积雪草苷对人骨肉瘤Saos-2细胞凋亡的影响

Effect of apoptosis on human osteosarcoma Saos-2 cells induced by asiaticoside

目的研究积雪草苷体外对人骨肉瘤细胞株Saos-2细胞生长、凋亡及氧化应激的影响。方法体外培养Saos-2细胞,MTT法检测不同时间(24、48、72 h)和不同浓度(20、40、80μmol/L)的积雪草苷对细胞生长的抑制作用;AnnexinV-FITC/PI双染流式细胞仪检测对细胞凋亡和细胞周期的影响;Western blotting法检测磷脂酰肌醇3-激酶(PI3Ks)、苏氨酸激酶(AKT)、p-AKT、糖原合成酶激酶3β(GSK-3β)、p-GSK-3β水平;Bradford法检测超氧化物歧化酶(SOD)、谷胱甘肽(GSH)和丙二醛(MDA)水平。结果与对照组比较,积雪草苷20、40、80μmol/L对Saos-2细胞的增殖抑制率逐渐增加,且具有时间和浓度相关性;与对照组比较,积雪草苷20、40、80μmol/L组对Saos-2细胞的凋亡率明显增加(P<0.01、0.001),且具有浓度相关性。与对照组比较,积雪草苷可剂量相关性的增加S期细胞比例(P<0.05、0.01)。与对照组比较,积雪草苷20、40、80μmol/L组PI3K、AKT、p-AKT、GSK-3β、p-GSK-3β表达均减少,且各蛋白水平随药物浓度的增加而逐渐降低。与对照组比较,积雪草苷20、40、80μmol/L组SOD和GSH活性均明显升高(P<0.05),而MDA水平则明显降低(P<0.05、0.01),均具有浓度相关性。结论积雪草苷具有抗人骨肉瘤Saos-2细胞活性的作用,通过调控PI3K/AKT/GSK-3β信号通路和抑制氧化应激抑制细胞生长,诱导细胞凋亡。

Objective To investigate the effects of asiaticoside on growth, apoptosis, oxidative stress of human osteosarcoma cell line Saos-2 in vitro. Methods Saos-2 cells were cultured in vitro, and MTT was performed to observe the inhibition of cell growth by asiaticoside at different times(24, 48, and 72 h) and different concentrations(20, 40, and 80 μmol/L). Annexin V-FITC/PI flow cytometry was used to determine the apoptosis rate and cell cycle. PI3K, AKT, p-AKT, GSK-3β, and p-GSK-3β contents were detected by Western blotting. Bradford was used to determine the contents of SOD, GSH, and MDA. Results Compared with the control group, the inhibition ratio of asiaticoside 20, 40, and 80 μmol/L on Saos-2 cells were increased, and had time and concentration correlation. Compared with the control group, the apoptosis rates of asiaticoside 20, 40, and 80 μmol/L on Saos-2 cells were significantly increased(P < 0.01, 0.001) with concentration correlation. Compared with the control group, asiaticoside could increase dose-dependent the ratio of S phase cell(P < 0.05, 0.01). Compared with the control group, PI3K, AKT, p-AKT, GSK-3β, and p-GSK-3β levels in asiaticoside 20, 40, and 80 μmol/L group were significantly decreased. And each protein level were decreased gradually with the increase of drug concentration. Compared with the control group, SOD and GSH levels in asiaticoside 20, 40, and80 μmol/L group were significantly increased(P < 0.05), while MDA level were significantly decreased(P < 0.05, 0.01), and they had concentration correlation. Conclusion Asiaticoside has an anti-osteosarcoma Saos-2 cell activity, and induces apoptosis by regulating PI3K/AKT/gsk-3 signaling pathways and inhibiting oxidative stress.