冬凌草甲素对人宫颈癌HeLa细胞线粒体凋亡途径的影响*

Effects of oridonin on mitochondrial apoptotic pathway of human cervical carcinoma Hela cells*

摘要:

目的 观察冬凌草甲素对人宫颈癌HeLa细胞凋亡的影响，并探讨其诱导HeLa细胞线粒体凋亡的机 制。方法 体外培养HeLa 细胞，分别以0 μmol/L （设为空白对照组）、5 μmol/L、10 μmol/L、25 μmol/L、 50 μmol/L、100 μmol/L 冬凌草甲素处理12 h、24 h、36 h、48 h，CCK-8 法检测HeLa 细胞活力，计算IC50。 以0 μmol/L、5 μmol/L、10 μmol/L、25 μmol/L 冬凌草甲素处理HeLa 细胞24 h，采用Annexin V/PI 双染法和 TUNEL染色分别检测HeLa细胞凋亡；JC-1染色检测线粒体膜电位（Δψm）改变；Western blotting检测细胞色 素c（Cyt c）、B细胞淋巴瘤-2因子（Bcl-2）、Bcl-2相关X蛋白（Bax）的表达；比色法检测半胱氨酸蛋白酶-3 （Caspase-3）、半胱氨酸蛋白酶-9（Caspase-9）活性。结果 冬凌草甲素抑制HeLa 细胞细胞活力呈剂量-时间 依赖效应（P <0.05），其12 h、24 h、36 h、48 h 的IC50分别为95.63 μmol/L、32.24 μmol/L、19.58 μmol/L 和 4.13 μmol/L；与空白对照组比较，5 μmol/L、10 μmol/L、25 μmol/L冬凌草甲素组HeLa细胞凋亡率和凋亡指数 升高（P <0.05）；与空白对照组比较，5 μmol/L、10 μmol/L、25 μmol/L 冬凌草甲素组HeLa 细胞Δψm 降低 （P <0.05）；与空白对照组比较，5 μmol/L、10 μmol/L、25 μmol/L冬凌草甲素组HeLa细胞Cyt c、Bax表达升高 （P <0.05），Bcl-2、Bcl-2/Bax 比值降低（P <0.05）；与空白对照组比较，5 μmol/L、10 μmol/L、25 μmol/L冬 凌草甲素组HeLa细胞Caspase-3、Caspase-9活性升高（P <0.05）。结论 冬凌草甲素可诱导HeLa细胞凋亡，其 机制可能是通过线粒体凋亡途径实现。

Abstract:

Objective To investigate effects of oridonin on apoptosis of human cervical carcinoma Hela cells with the purpose of analyzing the mitochondrial mechanisms of apoptosis. Methods The Hela cells were cultured in vitro and the viability of the cells was measured with cell counting kit-8 (CCK-8) at 12- , 24- , 36- and 48-hour oridonin treatment at different doses (0, 5, 10, 25, 50, 100 μmol/L) and the 50% inhibitory concentration (IC50) were measured. The Hela cells were treated with different doses of oridonin (0, 5, 10, 25 μmol/L) for 24 h. The apoptosis of the cells was determined with Annexin V/PI double staining and TUNEL staining. The mitochondrial membrane potential (ψm) was observed by JC-1 staining. The expression of cytochrome c (Cyt c), human B cell lymphoma factor-2 (Bcl-2) and human Bcl-2-related X protein (Bax) were detected by Western blotting. The activity of cysteinyl aspartate specific proteinase-3 (Caspase- 3) and cysteinyl aspartate specific proteinase-9 (Caspase- 9) were detected by colorimetric method. Results Oridonin inhibited the viability of cells in a dose- and time-dependent manner (P < 0.05), with IC50 of 95.63, 32.24, 19.58 and 4.13 μmol/L after 12, 24, 36 and 48 hours, respectively. Compared with those in the blank control group, increased apoptotic rate and apoptotic index (P < 0.05) were observed in the HeLa cells treated with 5, 10 and 25 μmol/L oridonin. Compared with those in the blank control group, decreased ψm (P < 0.05) were observed in the HeLa cells treated with 5, 10 and 25 μmol/L oridonin. Compared with those in the blank control group, increased expressions of Cyt c, decreased expressions of Bcl-2, increased expressions of Bax and decreased expressions of Bcl-2/Bax (P < 0.05) were observed in the HeLa cells treated with 5, 10 and 25 μmol/L oridonin. Compared with those in the blank control group, increased activity of Caspase-3 and Caspase-9 (P < 0.05) were observed in the HeLa cells treated with 5, 10 and 25 μmol/L oridonin. Conclusion Oridonin can induce apoptosis of the HeLa cells, and the possible mechanisms may be related to the mitochondrial apoptotic pathway.