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[摘要]
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、75 μmol/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-qPCR)法检测p53、Bax和Puma基因的mRNA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、45 μmol/L的迷迭香酸主要诱导HCT-8细胞早期凋亡(P<0.01),75 μmol/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mRNA表达上调,并呈浓度依赖性;45、75 μmol/L的迷迭香酸可以使p53和Bax的mRNA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;75 μmol/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。
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[Abstract]
Objective To investigate the effects of rosmarinic acid (RosA) 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 RosA for different time periods. The apoptosis rates of HCT-8 cells and the expression of related proteins were investigated after the treatment of RosA 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 mRNA expressions of p53, Bax, and Puma were detected by RT-qPCR. The protein levels of p53, Bax, Puma, and active Caspase-9 were detected by Western blotting. Results RosA could inhibit the proliferation of human colon cancer cells HCT-8 in a time-and dose-dependent manner. RosA at 15 and 45 μmol/L mainly induced early apoptosis (P < 0.01), RosA at 75 μmol/L mainly induced late apoptosis (P < 0.01). RosA could up-regulate the mRNA expression of Puma in a dose-dependent manner. RosA at 45 and 75 μmol/L increased the mRNA expression of p53 and Bax (P < 0.05). RosA could up-regulate the protein levels of Puma, Bax, and active Caspase-9 in a dose-dependent manner. RosA at 75 μmol/L could significantly increase the protein expression of p53 (P < 0.05). Conclusion RosA 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.
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[基金项目]
国家自然科学基金资助项目(81373633);浙江中医药大学基础医学院科技创新团队基金(711200E019)