世界中医药

目的：研究紫草素通过激活活性氧（ROS）自由基诱导大肠癌细胞凋亡的作用机制。方法：通过MTT测定法检测紫草素对人结肠癌细胞系HCT116和SW480细胞活力的影响，结合Annexin V-FITC/PI凋亡检测试剂盒及流式细胞术检测细胞凋亡情况；通过Caspase 3/9活性测定试剂盒检测半胱天冬酶活性，使用ROS检测试剂盒检测紫草素处理对HCT116和SW480细胞中ROS水平的影响，并借助Western Blotting检测Bcl-2和Bcl-xL蛋白表达情况。结果：细胞活力检测结果显示，人正常结肠黏膜上皮细胞系NCM460对紫草素干预不敏感，而紫草素对人结肠癌细胞系HCT116和SW480细胞活力表现出显著的抑制作用（均P<005）。在紫草素干预后，Western Blotting分析检测到细胞周期蛋白D、c-Myc、Bcl-2和Bcl-xL蛋白表达水平显著下调（均P<005）。同时，紫草素以剂量依赖性方式上调了Caspase3和Caspase9蛋白表达水平（均P<005）。流式细胞术检测结果表明，经10 μmol/L紫草素处理24 h后，HCT116和SW480细胞凋亡率增加至592%和656%，而几乎没有坏死细胞（Annexin V-，PI+）；并检测到HCT116和SW480细胞线粒体膜电位被去极化。ROS水平检测结果显示，紫草素以剂量依赖性方式上调了HCT116和SW480细胞ROS水平。结论：紫草素通过线粒体介导途径诱导结肠癌细胞凋亡，Bcl-2蛋白家族和细胞内ROS水平升高在该过程中发挥重要作用。

English Summary:

To study the molecular mechanism of shikonin by activating reactive oxygen species ROS to induce apoptosis of colorectal cancer cellsMethods:The effect of shikonin on the viability of human colon cancer cell lines HCT116 and SW480 was detected by MTT assay，combined with Annexin V-FITC/PI apoptosis detection kit and flow cytometry to detect cell apoptosis; by Caspase 3/9 activity assay kit and caspase activity were was detectedROS detection kit was used to detect the effect of shikonin treatment on the ROS levels in HCT116 and SW480 cells，and the expression of Bcl-2 and Bcl-xL proteins was detected by Western BlottingResults:Cell viability test results showed that human normal colonic mucosal epithelial cell line NCM460 was not sensitive to shikonin intervention，while shikonin showed a significant inhibitory effect on human colon cancer cell lines HCT116 and SW480 cells(P<005)Western Blotting detected cyclin D and c-Myc，and Bcl-2 and Bcl-xL protein levels were significantly down-regulated after shikonin intervention(P<005)Meanwhile，shikonin increased the expression levels of Caspase3 and Caspase9 protein in a dose-dependent manner(P<005)Flow cytometry results showed that after treated with 10 μmol/L shikonin for 24 h，the apoptosis rate of HCT116 and SW480 cells increased to 592% and 656%，with almost no necrotic cells(Annexin V-，PI+); Mitochondrial membrane potentials to HCT116 and SW480 cells were depolarizedROS levels showed that shikonin up-regulated ROS levels in HCT116 and SW480 cells in a dose-dependent mannerConclusion:Shikonin induces apoptosis of colon cancer cells through mitochondria-mediated pathway，and Bcl-2 protein family and intracellular ROS levels play an important role in this process.

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