黄芪甲苷对成纤维细胞的毒性研究

曹静; 罗时成; 谈笑; 程月

doi:10.13898/j.cnki.issn.1000-2200.2021.11.002

摘要:

目的探索黄芪甲苷（astragaloside-Ⅳ, AS-Ⅳ）对小鼠成纤维细胞L929的毒性及影响机制, 为AS-Ⅳ在皮肤创伤治疗中的应用提供实验依据。

方法体外培养L929细胞, 在倒置显微镜下观察各组细胞形态, 采用CCK8检测细胞增殖, Annexin V-FITC/PI双染法检测细胞凋亡, Western blotting检测细胞内cleaved Caspase-3和γ-H2AX蛋白表达, 细胞划痕实验检测细胞迁移能力。

结果与Control组相比, 30、60、120 μmol/L的AS-Ⅳ处理L929细胞12 h、24 h, 随给药浓度及给药时间增加, 圆形细胞逐渐增多, 悬浮细胞增多, 死亡细胞逐渐增多。30、60、90、120 μmol/L AS-Ⅳ处理L929细胞12 h和24 h, 其OD值均低于Control组（P < 0.05~P < 0.01）; 在同一处理时间, 随着给药浓度增加, 其OD值逐渐降低（P < 0.05~P < 0.01）; 同一浓度AS-IV处理L929细胞12 h和24 h, 其OD值均低于处理6 h（P < 0.05~P < 0.01）。30、60、120 μmol/L AS-Ⅳ干预L929细胞24 h, 细胞凋亡率均高于Control组（P < 0.05~P < 0.01）。与Control组相比, 60 μmol/L AS-Ⅳ干预L929细胞6 h、12 h、24 h均能升高细胞内cleaved Caspase-3及γ-H2AX蛋白表达（P < 0.05~P < 0.01）; 与Control组相比, 30、60、120 μmol/L AS-Ⅳ处理L929细胞12 h均能升高细胞内γ-H2AX和cleaved Caspase-3蛋白表达（P < 0.05~P < 0.01）。与Control组相比, 30、60、120 μmol/L AS-Ⅳ干预L929细胞12 h及24 h, 相对迁移率均下降（P < 0.05~P < 0.01）。

结论AS-Ⅳ浓度大于30 μmol/L时能抑制L929细胞增殖、迁移, 诱导其凋亡。

Abstract:

ObjectiveTo investigate the cytotoxicity and mechanism of astragaloside-Ⅳ(AS-Ⅳ) on L929 cells, and provide the experimental basis for the application of AS-Ⅳ in treating skin wounds.

MethodsThe L929 cells were cultured in vitro, and the cell morphology of each group was observed under inverted microscope.The cell proliferation was detected using CCK8, and the apoptosis was detected using Annexin V-FITC/PI double staining.The expression levels of cleaved Caspase-3 and γ-H2AX protein were detected using Western blotting, and the cell migration ability was detected using cell scratch test.

ResultsCompared with the control group, the L929 cells were treated with 30, 60 and 120 μmol/L AS-Ⅳ for 12 h and 24 h, with the increasing of drug concentration and time, the number of round cells, suspended cells and dead cells increased gradually.The OD value of L929 cells treated with 30, 60, 90 and 120 μmol/L AS-Ⅳ for 12 h and 24 h were lower than that of control group(P < 0.05 to P < 0.01).At the same treatment time, with the increasing of drug concentration, the OD value decreased gradually(P < 0.05 to P < 0.01).The OD value of L929 cells treated with the same concentration of AS-Ⅳ for 12 h and 24 h were lower than that of L929 cells treated for 6 h(P < 0.05 to P < 0.01).The apoptosis rate of L929 cells treated with 30, 60 and 120 μmol/L AS-Ⅳ for 24 h were higher than that of control group(P < 0.05 to P < 0.01).Compared with the control group, the expression levels of cleaved Caspase-3 and γ-H2AX protein in L929 cells treated with 60 μmol/L AS-Ⅳ for 6 h, 12 h and 24 h increased(P < 0.05 to P < 0.01).Compared with the control group, the expression levels of cleaved caspase-3 and γ-H2AX protein in L929 cells treated with 30, 60 and 120 μmol/L AS-Ⅳ for 12 h increased(P < 0.05 to P < 0.01).Compared with the control group, the relative mobility of L929 cells treated with 30, 60 and 120 μmol/L AS-Ⅳ for 12 h and 24 h decreased(P < 0.05 to P < 0.01).

ConclusionsWhen the concentration of AS-Ⅳ is higher than 30 μmol/L, it can inhibit the proliferation and migration, and induce the apoptosis of L929 cells.

黄芪甲苷对成纤维细胞的毒性研究

Study on the toxicity of astragaloside-Ⅳ to fibroblasts