西洛他唑通过Rb-p53-p21途径抑制大鼠血管平滑肌细胞增殖并诱导其凋亡

[Cilostazol inhibits proliferation and induces apoptosis in rat vascular smooth muscle cells through Rb-p53-p21 pathways].

作者信息

Wang Shou-li, Huang Ming-fang, Sun Fei, Yin Zhao, Chen Li-liang, Feng Guo-bin

机构信息

Department of Cardiology, People's Liberation Army 306 Hospital, Beijing, China.

出版信息

Zhonghua Xin Xue Guan Bing Za Zhi. 2013 Jan;41(1):48-53.

PMID:23651968

Abstract

OBJECTIVE

To explore the effects and related mechanisms of cilostazol on rat vascular smooth muscle cells (VSMCs)proliferation.

METHODS

VSMCs were treated with DMEM (control) and various doses of cilostazol (1.0×10(-7), 2.5×10(-7), 5.0×10(-7), 7.5×10(-7) and 1.0×10(-6) mol/L) for 13 d (cell counting) or 72 h. Proliferation of VSMCs was investigated by cell-counting, MTT and flow cytometry analysis. Cell apoptosis was determined by TUNEL staining. mRNA and protein expressions of cell cycle regulatory proteins, such as Rb, p53 and p21 were detected by RT-PCR and Western blot, respectively.

RESULTS

Cilostazol inhibited VSMCs proliferation and induced VSMCs arrest at G1 phase in a dose-dependent manner. High dose of cilostazol (7.5×10(-7) and 1.0×10(-6) mol/L) induced VSMCs apoptosis. p53 mRNA expression in 2.5×10(-7) mol/L to 7.5×10(-7) mol/L groups as well as 1.0×10(-6) mol/L group (3.22 ± 0.45 vs. 1.75 ± 0.32) and p53 protein expression in 7.5×10(-7) mol/L group and 1.0×10(-6) mol/L group (0.53 ± 0.11 vs. 0.18 ± 0.06) were significantly upregulated after 72 h culture (all P < 0.05 vs. control). Low dose of cilostazol (1.0×10(-7), 2.5×10(-7) and 5.0×10(-7) mol/L) significantly upregulated p21 mRNA expression compared to control group (1.86 ± 0.19, 2.20 ± 0.24 and 2.10 ± 0.18 vs. 1.210 ± 0.18, all P < 0.05). Similarly, Rb mRNA expression was significantly upregulated in 1.0×10(-7), 2.5×10(-7) and 5.0×10(-7) mol/L groups (0.89 ± 0.07 vs. 0.38 ± 0.04)compared with control group (all P < 0.05). However, high dose cilostazol (7.5×10(-7) and 1.0×10(-6) mol/L) significantly downregulated p21 mRNA expression (0.81 ± 0.09 vs. 1.21 ± 0.18, 0.36 ± 0.10 vs. 1.21 ± 0.18, all P < 0.05 vs. control) and Rb mRNA expression (0.12 ± 0.02 and 0.11 ± 0.02 vs. 0.38 ± 0.04, all P < 0.05 vs. control). p21 and Rb protein expressions also upregulated at low concentrations of cilostazol and downregulated at high concentrations of cilostazol.

CONCLUSION

Cilostazol could inhibit the proliferation of rat VSMCs through modulating Rb-p53-p21 pathway and induce VSMCs apoptosis through upregulating p53.

摘要

目的

探讨西洛他唑对大鼠血管平滑肌细胞（VSMCs）增殖的影响及其相关机制。

方法

用DMEM（对照组）和不同剂量的西洛他唑（1.0×10⁻⁷、2.5×10⁻⁷、5.0×10⁻⁷、7.5×10⁻⁷和1.0×10⁻⁶mol/L）处理VSMCs 13天（细胞计数）或72小时。通过细胞计数、MTT和流式细胞术分析研究VSMCs的增殖情况。通过TUNEL染色测定细胞凋亡。分别用RT-PCR和Western blot检测细胞周期调节蛋白如Rb、p53和p21的mRNA和蛋白表达。

结果

西洛他唑以剂量依赖性方式抑制VSMCs增殖并诱导VSMCs停滞于G1期。高剂量的西洛他唑（7.5×10⁻⁷和1.0×10⁻⁶mol/L）诱导VSMCs凋亡。培养72小时后，2.5×10⁻⁷mol/L至7.5×10⁻⁷mol/L组以及1.0×10⁻⁶mol/L组的p53 mRNA表达（3.22±0.45对1.75±0.32）和7.5×10⁻⁷mol/L组及1.0×10⁻⁶mol/L组的p53蛋白表达（0.53±0.11对0.18±0.06）均显著上调（与对照组相比，均P<0.05）。与对照组相比，低剂量的西洛他唑（1.0×10⁻⁷、2.5×10⁻⁷和5.0×10⁻⁷mol/L）显著上调p21 mRNA表达（1.86±0.19、2.20±0.24和2.10±0.18对1.210±0.18，均P<0.05）。同样，1.0×10⁻⁷、2.5×10⁻⁷和5.0×10⁻⁷mol/L组的Rb mRNA表达（0.89±0.07对0.38±0.04）与对照组相比也显著上调（均P<0.05）。然而，高剂量的西洛他唑（7.5×10⁻⁷和1.0×10⁻⁶mol/L）显著下调p21 mRNA表达（0.81±0.09对1.21±0.18，0.36±0.10对1.21±0.18，与对照组相比，均P<0.05）和Rb mRNA表达（0.12±0.02和0.11±0.02对0.38±0.04，与对照组相比，均P<0.05）。p21和Rb蛋白表达在低浓度西洛他唑时也上调，在高浓度西洛他唑时下调。