薯蓣皂苷对人结肠癌细胞凋亡的作用及机制研究

The effect and underlying mechanism of Dioscin on apoptosis of human colon cancer cells

目的:探究薯蓣皂苷(Dioscin)诱导人结肠癌HCT-116细胞、RKO细胞凋亡的作用及机制。方法:通过将体外培养的HCT-116细胞和RKO细胞分为对照组和不同浓度的药物组,采用CCK-8实验分析Dioscin对细胞增殖的抑制作用,可见分光光度法检测细胞LDH活性的变化;借助DNA含量检测法、Annexin V-FITC/PI双染法和流式细胞术检测细胞凋亡情况;DCFH-DA荧光探针法检测细胞活性氧簇(ROS)含量的变化;采用荧光探针法和可见分光光度法分别检测细胞超氧化物含量及细胞超氧化物歧化酶(SOD)活性的变化,Western印迹法检测凋亡相关蛋白的表达情况。结果:在24 h、48 h处理时间下,与对照组相比,HCT-116细胞Dioscin组(2、4、6、8、10μmol/L)、RKO细胞Dioscin组(5、10、15、20、25μmol/L)都明显抑制了结肠癌细胞的增殖活性(均P<0.05),且HCT-116、RKO细胞经Dioscin处理后,细胞膜破坏,细胞LDH释放量较对照组增多(P<0.05,P<0.01);与对照组相比,Dioscin作用HCT-116、RKO细胞后,DNA含量暴露增多(P<0.05,P<0.001),细胞凋亡率上调(P<0.001,P<0.01),ROS水平升高(P<0.05,P<0.001),超氧化物含量增多(均P<0.001),SOD表达降低(P<0.05,P<0.001);Western印迹结果表明,与对照组相比,Dioscin可以抑制HCT-116、RKO细胞蛋白p-Akt(P<0.01,P<0.001)、Nrf2(均P<0.001)、Bcl-2(均P<0.001)的表达,促进蛋白cleaved Caspase-9(P<0.001,P<0.01)的表达。结论:薯蓣皂苷可能通过PI3K/Akt/Nrf2信号通路激活氧化应激反应,从而抑制结肠癌细胞生长,促进其凋亡。

Objective:To investigate the effect of Dioscin on the apoptosis of human colon cancer HCT-116 cells and RKO cells and its mechanism.Methods:HCT-116 cells and RKO cells cultured in vitro were divided into control group and Dioscin groups with different concentrations.CCK-8 assay was conducted to analyze inhibitory effect of the cell proiferation,and visible spectrophotometry method was performed to measure the cell LDH activity.The cell apoptosis was detected by cell DNA content detection method,Annexin V-FITC/PI double staining method and flow cytometry.The content of reactive oxygen species(ROS)was detected by DCFH-DA fluorescent probe method.The content of superoxide and the activity of superoxide dismutase(SOD)were measured by fluorescent probe method and visible spectrophotometry method respectively.The expressions of apoptosis-related proteins were detected by Western blotting.Results:After 24 h and 48 h drug treated,compared with the control group,the proliferation activity of colon cancer cells was significantly inhibited in HCT-116 cell Dioscin group(2,4,6,8,10μmol/L)and RKO cell Dioscin group(5,10,15,20,25μmol/L)(all P<0.05),and after the HCT-116 and RKO cells were treated with Dioscin,the cell membrane was destroyed,and the cell LDH releasing amount increased than that in the control group(P<0.05,P<0.01).Compared with the control group,after the HCT-116,RKO cells were treated with Dioscin,the exposure of DNA content increased(P<0.05,P<0.001),and the cell apoptosis rate was up-regulated(P<0.001,P<0.01),and the intracellular ROS level was increased(P<0.05,P<0.001),and superoxide content were increased(all P<0.001),while the expression of SOD was decreased(P<0.05,P<0.001).Western blotting results showed that compared with the control group,Dioscin inhibited the HCT-116,RKO cells protein expression of p-Akt(P<0.01,P<0.001),Nrf2(all P<0.001),and Bcl-2(all P<0.001),and promoted the protein expression of cleaved Caspase-9(P<0.001,P<0.01).Conclusion:Dioscin may activate oxidative stress through PI3K/Akt/Nrf2 signaling pathway to inhibit the growth of colon cancer cells and promote their apoptosis.