丹参酚酸B量效相关性抑制大肠癌HCT-116细胞生长及其相关机制研究

Research on Inhibitory Effect of Salvianolic Acid B on Growth of Human Colorectal Cancer Cell Line HCT-116 and Its Mechanism

目的验证丹参酚酸B(Salvianolic acid B,SalB)浓度依赖性抑制人大肠癌HCT-116细胞增殖、促进其凋亡,并从活性氧簇(reactive oxygen species,ROS)水平进一步探究SalB抑制大肠癌生长的作用机制。方法以低、中、高剂量SalB干预HCT-116细胞48h,显微镜下观察细胞生长状态及形态学特征;平板克隆实验检测细胞克隆形成能力;流式细胞术分析细胞周期分布、细胞凋亡率以及细胞内ROS含量。结果随着SalB作用HCT-116细胞浓度增加,镜下可见细胞数量逐渐减少,细胞折光率逐渐减弱,体积变小,核固缩,核颜色加深,出现大量细胞碎片及漂浮细胞;平板克隆实验显示,中剂量和高剂量SalB显著抑制HCT-116细胞克隆形成能力(P<0.05或P<0.01),且随着药物浓度增加,抑制作用更强(P<0.01);中剂量和高剂量SalB处理后,GO/G1期细胞数量显著升高(P<0.01),S期和G2/M期细胞数量均显著降低(P<0.05或P<0.01),该周期阻滞作用在一定浓度范围内与SalB浓度呈正性相关;低、中、高浓度SalB均可诱导HCT-116细胞凋亡(P<0.05或P<0.01),浓度越高,细胞凋亡率越高(P<0.05或P<0.01);中、高剂量SalB浓度依赖性促进HCT-116细胞内ROS水平(P<0.05或P<0.01)。结论SalB通过上调HCT-116细胞内ROS含量,阻滞细胞周期于G0/G1期,进而量效相关性抑制细胞增殖并促进细胞凋亡。

Objective To verify that Salvianolic acid B(SalB)concentration-dependently inhibits the proliferation of human colorectal cancer HCT-116 cells and promotes its apoptosis,and further explores the inhibition mechanism of colorectal cancer growth by SalB by the level of intracellular Reactive oxygen species(ROS)level.Methods HCT-116 cells were treated with low,medium and high doses of SalB for 48 h.The growth status and morphological characteristics of HCT-116 cells were observed under microscope;Plate cloning test was used to detect cell proliferation;Cell cycle distribution,apoptosis rate and intracellular ROS content were analyzed by flow cytometry.Methods HCT-116 cells were treated with low,medium and high doses of SalB for 48 h.The growth status and morphological characteristics of HCT-116 cells were observed under microscope;Plate cloning test was used to detect cell proliferation;Cell cycle distribution,apoptosis rate and intracellular ROS content were analyzed by flow cytometry.Results With the increase of the concentration of SalB on HCT-116 cells,the number of cells decreased gradually,the refractive index decreased gradually,the volume became smaller,nuclear pyknosis occurred,the nuclear color deepened,and a large number of cell debris and floating cells appeared;The results of plate cloning assay showed that medium dose and high dose of SalB significantly inhibited the colony forming ability of HCT-116 cells(P<0.05 or P<0.01),and the inhibitory effect was stronger with the increase of drug concentration(P<0.01);After medium and high dose SalB treatments,the number of cells in GO/G1 phase increased significantly(P<0.01),with the number of cells in both S phase and G2/M phase significantly decreased(P<0.05 or P<0.01),and the cycle retardation effect was positively correlated with the concentration of SalB within a certain concentration range;Low,medium and high concentrations of SalB could induce apoptosis of HCT-116 cells(P<0.05 or P<0.01),the higher the concentration of SalB,the higher the apoptosis rate(P<0.05 or P<0.01);Medium and high dose of SalB promoted ROS levels in HCT-116 cells in a concentration dependent manner(P<0.05 or P<0.01).Conclusion SalB blocks the cell cycle in G0/G1 phase by up regulating ROS content in HCT-116 cells,thereby inhibits cell proliferation and promotes cell apoptosis in dose-dependent manner.