丹参酮ⅡA介导p38MAPK信号转导诱导人肝癌细胞凋亡

Tanshinone ⅡA induces apoptosis of liver cancer cells via p38MAPK signal transduction

目的:研究丹参酮ⅡA诱导人肝癌细胞凋亡和凋亡相关基因表达的p38MAPK信号转导通路,揭示其抗肝癌的部分机制.方法:4、8、16mg/L丹参酮ⅡA分别作用人肝癌SMMC-7721细胞48h后,免疫荧光染色观察细胞凋亡情况;琼脂糖凝胶电泳观察凋亡细胞特征性DNA条带;流式细胞仪法(Flow cytometry,FCM)检测细胞凋亡和细胞周期;荧光定量PCR检测Fas和Caspase-3基因mRNA的表达水平;并比较阻断p38MAPK信号通路后丹参酮ⅡA对肝癌细胞凋亡和Fas和Caspase-3基因mRNA的表达.结果:丹参酮ⅡA作用48h后,荧光显微镜下观察到经Hoechst染色的典型凋亡细胞.琼脂糖凝胶电泳可见凋亡细胞DNA呈规律性的梯状条带.4、8、16mg/L浓度丹参酮ⅡA作用人肝癌细胞后的细胞凋亡率分别为12.83%±1.51%,17.86%±2.70%和29.24%±7.58%,与对照组6.30%±2.08%比较均有显著性差异(P<0.01);阻断p38MAPK信号通路后,凋亡率和G0/G1期细胞比例明显降低(P<0.01).8mg/L丹参酮ⅡA作用人肝癌细胞48h后FasmRNA和Caspase-3mRNA的表达明显上升;阻断p38MAPK信号通路后,丹参酮ⅡA作用人肝癌细胞的FasmRNA和Caspase-3mRNA的表达明显下降.结论:丹参酮ⅡA能诱导人肝癌细胞株SMMC-7721凋亡,阻滞肝癌细胞于G0/G1期.通过p38MAPK信号转导通路上调Fas、Caspase-3mRNA的表达可能是其诱导肝癌细胞凋亡的重要机制.

AIM： To study the effect apoptosis via p38MAPK human liver cancer. of TS Ⅱ A on inducing signal transduction in METHODS： The apoptosis rate was assessed in liver cancer cells by immunofluorescence after treatment with TS Ⅱ A, and gel electrophoresis was used to observe the typical DNA ladder.Apoptosis and cell cycle were determined by flow cytometry （FCM）, the mRNA expression level of Fas and Caspase-3 were detected using fluorescent quantitation PCR. The mRNA expression level of Fas and Caspase-3 was detected after treatment with blocking agent. RESULTS： After SMMC-7721 cells were treated with 4, 8, 16 mg/L of TSIIA for 48 h, typical morphologic changes of apoptosis were observed by fluorescence microscopy using Hoechst staining. There were regular DNA ladders under agarose gel electrophoresis, after treatment with 4, 8, 16 mg/L of TSⅡA for 48 h, the cell apoptotic rates were respectively 12.83% ± 1.51%, 17.86% ± 2.70% and 29.24% ± 7.58%, showing significant difference （P 〈 0.01）. After signal transduction pathway of p38MAPK was blocked, the cell apoptotic rates and cell ratio of G0/G1 phase were decreased significantly （P 〈 0.01）. The mRNA expression of the Fas, Caspase-3 gene were increased obviously after treatment with 8 mg/L TS Ⅱ A for 48 h; whereas they were decreased significantly when the transduction pathway was blocked. CONCLUSION： TSIIA could induce the apoptosis of human liver cancer cells and arrest in G0/G1 phase. The mechanism might be related to up-regulated expression of Fas, Caspase-3 mRNA by regulating p38MAPK signal transduction pathway.