冬凌草甲素通过线粒体途径诱导人胆囊癌GBC-SD细胞凋亡

Oridonin-induced GBC-SD Cell Apoptosis through Mitochondrial Pathway

目的研究冬凌草甲素诱导人胆囊癌GBC-SD细胞的凋亡作用及其机制。方法 MTS法检测冬凌草甲素对GBC-SD细胞的生长抑制作用;瑞氏染色法观察细胞的形态学变化;流式细胞仪检测细胞凋亡率、线粒体膜电位改变、caspase-3活性变化;Western blot检测caspase-9活化情况。结果冬凌草甲素能显著抑制GBC-SD细胞增殖,呈时间剂量依赖性(P<0.05)。细胞形态学观察可见冬凌草甲素可诱导细胞发生凋亡。流式细胞仪检测结果显示28μmol/L药物处理GBC-SD细胞24h后,细胞凋亡率为51.28%±2.65%。随着药物浓度增加,GBC-SD细胞线粒体膜电位逐渐下降而caspase-3活性逐渐增强,56μmol/L组caspase-3活性是对照组的11倍。Western blot分析结果显示caspase-9酶原被激活,且活化条带随药物浓度的增加而增强。结论 冬凌草甲素可能通过细胞线粒体膜电位下降激活caspase-3并最终诱导GBC-SD细胞凋亡。

Objective To study the mechanisms of apoptosis of human gallbladder carcinoma GBC - SD cell induced by oridonin and the role of mitochondrial pathway in the apoptotic process. Methods MTS assay was uesd to examine the oridonin - induced growth inhibition of human gallbladder carcinoma GBC - SD cell. Wright＇s staining was used to observe the cell morphologic changes of apoptosis induced by oridonin. With a kind of double - dye assay of Annexin V ＋/PI,the rates of apoptotic cell were detected next to flow cytome- try. Similarly,the mitoehondria membrane potential and positive rate of caspase - 3 was also detected by flow cytometry. The expression of caspase - 9 was detected by Western blot. Results Oridonin obviously inhibited the growth of GBC - SD ceils in a time - and dose - de- pendent manners. Morphological changes indicated that oridonin induced GBC - SD cells apoptosis. Flow cytometry revealed the apoptosis rate values of 28p^mol/L oridonin 24h after administration were 51.28% ＋ 2.65%. The mitochondrial membrane potential of GBC - SD ceils gradually decreased while caspase - 3 activity gradually increased with the increase concentration of oridonin. The activity of caspase -3 was increased after treatment with oridonin （56~mol/L） about 11 times as much as the control value. Caspase -9 zymogen was acti- vated according to Western blot analysis. Conclusion Oridonin could induce GBC - SD cells apoptosis by the mechanism of mitochondri- al- regulated caspase pathway.