姜黄素诱导人肝癌细胞系Huh7自噬和凋亡

Curcumin inducing autophagy and apoptosis of human hepatocarcinoma cell line Huh7

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、40μmol/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5～40μmol/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20μmol/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20μmol/L姜黄素后自噬小体增多。与单独使用20μmol/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5～40μmol/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20μmol/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。

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 40μmol/L)-contained medium,and the proliferation ability was detected by CCK-8 kit.After culturing Huh7 cells using 5-40μmol/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 5mmol/L autophagy inhibitor 3-methyladenine(3-MA) and 20μmol/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 20μmol/L curcumin.Compared with the Huh7 cells cultured with the medium containing curcumin alone at 20μmol/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 5-40μmol/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 20μmol/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.