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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 investigate the effect of oleanolic acid on mitochondrial function and energy metabolism in the process of inducing apoptosis of SMMC-7721 tumor cells. Methods The cells in logarithmic growth phase were divided into blank control group (0.01% DMSO) and three concentrations of experimental group (oleanolic acid, 15, 30, 60μmol·L -1). Using oleanolic acid for 24 h, the inhibitory effect of oleanolic acid on the proliferation of hepatoma cells was detected by CCK-8 (Cell Counting Kit-8). The apoptosis of hepatoma cells was observed by Annexin V-FITC double staining. 1 were used to detect the mitochondrial membrane potential. The activities of mitochondrial respiratory chain enzymes Ⅰ, Ⅱ, Ⅲ and Ⅳ were measured by enzyme-linked immunosorbent assay. The activities of ATPase and ATP were detected by colorimetric assay. The expressions of apoptosis related protein The change. Results Oleanolic acid could significantly inhibit the proliferation of SMMC-7721 hepatocellular carcinoma cell lines. The inhibitory rates of the three concentrations of low, medium and high concentrations were (21.96 ± 3.37)%, (55.62 ± 3.45)%, (89.56 ± 2.11)%, (3.45 ± 0.17)% in the control group, the difference was statistically significant compared with the blank control group (all P <0.01); the total apoptotic rate of the cells in low, medium and high concentrations after oleanolic acid treatment for 24 h were (18.43 ± 2.16%, 46.75 ± 5.29%, and 72.03 ± 4.07%, respectively. Compared with the blank control group, the total apoptosis rate was (2.18 ± 1.11)%, the difference was statistically significant (all P <0.01); Low, medium and high concentrations of oleanolic acid decreased mitochondrial membrane potential, the percentage of cells with low fluorescence was (23.52 ± 3.65)%, (67.45 ± 5.12)% and (89.52 ± 3.75)%, respectively, (8.54 ± 1.17)%, the difference was statistically significant (P <0.01). Oleanolic acid could inhibit the activities of mitochondrial respiratory chain synthase Ⅰ, Ⅱ, Ⅲ and Ⅳ in middle and high concentrations of 2 concentrations, + -K ~ + -ATPase and Ca ~ (2 +) - Mg ~ (2 +) - ATPase decreased and ATP level decreased. Oleanolic acid reduced the expression of Bcl-2, Bax, Cyt C in mitochondria, increased in cytoplasm and increased the expression of cleaved-caspase-9 and cleaved-caspase-3. Conclusion Oleanolic acid can significantly inhibit the growth of hepatoma cells and induce apoptosis, which may be related to its effects on mitochondrial function and energy metabolism.