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  • 尹煜鹏,孙树汉*.姜黄素诱导人肝癌细胞系Huh7自噬和凋亡[J].第二军医大学学报,2019,40(1):38-42    [点击复制]
  • YIN Yu-peng,SUN Shu-han*.Curcumin inducing autophagy and apoptosis of human hepatocarcinoma cell line Huh7[J].Acad J Sec Mil Med Univ,2019,40(1):38-42   [点击复制]
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姜黄素诱导人肝癌细胞系Huh7自噬和凋亡
尹煜鹏,孙树汉*
0
(海军军医大学(第二军医大学)基础医学院医学遗传学教研室, 上海 200433
*通信作者)
摘要:
目的 探讨姜黄素诱导人肝癌细胞Huh7自噬和凋亡的作用,以及抑制自噬对姜黄素诱导凋亡的影响。方法 将姜黄素(5、10、20、40 μmol/L)加入人肝癌细胞系Huh7的培养液中,用CCK-8检测细胞增殖水平变化。Huh7细胞用5~40 μmol/L姜黄素培养48 h,通过蛋白质印迹法检测自噬微管相关蛋白1轻链3(LC3)-Ⅱ/LC3-Ⅰ的比值,免疫荧光法检测自噬小体的形成,评估Huh7细胞自噬水平的变化。通过流式细胞术检测Huh7细胞凋亡情况。加入5 mmol/L自噬抑制剂3-甲基嘌呤(3-MA)和20 μmol/L姜黄素培养Huh7细胞,检测Huh7细胞凋亡水平和自噬水平的变化。结果 CCK-8检测显示姜黄素能抑制Huh7细胞的增殖(P<0.05,P<0.01),且细胞凋亡率随着姜黄素浓度的升高而上升;蛋白质印迹检测显示加入姜黄素后LC3-Ⅱ/LC3-Ⅰ的比值上升(P<0.05,P<0.01),免疫荧光检测显示加入20 μmol/L姜黄素后自噬小体增多。与单独使用20 μmol/L姜黄素培养的Huh7细胞相比,姜黄素联用3-MA培养的Huh7细胞LC3-Ⅱ/LC3-Ⅰ的比值下降(P<0.01),自噬小体减少。流式细胞术检测发现5~40 μmol/L姜黄素促进了Huh7细胞凋亡(P<0.05,P<0.01),而联用3-MA后与单独使用20 μmol/L姜黄素培养相比引起的Huh7细胞凋亡减少(P<0.05)。结论 姜黄素能诱导Huh7细胞凋亡、抑制细胞增殖并促进细胞自噬,抑制自噬能减弱姜黄素对Huh7细胞的诱导凋亡效应。
关键词:  肝细胞癌  姜黄素  自噬  细胞凋亡
DOI:10.16781/j.0258-879x.2019.01.0038
投稿时间:2018-08-24修订日期:2018-11-23
基金项目:国家自然科学基金(81830085,8177110318).
Curcumin inducing autophagy and apoptosis of human hepatocarcinoma cell line Huh7
YIN Yu-peng,SUN Shu-han*
(Department of Medical Genetics, College of Basic Medical Sciences, Naval Medical University(Second Military Medical University), Shanghai 200433, China
*Corresponding author)
Abstract:
Objective To explore the role of curcumin in inducing autophagy and apoptosis of human hepatocarcinoma cell line Huh7, and the effect of autophagy inhibition on curcumin-induced apoptosis.Methods Human hepatocarcinoma cell line Huh7 was cultured using curcumin (5, 10, 20, and 40 μmol/L)-contained medium, and the proliferation ability was detected by CCK-8 kit. After culturing Huh7 cells using 5-40 μmol/L curcumincontained medium for 48 h, the expression levels of microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ and LC3-Ⅰ were measured by Western blotting, and the ratio of LC3-Ⅱ to LC3-Ⅰ was calculated. The autophagosome was observed under fluorescence microscope. The apoptosis level of Huh7 cells was measured by flow cytometry. Then Huh7 cells were cultured using the medium containing 5 mmol/L autophagy inhibitor 3-methyladenine (3-MA) and 20 μmol/L curcumin, and the apoptotic and autophagic levels were detected.Results CCK-8 assay showed that curcumin could significantly inhibit the proliferation of Huh7 cells in a dose dependent manner (P<0.05, P<0.01). Western blotting analysis showed that curcumin significantly increased the ratio of LC3-Ⅱ to LC3-Ⅰ (P<0.05, P<0.01). Immunofluorescence microscopy showed that the number of autophagosome increased after adding 20 μmol/L curcumin. Compared with the Huh7 cells cultured with the medium containing curcumin alone at 20 μmol/L, the the ratio of LC3-Ⅱ to LC3-Ⅰ was significantly decreased in the Huh7 cells cultured with the medium containing curcumin and 3-MA (P<0.01), and the number of autophagosome decreased. Flow cytometry showed that the 5-40 μmol/L curcumin significantly induced the apoptosis of Huh7 cells (P<0.05, P<0.01), and 3-MA combined with curcumin could significantly decrease the apoptosis of Huh7 cells compared with 20 μmol/L curcumin alone (P<0.05).Conclusion Curcumin induces the apoptosis of Huh7 cells, inhibits proliferation and increases autophagy level, and inhibition of autophagy can attenuate the apoptotic effect of curcumin on Huh7 cells.
Key words:  hepatocellular carcinoma  curcumin  autophagy  apoptosis