齐墩果酸经线粒体损伤诱导SMMC-7721细胞凋亡的作用

Function of oleanolic acidinduced apoptosis viamitochondrial damage in SMMC-7721 cells

目的研究齐墩果酸在诱导SMMC-7721肿瘤细胞凋亡过程中对线粒体功能及能量代谢的影响。方法取对数生长期细胞,分为空白对照组(0.01%二甲基亚砜)和3个浓度实验组(齐墩果酸,15,30,60μmol·L^(-1))。用齐墩果酸作用24 h,以CCK-8(Cell Counting Kit-8)法检测齐墩果酸对肝癌细胞增殖的抑制作用;以Annexin V-FITC双染法观察细胞凋亡;用JC-1染色检测线粒体膜电位;以酶联免疫吸附法测定线粒体呼吸链复合酶Ⅰ、Ⅱ、Ⅲ、Ⅳ活性;用比色法检测ATP酶活力和ATP水平;以免疫印迹法检测凋亡相关蛋白表达的变化。结果齐墩果酸能显著抑制SMMC-7721肝癌细胞株增殖,低中高3个浓度对细胞的抑制率分别为(21.96±3.37)%,(55.62±3.45)%,(89.56±2.11)%,空白对照组为(3.45±0.17)%,与空白对照组比较差异有统计学意义(均P<0.01);齐墩果酸作用24 h后,低中高3个浓度细胞总凋亡率分别为(18.43±2.16)%,(46.75±5.29)%,(72.03±4.07)%,与空白对照组的细胞总凋亡率为(2.18±1.11)%比较,差异有统计学意义(均P<0.01);低中高3个浓度齐墩果酸可降低线粒体膜电位,低荧光部分细胞所占百分比分别为(23.52±3.65)%,(67.45±5.12)%,(89.52±3.75)%,与空白对照组的(8.54±1.17)%比较,差异有统计学意义(P<0.01);在中高2个浓度齐墩果酸可显著抑制线粒体呼吸链复合酶Ⅰ、Ⅱ、Ⅲ和Ⅳ活性,引起细胞内Na^+-K^+-ATP酶和Ca^(2+)-Mg^(2+)-ATP酶活力降低,ATP水平下降。齐墩果酸可使Bcl-2表达减少,Bax表达增加,Cyt C在线粒体中表达降低而在胞浆中表达增加,cleaved-caspase-9和cleaved-caspase-3蛋白表达增加。结论齐墩果酸能明显抑制肝癌细胞的生长,诱导细胞凋亡,可能与其影响线粒体功能和能量代谢有关。

Objective To examine the effects of oleanolic acid（ OA）on mitochondrial function and energy metabolism during the process of apoptosis induction in human hepatocellular carcinoma SMMC- 7721 cells. Methods The logarithmic growth phase cells were divided into blank control group（ 0. 01% DMSO） and three concentration experimental groups（ oleanolic acid,15,30,60 μmol·L^（-1）） and treated for 24 h. The inhibitory effects of oleanolic acid（ OA） on the viability of SMMC- 7721 cells were examined using Cell Counting Kit- 8（ CCK- 8） assay. Apoptosis was determined by Annexin V- FITC staining and PI labeling. Quantitative changes of mitochondrial membrane potential at the early stage of the cell apoptosis were measured by JC-1 probe. The activities ofmitochondrial complexⅠ,Ⅱ,Ⅲ,Ⅳ were measured by human mitochondrial respiratory chain complexes by ELISA Kit.Na~＋- K~＋- ATPase and Ca^（2＋）-Mg^（2＋）-ATPase were detected by the colorimetric method. The expressions of apoptotic related proteins were determined by Western blotting. Results The results showed that OA strongly inhibited human hepatoma cells proliferation. The 15,30,60 μmol·L- 1oleanolic acid on the inhibitory rate were（ 21. 96 ± 3. 37） %,（ 55. 62 ± 3. 45） %,and（ 89. 56 ± 2. 11） % while the blank control group was（ 3. 45 ± 0. 17） %. There was significant difference between groups（ P〈0. 01）. When SMMC- 7721 cells were pretreated with OA for 24 h,OA induced apoptosis in a concentration- dependent manner. Oleanolic acid（ 15,30,60 μmol·L^（-1）） on the apoptosis rate were（ 18. 43 ± 2. 16） %,（ 46. 75 ± 5. 29） % and（ 72. 03 ± 4. 07） % while the blank control group was（ 2. 18 ± 1. 11） %. There was significant difference compared with blank control group（ P〈0. 01）. Compared with the corresponding blank control group（ 8. 54 ± 1. 17） %,OA caused an obvious decrease of mitochondrial membrane potential（ P〈0. 01）; the percentage of low fluorescence cells were（ 23. 52 ± 3. 65） %,（ 67. 45 ± 5. 12） %,（ 89. 52 ± 3. 75） % in experimental groups. OA inhibited the activities of mitochondrial complex Ⅰ,Ⅱ,Ⅲ and Ⅳ in SMMC- 7721 cells at middle and high concentration in experimental groups. The activity of ATPase and intracellular ATP level were decreased significantly in OA- treated SMMC- 7721 cells. Cytochrome C（ Cyt C） was accumulated in the cytosol and simultaneously decreased in mitochondria in a concentration- dependent manner. In addition,OA significant decreased of Bcl- 2 protein expression and increased of Bax protein expression,cleaved- caspase- 9 and cleaved- caspase- 3 activity. Conclusion The inhibitory effects and apoptosis induction of OA are related with its disruption of mitochondrial functions and energy metabolism.