自噬在熊果酸抑制人宫颈癌HeLa细胞增殖中的作用

Role of Autophagy in Ursolic Acid Inhibiting Human Cervical Cancer HeLa Cells Proliferation

[目的]分析自噬在熊果酸抑制人宫颈癌He La细胞增殖中的作用。[方法]体外培养人宫颈癌He La细胞,不同浓度(5、10、20μmol/L)的熊果酸处理48h后,采用MTT法检测He La细胞增殖抑制率的变化;透射电镜观察细胞自噬超微结构的改变;Western blotting检测自噬相关蛋白p62及Beclin-1的表达情况;微管相关蛋白1轻链3A/B(microtubule-associated protein 1 light chain 3 A/B,LC3A/B)免疫荧光检测熊果酸对He La细胞自噬水平的影响;检测自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)抑制自噬前后对熊果酸增殖抑制作用的影响。[结果]不同浓度(5、10、20μmol/L)的熊果酸处理组对He La细胞的抑制率分别为20.1%±1.3%、35.6%±2.6%和49.0%±1.0%,熊果酸可抑制He La细胞增殖并呈剂量依赖性(F=446.177,P<0.001);熊果酸能诱导He La细胞发生自噬:透射电镜观察发现经熊果酸处理后He La细胞中自噬囊泡明显增加;Western blotting分析表明熊果酸可呈剂量依赖性增加Beclin-1的表达,降低p62的表达;免疫荧光检测显示熊果酸处理后,He La细胞的荧光强度与对照组相比明显增强;3-MA与熊果酸联合作用于He La细胞时可抑制细胞增殖。[结论]熊果酸抑制He La细胞增殖,并诱导其发生自噬,自噬抑制剂3-MA能够增强熊果酸对He La细胞的增殖抑制作用。

[Purpose] To investigate the role of autophagy in the proliferation inhibition to He La cells by ursolic acid（UA）.[Methods]Human cervical cancer He La cells were cultured in vitro with various concentrations（5,10,20 μmol/L） of UA for 48 h and the proliferation inhibition rate of He La cells was detected by MTT method. The change of ultrastructure was observed under trans-mission electronic microscope（TEM）. The expressions of autophagy-associated proteins in He La cells treated with UA were determined by fluorescent staining of microtubule-associated protein 1light chain 3A/B（LC3A/B）. The protein levels of autophagy-associated protein p62 and Beclin-1were detected by Western blotting analysis. Detect the impact to cell proliferation inhibition after autophagy inhibitor 3-methyladenine（3-MA） inhibited autophagy in He La cells. [Results]The inhibition rate in 5,10 and 20μmol/L UA group was 20.1%±1.3%,35.6%±2.6% and 49.0%±1.0%,UA at various concentrations showed significantly proliferationinhibition of He La cells in a dose-dependent manner. Autophagy was induced in He La cells treated with UA as observed by TEM that autophagic vesicles were strikingly increased in He La cells. Western blotting analysis showed that UA aroused a dose-dependent increase in the expression of Beclin-1 and reduced the expression of p62. The fluorescent density of LC3A/B positive in He La cells increased significantly as compared with those in control group. 3-MA＋UA remarkablely decreased He La cell viability. [Conclusions]UA can inhibit He La cell proliferation and induce autophagy. Autophagy inhibitor 3-MA can enhance the proliferation inhibition of UA to He La cells.