Shikonin inhibits the proliferation, promotes the apoptosis and blocks cell cycle progression of VSMC. These effects are associated with the expression changes of cell cycle regulatory proteins.
研究紫草素对体外培养的大鼠血管平滑肌细胞（VSMC）的抗增殖、促凋亡及细胞周期阻滞作用。
采用组织块法原代培养雄性SD大鼠胸主动脉VSMC。加入不同浓度（4、2、1、0.5、0.25和0 μmol/L）的紫草素。采用MTT法检测细胞活力。用台盼蓝排斥法绘制细胞生长曲线。采用³H-胸腺嘧啶核苷掺入法计算DNA合成抑制率。用流式细胞术检测细胞周期。通过荧光显微镜观察细胞凋亡。采用蛋白质印迹法检测细胞凋亡及细胞周期调节蛋白如细胞周期蛋白D₁、E、增殖细胞核抗原（PCNA）、p21（waf1/cip1）、p27（kip1）和p53的表达。
与对照组相比，紫草素在0.25 - 1 μmol/L浓度时对细胞活力无明显细胞毒性作用（P > 0.05）。但它能时间和剂量依赖性地抑制VSMC生长，在72 h时1 μmol/L的抑制作用最显著（1.9×10⁵/孔 vs 5.8×10⁵/孔，P < 0.05），并且DNA合成也以时间和剂量依赖性方式被显著抑制，抑制率在33%至98%之间（P < 0.05或P < 0.01）。1 μmol/L紫草素显著阻滞增殖期VSMC的细胞周期进程，使S期、G₂/M期细胞减少（P < 0.05），G₀/G₁期细胞增加（P < 0.05）至静止水平，48 h时出现亚G₁期凋亡分布（10.9% ± 0.3%）。1 - 2 μmol/L紫草素与对照组相比，能时间和剂量依赖性地显著增加凋亡细胞百分比（2.8% - 23.7% vs 0.2% - 0.4%，P < 0.05），并观察到典型的凋亡核形态变化。1 μmol/L紫草素显著下调细胞周期蛋白D₁、E和PCNA表达，上调p21（wif1/cip1）表达，对p27（kip1）和p53表达无明显影响。
紫草素抑制VSMC增殖，促进其凋亡并阻滞细胞周期进程。这些作用与细胞周期调节蛋白的表达变化有关。
