地榆皂苷Ⅱ通过磷脂酰肌醇-3激酶/蛋白激酶B信号对神经母细胞瘤SK-N-SH细胞的作用研究

Study on the effect of Ziyuglycoside Ⅱ on neuroblastoma SK-N-SH cells via phosphoinositide 3-kinase/protein kinase B signaling

目的探讨地榆皂苷Ⅱ通过磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(AKT)信号调控线粒体凋亡途径诱导神经母细胞瘤SK-N-SH细胞凋亡的机制。方法将神经母细胞瘤SK-N-SH细胞分成对照组、低剂量实验组(40μmol·L^(-1)地榆皂苷Ⅱ处理)、高剂量实验组(80μmol·L^(-1)地榆皂苷Ⅱ处理)、低剂量实验组+胰岛素样生长因子-1(IGF-1)组(40μmol·L^(-1)地榆皂苷Ⅱ+10 ng·mL^(-1) IGF-1处理)、高剂量实验组+IGF-1组(80μmol·L^(-1)地榆皂苷Ⅱ+10 ng·mL^(-1) IGF-1处理)。用蛋白质印迹法检测PI3K/AKT信号相关蛋白和胞浆、线粒体中细胞色素C(CytC)蛋白表达水平,用细胞计数试剂盒-8(CCK-8)实验检测细胞增殖情况,用流式细胞术检测细胞凋亡情况,用线粒体膜电位检测试剂盒(JC-1)法检测线粒体膜电位水平。结果对照组、低、高剂量实验组、低剂量实验组+IGF-1组、高剂量实验组+IGF-1组细胞中p-PI3K/PI3K蛋白表达水平分别为0.85±0.09,0.63±0.06,0.36±0.04,0.77±0.06和0.53±0.05,p-AKT/AKT蛋白表达水平分别为0.75±0.07,0.56±0.05,0.32±0.03,0.68±0.06和0.49±0.04,细胞存活率分别为(100.00±11.47)%,(80.65±7.15)%,(62.95±4.81)%,(92.68±6.84)%和(75.94±7.82)%,细胞凋亡率分别为(4.56±0.45)%,(8.94±0.86)%,(13.84±1.12)%,(5.95±0.52)%和(8.62±0.61)%,胞浆中CytC蛋白表达水平分别为0.30±0.04,0.42±0.05,0.59±0.05,0.37±0.04和0.44±0.06,线粒体中CytC蛋白表达水平分别为0.45±0.05,0.33±0.02,0.23±0.03,0.38±0.03和0.30±0.04,线粒体膜电位分别为1.00±0.09,0.75±0.07,0.60±0.06,0.87±0.08和0.72±0.07。对照组的上述指标与低、高剂量实验组比较,低剂量实验组的上述指标与高剂量实验组、低剂量实验组+IGF-1组比较,高剂量实验组的上述指标与高剂量实验组+IGF-1组比较,差异均有统计学意义(均P<0.05)。结论地榆皂苷Ⅱ通过抑制PI3K/AKT信号激活线粒体凋亡途径促进神经母细胞瘤SK-N-SH细胞凋亡。

Objective To explore the mechanism of Ziyuglycoside Ⅱ inducing neuroblastoma SK-N-SH cell apoptosis through phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)signaling to regulate mitochondrial apoptosis.Methods Neuroblastoma SK-N-SH cells were divided into control group,experimental-L(40μmol·L^(-1) Ziyuglycoside Ⅱ treatment),experimental-H group(80μmol·L^(-1) Ziyuglycoside Ⅱ treatment),experimental-L+insulin-like growth factor-1(IGF-1)group(40μmol·L^(-1) Ziyuglycoside Ⅱ,IGF-1 treatment),experimental-H+IGF-1 group(80μmol·L^(-1) Ziyuglycoside Ⅱ,IGF-1 treatment).The expression changes of PI3K/AKT signal-related proteins and cytochrome C(CytC)protein in cytoplasm and mitochondria were analyzed by Western blot.Cell proliferation was examined by cell counting kit-8 assay.Apoptosis was detected by flow cytometry.Mitochondrial membrane potential was measured by JC-1 assay.Results The expression levels of p-PI3K/PI3K protein in the control group,experimental-L group,experimental-H group,experimental-L+IGF-1 group and experimental-H+IGF-1 group were 0.85±0.09,0.63±0.06,0.36±0.04,0.77±0.06 and 0.53±0.05;p-AKT/AKT protein expression were 0.75±0.07,0.56±0.05,0.32±0.03,0.68±0.06 and 0.49±0.04;the cell survival rate were(100.00±11.47)%,(80.65±7.15)%,(62.95±4.81)%,(92.68±6.84)%and(75.94±7.82)%;the apoptosis rate were(4.56±0.45)%,(8.94±0.86)%,(13.84±1.12)%,(5.95±0.52)%and(8.62±0.61)%;cytoplasmic CytC protein expression were 0.30±0.04,0.42±0.05,0.59±0.05,0.37±0.04 and 0.44±0.06;mitochondrial CytC protein expression were 0.45±0.05,0.33±0.02,0.23±0.03,0.38±0.03 and 0.30±0.04;mitochondrial membrane potential were 1.00±0.09,0.75±0.07,0.60±0.06,0.87±0.08 and 0.72±0.07.Compared the control group with the experimental-L group and experimental-H group,the difference of the above indicators were statistically significant(all P<0.05);compared the experimental-L group with the experimental-H group,the difference were statistically significant(all P<0.05);compared the experimental-L group with the experimental-L+IGF-1 group,the difference were statistically significant(all P<0.05);the experimental-H group compared with the experimental-H+IGF-1 group,the difference were statistically significant(P<0.05).Conclusion Ziyuglycoside Ⅱ promote neuroblastoma SK-N-SH cell apoptosis by inhibiting PI3K/AKT signal to activate mitochondrial apoptosis pathway.