迷迭香酸对人结肠癌HCT-8细胞增殖和凋亡的影响及分子机制研究

Effects of rosmarinic acid on proliferation and apoptosis of HCT-8 cells and its molecular mechanisms

目的研究迷迭香酸对人结肠癌HCT-8细胞增殖和凋亡的影响,并探究相关机制。方法采用CCK-8法检测不同浓度迷迭香酸作用不同时间后HCT-8细胞的增殖情况;根据CCK-8法结果确定迷迭香酸15、45、75μmol/L作用72 h,考察HCT-8细胞的凋亡和相关基因及蛋白的表达;通过流式细胞术检测HCT-8细胞的凋亡率;采用实时荧光定量PCR(RT-q PCR)法检测p53、Bax和Puma基因的m RNA表达;通过Western blotting法检测p53、Bax、Puma和active Caspase-9的蛋白表达水平。结果迷迭香酸能够抑制HCT-8细胞的增殖,并呈时间和浓度依赖性。15、45μmol/L的迷迭香酸主要诱导HCT-8细胞早期凋亡(P<0.01),75μmol/L的迷迭香酸主要诱导HCT-8细胞晚期凋亡(P<0.01)。迷迭香酸可以使Puma的m RNA表达上调,并呈浓度依赖性;45、75μmol/L的迷迭香酸可以使p53和Bax的m RNA表达升高(P<0.05)。迷迭香酸可以上调Puma、Bax和active Caspase-9的蛋白表达水平,并呈浓度依赖性;75μmol/L的迷迭香酸可以使p53的蛋白表达明显升高(P<0.05)。结论迷迭香酸对HCT-8细胞具有明显的增殖抑制作用,并且是通过诱导凋亡实现的,促凋亡蛋白p53、Puma、Bax和active Caspase-9诱导了凋亡的发生。

Objective To investigate the effects of rosmarinic acid（Ros A） on the proliferation and apoptosis of human colon cancer HCT-8 cells, and explore the related mechanisms. Methods The proliferation of HCT-8 cells was detected by CCK-8 assay at different concentrations of Ros A for different time periods. The apoptosis rates of HCT-8 cells and the expression of related proteins were investigated after the treatment of Ros A at 15, 45, and 75 μmol/L for 72 h according to the CCK-8 results. The apoptosis rates of HCT-8 cells were detected by FCM. The m RNA expressions of p53, Bax, and Puma were detected by RT-q PCR. The protein levels of p53, Bax, Puma, and active Caspase-9 were detected by Western blotting. Results Ros A could inhibit the proliferation of human colon cancer cells HCT-8 in a time-and dose-dependent manner. Ros A at 15 and 45 μmol/L mainly induced early apoptosis（P 〈0.01）, Ros A at 75 μmol/L mainly induced late apoptosis（P 〈0.01）. Ros A could up-regulate the m RNA expression of Puma in a dose-dependent manner. Ros A at 45 and 75 μmol/L increased the m RNA expression of p53 and Bax（P 〈0.05）. Ros A could up-regulate the protein levels of Puma, Bax, and active Caspase-9 in a dose-dependent manner. Ros A at 75 μmol/L could significantly increase the protein expression of p53（P 〈0.05）. Conclusion Ros A can significantly inhibit the proliferation of HCT-8 cells by inducing the apoptosis. The apoptosis-inducing proteins of p53, Puma, Bax, and active Caspase-9 induce the apoptosis of cells.