积雪草酸通过细胞周期阻滞和诱导凋亡抑制人肝癌Huh7细胞

Inhibitory effect of asiatic acid on human liver carcinoma cells via cell cycle blockage and apoptosis induction

目的:探究积雪草酸(asiatic acid,AA)对人肝癌细胞恶性生物学行为的抑制作用及其机制。方法:选择人肝癌Huh7细胞作为研究对象,体外培养过程中分别给予0、5、10、25、50、100μmol/L的AA处理,采用CCK-8法和EdU实验测定其对细胞增殖的影响,流式细胞术检测凋亡和细胞周期的变化,WB检测凋亡相关蛋白p-AKT、p-ERK1/2、p-p38、cleaved-caspase3、cleavedcaspase9、BAX、Bcl-2、AKT、ERK、p38、pro-caspase3和pro-caspase9表达的变化。结果:AA能够以剂量和时间依赖的方式抑制Huh7细胞的增殖(均P<0.05);10μmol/L AA孵育Huh7细胞24 h后细胞增殖被显著抑制(P<0.05)、凋亡率显著升高(P<0.05)、细胞周期阻滞于G1期(P<0.05)。AA可诱导p-p38的表达,但以剂量依赖的方式抑制p-AKT和p-ERK的表达(均P<0.05);随着AA浓度的增加,cleaved-caspase3、cleaved-caspase9、BAX的水平增加,而Bcl-2的水平降低(均P<0.05)。结论:AA抑制人肝癌细胞的增殖并促使其凋亡,该作用与MAPK和PI3K/AKT通路有关。

Objective:To investigate the inhibitory effect of asiatic acid(AA)on malignant biological behaviors of human liver cancer cells and to explore the mechanism.Methods:Human liver cancer cell line(Huh7)was used as research subject,and treated with different concentrations of AA(0,5,10,25,50,100μmol/L)in vitro.The effect of AA on cell proliferation was determined by CCK-8 and EdU assay;the apoptosis and cell cycle distribution were detected by flow cytometry,while the expressions of apoptosis-related proteins(AKT,P-ERK 1/2,p38,cleaved-caspase3,cleaved-caspase9,BAX,Bcl-2,AKT,ERK,p38,pro-caspase 3 and pro-caspase 9)were examined by WB.Results:AA could inhibit the proliferation of Huh7 cells in a dose-and time-dependent manner(all P<0.05).After being incubated with 10μmol/L AA for 24 h,the proliferation of Huh7 cells was significantly inhibited(P<0.05),the apoptosis rate was significantly increased(P<0.05),and cell cycle was arrested in G1 phase(P<0.05).AA induced p-p38 expression,but inhibited the expression of p-AKT and p-ERK in a dose-dependent manner(all P<0.05).In addition,as the concentration of AA increased,the levels of cleaved-caspase 3,cleaved-caspase 9 and BAX increased,while the level of Bcl-2 decreased(all P<0.05).Conclusion:AA inhibits the proliferation of human liver cancer cells and promotes its apoptosis,which is associated with the MAPK and PI3 K/AKT pathways.