牛耳枫生物碱F21对HepG-2细胞的抑制作用及机制

Inhibitory effect of F21 from Daphniphyllum calycinum on HepG-2 cells and its mechanism

目的探讨牛耳枫生物碱F21对HepG-2增殖的影响及抗肿瘤作用机制。方法 F21 6.25～100 mg.L-1作用24,48和72 h,MTT法检测细胞抑制率并计算IC50。F21 10～100 mg.L-1作用48 h,瑞姬氏染色光学显微镜下观察细胞形态,琼脂糖凝胶电泳观察DNA的梯形条带,流式细胞仪检测细胞凋亡。F2110,30 mg.L-1+Z-VAD-FMK(胱天蛋白酶抑制剂)20μmol·L-1作用48 h,MTT法检测抑制率。结果 F21可明显抑制HepG-2细胞增殖,其于24,48和72 h的IC50值分别为5.3±0.1,1.3±0.1和(0.13±0.1)mg.L-1,且具有量效(F=232.39,P<0.05)和时效(F=65.59,P<0.05)关系。F21 10～30 mg.L-1可使HepG-2细胞体积增大、胞质内细胞核周围出现大量空泡、细胞膜完整、细胞核破碎甚至细胞形态消失。琼脂糖凝胶电泳未观察到典型的凋亡细胞DNA梯形条带。流式细胞术分析显示,与正常对照组比较,F21 10,30和100 mg.L-1处理HepG-2细胞48 h后,晚期凋亡率分别(17.34±0.01)%,(25.45±0.01)%和(43.67±0.03)%(P<0.05)。Z-VAD-FMK可明显抑制星形孢菌素诱导的细胞死亡。F21 10和30 mg.L-1组的增殖抑制率分别为(18.42±0.09)%和(42.77±0.01)%,而F21 10,30 mg.L-1+Z-VAD-FMK组的增殖抑制率分别为(16.46±0.01)%和(44.01±0.01)%,与相应的F21组相比无统计学差异。结论 F21在体外对HepG-2细胞具有显著的抑制作用,其作用机制并非通过激活胱天蛋白酶途径而诱导肿瘤细胞的凋亡。

OBJECTIVE To explore the inhibitory effect of F21 from Daphniphyllum calycinum on HepG-2 cells and its possible mechanism.METHODS The proliferation inhibition rate and IC50 of F21 were measured by MTT assay in vitro after HepG-2 cells were treated with F21 6.25-100 mg·L-1 for 24,48 and 72 h,respectively.Cell morphologic changes with Wright-Giemsa staining were observed by optical microscopy after HepG-2 cells were treated by F21 10-100 mg·L-1 for 48 h.DNA ladders were measured by agarose gel electrophoresis under the same condition.Apoptosis rates in HepG-2 cells were assessed by flow cytometry after HepG-2 cells were treated with F21 for 48 h.The inhibition pathway of F21 was analyzed by adding apoptosis inhibitor Z-VAD-FMK 20 μmol·L-1 by MTT.RESULTS MTT assay results revealed that F21 could obviously inhibit proliferation of HepG-2 cells and inhibitory rates significantly increased in a concentration-and time-dependent manner.The IC50 was 5.3±0.1,1.3±0.1 and（0.13±0.1）mg·L-1 after HepG-2 cells were treated with F21 for 24,48,and 72 h,respectively.The cell morphologic changes were significant as shown by the increase of the cell volume and many vacuoles in cytoplasm.The cell nucleus and membrane were complete after HepG-2 cells were treated with F21 10 mg·L-1.Typical DNA ladders were not observed.Compared with control group,the late apoptotic rate was（17.34±0.01）%,（25.45±0.01）% and（43.67±0.03）% when HepG-2 cells were cultured with F21 10,30 and 100 mg·L-1 for 48 h respectively.The inhibitory rate was（18.42±0.09）% in F21 10 mg·L-1 group and（16.46±0.01）% in F21 10 mg·L-1＋Z-VAD-FMK group.The inhibitory rate in F21 30 mg·L-1 group was（42.77±0.01）% and（44.01±0.01）% in F21 10,30 mg·L-1＋Z-VAD-FMK groups.There was no statistical significant difference between the groups.CONCLUSION F21 can inhibit proliferation of HepG-2 cells and the mechanism is not by activating caspase3.