半枝莲提取物对肝癌HuH-7细胞自噬的影响及其作用机制

Effect of Scutellaria barbata extract on autophagy of hepatoma cell line HuH-7 and its mechanism

目的探讨半枝莲提取物对肝癌HuH-7细胞自噬的影响,并分析自噬在半枝莲提取物调节HuH-7细胞生长抑制中的作用机制。方法分别用100、200、500、1000和1500μg/mL浓度的半枝莲提取物以及500μg/mL半枝莲提取物+5 mol/L 3-Methyladenine(3-MA)处理HuH-7细胞24 h,设不加半枝莲提取物对照组。CCK-8试验检测各组细胞活力,确定最适半枝莲提取物处理浓度,并检测半枝莲提取物及3-MA逆转半枝莲提取物对HuH-7细胞活力的影响;激光共聚焦试验和Western blot法检测半枝莲提取物及3-MA逆转半枝莲提取物对HuH-7细胞自噬的影响;流式细胞术检测半枝莲提取物及3-MA逆转半枝莲提取物对HuH-7细胞凋亡的影响;Western blot法检测半枝莲提取物对PI3K/AKT/mTOR信号通路的影响。结果500μg/mL半枝莲提取物抑制HuH-7细胞活性40%左右,作用浓度适中,选择该浓度处理HuH-7细胞。与对照组相比,500μg/mL半枝莲提取物可明显诱导LC3B斑点的形成(F=2.597,P<0.01);LC3B-Ⅱ蛋白表达明显升高(F=2.127,P<0.01),而P62蛋白表达明显降低(F=0.589,P<0.01)。与500μg/mL半枝莲提取物组相比,500μg/mL半枝莲提取物+5 mol/L 3-MA细胞LC3B斑点形成明显降低(F=1.180,P<0.01);LC3B-Ⅱ蛋白表达明显降低(F=2.315,P<0.01),而P62蛋白表达明显升高(F=0.153,P<0.01);细胞活性明显恢复(F=1.462,P<0.01),且细胞凋亡率明显降低(F=0.119,P<0.01)。与对照组相比,500μg/mL半枝莲提取物可明显抑制PI3K/AKT/mTOR信号通路的活性。结论半枝莲提取物可通过激活自噬抑制肝癌HuH-7细胞生长,其机制可能与对PI3K/AKT/mTOR信号通路的抑制相关。

Objective To investigate the effect of Scutellaria barbata extract on autophagy of hepatoma cell line HuH-7 and analyze the mechanism of autophagy in the inhibition of HuH-7 cell growth regulated by Scutellaria barbata extract.Methods HuH-7 cells were treated with 100,200,500,1000 and 1500μg/mL Scutellaria barbata extract or 500μg/mL Scutellaria barbata extract with 5 mol/L 3-Methyladenine(3-MA)for 24 h respectively,using those untreated with Scutellaria barbata extract as control group.Cell viability of various groups were detected by CCK-8 assay to determine the optimal concentration of Scutellaria barbata extract as well as the effects of Scutellaria barbata extract and 3-MA reverse Scutellaria barbata extract on the viability of HuH-7 cells.The effects of Scutellaria barbata extract and 3-MA reverse Scutellaria barbata extract on autophagy of HuH-7 cells were determined by laser scanning confocal microscopy and Western blotting,while the effects on cell apoptosis were determined by Flow cytometry(FCM).The effect of Scutellaria barbata extract on PI3K/AKT/mTOR signal pathway was determined by Western blotting.Results 500μg/mL of Scutellaria barbata extract inhibited the activity of HuH-7 cells by about 40%and the effect concentration was moderate,which was selected to treat HuH-7 cells.Compared with control group,500μg/mL Scutellaria barbata extract significantly induced the formation of LC3B spots(F=2.597,P<0.01);The expression of LC3B-Ⅱprotein significantly increased(F=2.127,P<0.01),while the expression of P62 protein significantly decreased(F=0.589,P<0.01).Compared with those in 500μg/mL Scutellaria barbata extract group,the formation of LC3B spot in 500μg/mL Scutellaria barbata extract+5 mol/L 3-MA group significantly decreased(F=1.180,P<0.01);The expression of LC3B-Ⅱprotein significantly decreased(F=2.315,P<0.01),while the expression of P62 protein significantly increased(F=0.153,P<0.01);The cell activity was significantly restored(F=1.462,P<0.01),and apoptosis rate significantly decreased(F=0.119,P<0.01).Compared with control group,500μg/mL Scutellaria barbata extract significantly inhibited the activity of PI3K/AKT/mTOR signal pathway.Conclusion Scutellaria barbata extract inhibited the growth of HuH-7 cells by activating autophagy,and the mechanism may be related to the inhibition of PI3K/AKT/mTOR signal pathway.