摘要
目的探讨双氢青蒿素在体外诱导人肝癌细胞SMMC-7721凋亡作用,并初步研究其可能分子机制。方法 25,50,100,200和400μmol·L^(-1)双氢青蒿素作用SMMC-7721细胞24 h后,采用四甲基偶氮唑蓝(MTT)法检测双氢青蒿素对SMMC-7721细胞增殖的抑制作用,流式细胞术检测细胞周期和凋亡,激光共聚焦显微镜检测凋亡形态的变化,Rho123染色法检测细胞线粒体膜电位变化;Western blot法检测细胞内Bcl-2,Bax,Cleaved Caspase-3,Cleaved Caspase-9和Cyto C蛋白表达变化。结果 MTT实验结果表明,25~400μmol·L^(-1)双氢青蒿素对SMMC-7721细胞增殖具有明显的抑制作用。流式细胞仪检测周期结果显示,双氢青蒿素可以使SMMC-7721细胞周期阻滞于G2/M期。Hochest 333258染色结果显示,给药组细胞核出现染色质不均匀,核浓缩聚集、碎裂的凋亡形态。流式细胞仪检测凋亡结果显示,双氢青蒿素给药组细胞凋亡随药物浓度呈现升高趋势(P<0.01)。Rho 123染色结果显示,双氢青蒿素给药组细胞线粒体膜电位呈现明显的下降趋势(P<0.01)。Western blot结果显示,双氢青蒿素给药组细胞内Bcl-2蛋白表达下调,Bax蛋白表达上调,Bax/Bcl-2蛋白表达比例升高,Cleaved Caspase-3,Cleaved Caspase-9和Cyto C蛋白表达升高。结论双氢青蒿素能诱导SMMC-7721细胞凋亡,其凋亡机制可能与线粒体途径相关。
OBJECTIVE To investigate the apoptosis effect of human hepatocellular carcinoma cell line SMMC-7721 induced by dihydroartemisinin in vitro and the possible mechanism. METHODS After treatment with 25, 50, 100, 200, and 400 μmol·L^-1 dihydroartemisinin for 24 h. The proliferation inhibitory effect of dihydroartemisinin on SMMC-7721 cell was detected by MTT assay. Cell cycle and apoptosis were detected by flow cytometry. The change of apoptotic morphology was detected by confocal laser scanning microscopy. Rho 123 staining method was used to detect the changes of mitochondrial membrane potential. Western blot was used to detect expression of Bcl-2, Bax, Cleaved Caspase-3, Cleaved Caspase-9 and Cyto C. RESULTS MTT results showed that 25 -400μmol·L^-1 dihydroartemisinin can inhibit the proliferation of SMMC-7721 cells obviously. The cell cycle detection results of flow cy- tometry showed that dihydroartemisinin could block SMMC-7721 cell cycle in GJM phase. The results of Hochest 333258 staining showed that the nuclei were heterogeneous, condensed and fragmented in the DHA treatment group. The cell apoptosis detection results of flow cytometry showed that the apoptosis rate of dihydroartemisinin treated groups were increased obviously （P 〈 0. 01 ）. The results of Rho 123 staining showed that the mitochondrial membrane potential was decreased significantly （P 〈 0. 01 ）. Western blot results showed that the expression of Bcl-2 was down-regulated, expression of Bax was up-regulated, the ration of Bax/Bcl-2 was increased and the expression of Cleaved Caspase-3, Cleaved Caspase-9 and Cyto C were up-regulated. CONCLUSION Dihydroartemisinin can induce apoptosis of SMMC-7721 cells, on the mechanism of apoptosis may be related to mitochondrial pathway.
出处
《中国药学杂志》
CAS
CSCD
北大核心
2018年第3期187-192,共6页
Chinese Pharmaceutical Journal
基金
吉林省科技发展计划项目资助(20140204002YY)
吉林市科技计划项目资助(20156428)
吉林省卫计委青年科研课题资助(2015Q041)
关键词
双氢青蒿素
细胞凋亡
线粒体膜电位
肝癌细胞
G
/M期阻滞
dihydroartemisinin
apoptosis
mitochondrial membrane potential
human hepatoma cell
G2/ M arrest
