马鞭草C部位单体4′-甲醚-黄芩素对人绒毛膜癌细胞增殖的抑制作用

Inhibitory effect of 4'-methylether-scutellarein on human choriocarcinoma JAR cells and the possible mechanism

目的:探讨马鞭草有效成分所提取的单体4′-甲醚-黄芩素(4′-methylether-scutellarein,4-MS)对人绒毛膜癌JAR细胞的增殖抑制作用及其相关机制。方法:JAR细胞传代后加入不同浓度4-MS作用一定时间,应用MTT法检测对细胞增殖的抑制作用,流式细胞术测定细胞凋亡率及细胞周期变化,AO/EB双染法区分早、晚期凋亡细胞和坏死细胞,RT-PCR技术分析4-MS对人绒毛膜癌JAR细胞凋亡相关基因Caspase3、p38MAPK及Survivin表达的影响,并进一步用Western blotting测定Caspase3、p38MAPK及Survivin在用药前后蛋白水平的表达差异。结果:不同质量浓度(10、20、40mg/L)的4-MS对JAR细胞均有增殖抑制作用,并随药物浓度和作用时间的增加而不断增强(P<0.05,P<0.01);流式细胞术显示,随着药物浓度的增加细胞凋亡率亦逐渐升高,G2/M期细胞所占比例增大(P<0.05);AO/EB双染后发现,随着4-MS剂量的增加,晚期凋亡细胞逐渐增多;20和40mg/L的4-MS作用48h后,JAR细胞中p38MAPK及Caspase3mRNA表达下降,Survivin mRNA表达上升,与对照组相比均有统计学意义(P<0.05);Western blotting证实,20mg/L和40mg/L4-MS作用48h后Survivin蛋白表达量显著下降,p38磷酸化水平升高,Caspase3被明显激活。结论:4-MS能抑制人绒毛膜癌JAR细胞的增殖并诱导凋亡,可能与其阻滞细胞生长于G/M期、抑制Survivin抗凋亡活性,直接激活p38MAPK信号通路和Caspase3活化有关。

Objective： To investigate the inhibitory, effect of 4＇-methylether-scutellarein （4-MS）, an extract from Verbena officinalis, on human choriocarcinoma JAR cell line and the possible mechanism. Methods： JAR cells were exposed to 4＇-methylether-scutellarein of different concentrations for 48 h. MTT assay was used to examine the anti-proliferative effect of 4＇-methylether-scutellarein. Flow cytometry was used to investigate the apoptosis and the changes of cell cycle. AO/EB double staining was applied to discriminate the apoptotic cells from dead ones. The changes of Survivin, p38-MAPK and Caspase 3 mRNA expressions were detected by RT-PCR in JAR ceils treated with 4-MS. Furthermore, Western blotting assay was used to determine Survivin protein expression, phosphorylation level of p38 and Caspase 3 in JAR cells before and after 4-MS treatment. Results：4-MS inhibited the proliferation of JAR cells in a dose- and timedependent manner （ P 〈 0.05, P 〈 0.01 ）. 4-MS treatment also induced apoptosis in JAR cells in a concentration-dependent manner, and percentage of cell cycle progression in G2/M phase increased dramatically compared with the control group （P 〈 0.05）. The result of AO/EB double staining showed that there were more viable apoptotic cells in 4-MS treated groups than in the control group and the number of non-viable apoptotic cells and dead cells increased with dose. Phosphorylated p38 and Caspase 3 expressions in 4-MS treated ceils were increased at both mRNA and protein levels according to RT-PCR and Western blotting results, while Survivin expression was down-regulated; there were significant differences between the 4-MS group and the control group （P 〈 0.05）. Conclusion ：4-MS can inhibit proliferation of JAR ceils and induce their apoptosis, which might be related to cell arrest at G2/M, down-regulation of Survivin activity, and direct activation of p38 MAPK pathway and Caspase 3.