人参皂苷Rb1介导线粒体自噬途径对肝癌细胞凋亡的影响

Effect of Ginsenoside Rb1 mediated mitochondrial autophagy pathway on apoptosis of hepatocellular carcinoma cells

目的 研究人参皂苷Rb1对肝癌细胞凋亡及线粒体自噬的影响。方法 体外培养人肝癌Huh7细胞,将细胞随机分为对照组(正常培养的细胞)、低剂量实验组(0.1 mg·mL^(-1)的人参皂苷Rb1处理细胞)、中剂量实验组(0.2 mg·mL^(-1)的人参皂苷Rb1处理细胞)、高剂量实验组(0.4 mg·mL^(-1)的人参皂苷Rb1处理细胞)、抑制药组[10 mmol·L^(-1)的自噬抑制药3-甲基腺嘌呤(3-MA)处理细胞]和高剂量+抑制药组(0.4 mg·mL^(-1)的人参皂苷Rb1+10 mmol·L^(-1)的3-MA处理细胞)。用噻唑蓝(MTT)法检测各组细胞存活率,用流式细胞术检测各组细胞凋亡情况、JC-1绿/红荧光比值和活性氧(ROS)水平,用蛋白质印迹法检测各组细胞相关蛋白表达水平。结果 对照组、低剂量实验组、中剂量实验组、高剂量实验组、高剂量+抑制药组的细胞存活率分别为(100.00±2.01)%、(78.12±6.86)%、(66.84±6.42)%、(52.03±1.14)%和(74.23±6.35)%。对照组、低剂量实验组、中剂量实验组、高剂实验量组的细胞凋亡率分别为(3.59±0.28)%、(11.37±0.83)%、(16.66±1.22)%和(26.49±1.11)%;JC-1绿/红荧光比值分别为1.00±0.12、1.20±0.07、1.59±0.04和2.46±0.19;ROS水平分别为(11.33±1.04)%、(13.74±0.93)%、(21.33±2.03)%和(33.19±2.02)%;PTEN诱导假定激酶1(PINK1)蛋白水平分别为0.35±0.06、0.53±0.06、0.78±0.04和1.08±0.11;LC3Ⅱ/LC3Ⅰ蛋白水平分别为0.25±0.03、0.41±0.05、0.62±0.03和1.12±0.07。以上指标,低剂量实验组、中剂量实验组、高剂实验量组分别与对照组比较,差异均有统计学意义(均P<0.05);中剂量实验组、高剂量实验组分别与低剂量实验组比较,差异均有统计学意义(均P<0.05);中剂量实验组与高剂量实验组比较,高剂量实验组与高剂量+抑制药组比较,差异均有统计学意义(均P<0.05)。结论 人参皂苷Rb1可介导线粒体自噬诱导细胞凋亡进而抑制肝癌的进展。

Objective To investigate the effects of Ginsenoside Rbl on apoptosis and mitochondrial autophagy of hepatocellular carcinoma cells.Methods Human hepatocellular carcinoma Huh7 cells were cultured in vitro.The cells were randomly divided into control(normal cultured cells),experimental-L(0.1 mg·mL^(-1)Ginsenoside Rbl),experimental-M(0.2 mg·mL^(-1) Cinsenoside Rb1),experimental-H(0.4 mg·mL^(-1) Ginsenoside Rb1),3-methyladenine(3-MA,10 mmol·L^(-1)autophagy inhibitor 3-MA)and experimental-H+3-MA groups(0.4 mg·mL^(-1)ginsenoside Rbl+10 mmol·L^(-1)3-MA).Cell survival rate of each group was detected by methyl thiazolyl tetrazolium(MTT)assay;cell apoptosis,JC-1 green/red fluorescence ratio and reactive oxygen species(ROS)level were detected by flow cytometry;expression levels of related proteins were detected by Western blot.Results Cell survival rates in control,experimental-L,experimental-M and experimental-H and experimental-H+3-MA groups were(100.00±2.01)%,(78.12±6.86)%,(66.84±6.42)%,(52.03±1.14)%and(74.23±6.35)%.The apoptosis rates in control,experimental-L,experimental-M and experimental-H groups were(3.59±0.28)%,(11.37±0.83)%,(16.66±1.22)%and(26.49±1.11)%,respectively;JC-1 green/red fluorescence ratios were 1.00±0.12,1.20±0.07,1.59±0.04 and 2.46±0.19,respectively;R0S levels were(11.33±1.04)%,(13.74±0.93)%,(21.33±2.03)%and(33.19±2.02)%,respectively;PTEN induced postulated kinase 1(PINK1)protein levels were 0.35±0.06,0.53±0.06,0.78±0.04 and 1.08±0.11,respectively;the protein levels of LC3 II/LC3 I were 0.25±0.03,0.41±0.05,0.62±0.03 and 1.12±0.07,respectively.Compared with control group,the differences of the above indicators in experimental-L,experimental-M and experimental-H groups were statistically significant(all P<0.05);the experimental-M and experimental-H groups were compared with experimental-L group,the differences were statistically significant(all P<0.05);the differences between experimental-M group and experimental-H group,experimental-H group and experimental-H+3-MA group were statistically significant(all P<0.05).Conclusion Ginsenoside Rbl can mediate mitochondrial autophagy to induce cell apoptosis and inhibit the progression of liver cancer.