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Effect of atractylone on the viability and apoptosis of hepatoma HepG2 cells and related mechanism
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摘要:
目的 观察苍术酮对肝癌HepG2细胞活性、凋亡的影响，并探讨其作用机制。 方法 以肝癌HepG2细胞为研究对象，分为苍术酮低、中、高剂量组(5、10、20 μmol/L)，对照组加入等体积的DMSO。MMT比色法检测不同浓度苍术酮处理后HepG2细胞活性。流式细胞仪检测HepG2细胞凋亡率及线粒体膜电位。DCFH-DA荧光探针标记法检测HepG2细胞内ROS水平。Transwell实验检测苍术酮对HepG2细胞迁移能力的影响。蛋白质印迹法检测Bcl-2、Bax和Cleaved caspase-3蛋白表达水平。计量资料多组间比较采用单因素方差分析，组间的两两比较采用LSD-t检验。 结果 在苍术酮处理细胞24、48 h后，与对照组同一时间相比，苍术酮低、中、高剂量组的细胞活性呈下降趋势，且差异均有统计学意义(P值均＜0.05)，苍术酮处理HepG2细胞72 h的半数抑制率为26.19 μmol/L。苍术酮低、中、高剂量组的细胞凋亡率分别为14.34%、29.32%和50.12%，均高于对照组(0.32%)(P值均＜0.05)。与对照组比较，苍术酮低、中、高剂量组HepG2细胞中ROS的荧光强度明显升高(P值均＜0.05)。在苍术酮处理HepG2细胞48 h后，与对照组比较，苍术酮低、中、高剂量组的细胞迁移数量明显降低(132.67 ± 18.36、57.00 ± 9.26、31.00 ± 2.45 vs 258.11 ± 38.54，P值均＜0.05)；与对照组比较，苍术酮低、中、高剂量组能显著抑制抗凋亡因子Bcl-2的表达，促进凋亡因子Bax和Cleaved caspase-3的表达，差异均有统计学意义(P值均＜0.05)。 结论 苍术酮能够诱导HepG2细胞凋亡，并抑制其迁移，为进一步开发利用苍术酮提供一定的实验基础。 Abstract:Objective To investigate the effect of atractylone on the viability and apoptosis of hepatoma HepG2 cells and its mechanism of action. Methods Hepatoma HepG2 cells were selected and divided into low-, middle-, and high-dose atractylone groups (5, 10, and 20 μmol/L), and the cells in the control group were added with an equal volume of DMSO. MTT colorimetry was used to measure the viability of HepG2 cells after treatment with different concentrations of atractylone; flow cytometry was used to measure the apoptosis rate and mitochondrial membrane potential of HepG2 cells; the DCFH-DA fluorescent probe labeling method was used to measure the level of reactive oxygen species (ROS) in HepG2 cells; Transwell assay was used to evaluate the effect of atractylone on the migration ability of HepG2 cells; Western blot was used to measure the protein expression levels of Bcl-2, Bax, and cleaved caspase-3. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for comparison between two groups. Results After 24 and 48 hours of treatment with atractylone, compared with the control group, the low-, middle-, and high-dose atractylone groups had a tendency of reduction in cell viability (all P < 0.05), with a half inhibitory concentration of 26.19 μmol/L in atractylone treatment of HepG2 cells for 72 hours. The low-, middle-, and high-dose atractylone groups had a significantly higher apoptosis rate than the control group (14.34%/29.32%/50.12% vs 0.32%, all P < 0.05). Compared with the control group, the low-, middle-, and high-dose atractylone groups had a significant increase in the fluorescence intensity of ROS in HepG2 cells (all P < 0.05). After 48 hours of treatment with atractylone, compared with the control group, the low-, middle-, and high-dose atractylone groups had a significant reduction in the number of migrated cells (132.67±18.36/57.00±9.26/31.00±2.45 vs 258.11±38.54, P < 0.05). Compared with the control group, the low-, middle-, and high-dose atractylone groups had a significant reduction in the expression of the anti-apoptotic factor Bcl-2 and significant increases in the expression of the apoptotic factors Bax and cleaved caspase-3 (all P < 0.05). Conclusion Atractylone can induce the apoptosis and inhibit the migration of HepG2 cells, which provides an experimental basis for further development and utilization of atractylone. -
Key words:
- Atractylone /
- Liver Neoplasms, Experimental /
- Cellular Activity /
- Apoptosis
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注: a, 对照组；b，5 μmol/L组；c, 10 μmol/L组；d, 20 μmol/L组，e，苍术酮对HepG2细胞内ROS水平定量分析。
图 4 苍术酮对HepG2细胞中ROS水平的影响
表 1 苍术酮对HepG2细胞迁移的影响
组别 迁移数量 对照组 258.11±38.54 5 μmol/L组 132.67±18.361) 10 μmol/L组 57.00±9.261)2) 20 μmol/L组 31.00±2.451)2) 注：与对照组比较，1)P＜0.05；与5 μmol/L组比较，2)P＜0.05。 
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