土槿皮乙酸对胶质瘤C6细胞生长的抑制作用

Inhibition of pseudolaric acid B on growth of glioma C6 cells

目的研究土槿皮乙酸(PAB)对大鼠胶质瘤C6细胞生长的抑制作用及其分子机制。方法采用MTT法测定不同质量浓度(0、0.1、0.2、0.4、0.8、1.6、8.0、16.0μg/mL)PAB对C6细胞增殖能力的影响。以0(对照组)、0.15、0.3、0.6μg/mL PAB处理后,采用Annexin V-FITC/PI双染色法经流式细胞仪检测C6细胞的凋亡情况,RT-PCR和Western blotting法检测凋亡抑制蛋白Survivin、自噬标志性分子微管相关蛋白1轻链3-β(LC3)和凋亡剪切产物聚腺苷二磷酸核糖聚合酶(Cleaved-PARP)的表达。结果 MTT法测定结果显示,PAB呈时间剂量依赖性抑制C6细胞的生长,PAB处理12、24、36、72 h后,PAB对C6细胞的半数致死剂量(IC50)分别为1.23、1.05、0.30、0.09μg/mL。流式细胞仪检测结果显示,0.15、0.30、0.60μg/mL PAB作用C6细胞36 h后细胞出现凋亡,并且随着PAB浓度的升高,细胞凋亡率呈上升趋势,分别为(24.89±0.92)%、(31.38±0.36)%和(62.84±2.22)%,与对照组比较差异均有统计学意义(P<0.05)。RT-PCR和Western blotting检测结果显示:PAB处理后的C6细胞内Survivin表达下降,LC3和Cleaved-PARP表达升高,与对照组相比差异均有统计学意义(P<0.05或P<0.01);且随着PAB浓度的升高,细胞内Survivin、LC3、Cleaved-PARP的表达变化呈剂量依赖性。结论 PAB可抑制大鼠胶质瘤C6细胞增殖,并促进其凋亡;下调Survivin的表达,上调LC3和Cleaved-PARP的表达为PAB可能的作用机制。

Objective To investigate the inhibitory effect of pseudolaric acid B（PAB） on the growth of glioma C6 cells,and explore the possible molecular mechanism.Methods The effects of different mass concentrations of PAB（0,0.1,0.2,0.4,0.8,1.6,8.0 and 16.0 μg/mL） on the growth of C6 cells were analysed by MTT assay.After treatment with 0（control）,0.15,0.3 and 0.6 μg/mL PAB,the apoptosis of C6 cells was detected by flow cytometry with Annexin V-FITC/PI double staining,and the expression of Survivin（apoptosis inhibition protein）,LC3（microtubule-associated protein 1 light chain 3-β） and cleaved poly（ADP-ribose） polymerase（Cleaved-PARP） was determined by RT-PCR and Western blotting.Results MTT assay revealed PAB inhibited the growth of C6 cells in a dose and time dependent manner.The median lethal dosages（IC50） of PAB on C6 cells were 1.23,1.05,0.30 and 0.09 μg/mL after treatment with PAB for 12,24,36,and 72 h respectively.Flow cytometry indicated that apoptosis of C6 cells occurred 36 h after treatment with 0.15,0.30 and 0.60 μg/mL PAB,and the apoptosis rates were（24.89±0.92）%,（31.38±0.36）% and（62.84±2.22）% respectively,which were significantly different from that of control group（P〈0.05）.RT-PCR and Western blotting demonstrated that the expression of Survivin in C6 cells decreased,and the expression of LC3 and Cleaved-PARP increased after treatment with PAB,which was significantly different from that of control group（P〈0.05 or P〈0.01）.With the concentration increase of PAB,the expression of Survivin,LC3 and Cleaved-PARP in cells changed in a dose dependent manner.Conclusion PAB can inhibit the growth of glioma C6 cells and induce cell apoptosis,which may be associated with the decreased expression of Survivin and increased expression of LC3 and Cleaved-PARP.