重楼皂苷Ⅵ对肝癌HepG2细胞凋亡的作用
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摘要
目的:研究重楼皂苷Ⅵ(PⅥ)对肝癌HepG2细胞增殖、周期及凋亡的影响并初探其机制。方法:采用MTT法检测不同浓度(2、4、8μmol/L)PⅥ对肝癌HepG2细胞的增殖抑制作用;采用DAPI染色观察HepG2细胞形态学变化;利用流式细胞仪检测PⅥ对HepG2细胞内活性氧和线粒体膜电位变化;利用Annexin V-FITC/PI双染检测细胞凋亡变化;利用细胞周期检测试剂盒检测细胞周期变化。结果:MTT结果显示PⅥ能明显抑制肝癌HepG2细胞活力,呈时间与剂量依赖关系;DAPI染色发现随着药物浓度升高,细胞形态发生明显变化,出现核固缩,形成凋亡小体等。流式结果表明PⅥ能使HepG2细胞内活性氧水平升高,线粒体膜电位水平下降;与空白对照组比较,给药组HepG2细胞出现明显的凋亡细胞,且细胞凋亡率随PⅥ浓度增大而逐渐升高;给药组HepG2细胞处于G0/G1期的细胞数减少,S与G2/M期的细胞增多。结论:PⅥ在体外对肝癌HepG2细胞的增殖具有明显抑制作用,可能通过增加活性氧,使线粒体功能受损,改变细胞周期分布,诱导细胞凋亡。
Objective: To investigate the effects of polyphyllin Ⅵ(PⅥ) on the proliferation, cycle and apoptosis of hepatocellular carcinoma HepG2 cellsand to explore its mechanism. Methods: The inhibitory effect of PⅥ at different concentrations(2, 4, 8μmol/L) on the HepG2 cells wasevaluated by MTT method. The cell morphology changes were observed by DAPI staining.The changes of reactive oxygen species(ROS) and mitochondrial membrane potential(MMP) in HepG2 cells were determined by flow cytometry. The changes of apoptosis were analyzed by Annexin V-FITC/PI double staining, and cell cycle was detected by cell cycle kit. Results: MTT results showed that PⅥ could significantly inhibit the activity of HepG2 cells in a time-dose dependent manner. DAPI staining showed that, with the increase of drug concentration, cell morphology changed significantly,and karyopyknosis appeared, and formed apoptotic bodies and so on. When compared with the control group, HepG2 cells in the administration group showed obvious apoptosis, and the apoptosis rate increased with the increase of PⅥ concentration. The number of HepG2 cells in G0/G1 phase decreased in the administration group, while the number of cells in S and G2/M phase increased.Conclusion: PⅥ has a significant inhibitory effect on the proliferation of hepatocellular carcinoma HepG2 cells in vitro, which may damage mitochondrial function, change cell cycle distribution and induce apoptosis by increasing ROS.
Objective: To investigate the effects of polyphyllin Ⅵ(PⅥ) on the proliferation, cycle and apoptosis of hepatocellular carcinoma HepG2 cellsand to explore its mechanism. Methods: The inhibitory effect of PⅥ at different concentrations(2, 4, 8μmol/L) on the HepG2 cells wasevaluated by MTT method. The cell morphology changes were observed by DAPI staining.The changes of reactive oxygen species(ROS) and mitochondrial membrane potential(MMP) in HepG2 cells were determined by flow cytometry. The changes of apoptosis were analyzed by Annexin V-FITC/PI double staining, and cell cycle was detected by cell cycle kit. Results: MTT results showed that PⅥ could significantly inhibit the activity of HepG2 cells in a time-dose dependent manner. DAPI staining showed that, with the increase of drug concentration, cell morphology changed significantly,and karyopyknosis appeared, and formed apoptotic bodies and so on. When compared with the control group, HepG2 cells in the administration group showed obvious apoptosis, and the apoptosis rate increased with the increase of PⅥ concentration. The number of HepG2 cells in G0/G1 phase decreased in the administration group, while the number of cells in S and G2/M phase increased.Conclusion: PⅥ has a significant inhibitory effect on the proliferation of hepatocellular carcinoma HepG2 cells in vitro, which may damage mitochondrial function, change cell cycle distribution and induce apoptosis by increasing ROS.
引文
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[19]张泰魏,李燕,许露,等.逍遥散加减方对HPRL模型大鼠卵巢颗粒细胞凋亡及相关蛋白的影响.中华中医药杂志,2018,33(9):3909-3912
[20]孙超,吴铭杰,江泽群,等.白花蛇舌草有效成分2-羟基-3-甲基蒽醌通过IL-6/STAT3信号通路诱导肝癌细胞凋亡作用机制.中华中医药杂志,2018,33(12):5346-5350
[2]Jemal A,Bray F,Center M M,et al.Global cancer statistics.CA:A Cancer Journal for Clinicians,2011,61(2):69-90
[3]Jung K H,Noh J H,Kim J K,et al.Histone deacetylase 6 functions as a tumor suppressor by activating c-Jun NH2-terminal kinasemediated beclin 1-dependent autophagic cell death in liver cancer.Hepatology,2012,56(2):644-657
[4]Xu Y,Luo Q,Lin T,et al.U12,a UDCA derivative,acts as an antihepatoma drug lead and inhibits the mTOR/S6K1 and cyclin/CDK complex pathways.PloS One,2014,9(12):e113479
[5]Zhang Z,Zhang C,Ding Y,et al.The activation of p38 and JNK by ROS,contribute to OLO-2-mediated intrinsic apoptosis in human hepatocellular carcinoma cells.Food and Chemical Toxicology,2014,63:38-47
[6]国家药典委员会.中华人民共和国药典.一部.北京:中国医药科技出版社,2015
[7]Huang X,Gao W,Man S,et al.Isolation and identification of compounds present in rhizomes of Paris axialis H.Li and study of their cytotoxic effects.Latin American Journal of Pharmacy,2011,30(3):540-545
[8]Wang H,Zhai Z,Li N,et al.Steroidal saponin of Trillium tschonoskii.Reverses multidrug resistance of hepatocellular carcinoma.Phytomedicine,2013,20(11):985-991
[9]Lin Z,Liu Y,Li F,et al.Anti-lung cancer effects of polyphyllinⅥandⅦpotentially correlate with apoptosis in vitro and in vivo.Phytotherapy Research,2015,29(10):1568-1576
[10]Xue J,Chen F,Wang J,et al.Emodin protects against concanavalin A-induced hepatitis in mice through inhibiting activation of the p38MAPK-NF-u03ba B signaling pathway.Cellular Physiology&Biochemistry,2015,35(4):1557-1570
[11]Dong X,Jing F,Yin X,et al.Induction of apoptosis in heparg cell line by aloe-emodin through generation of reactive oxygen species and the mitochondrial pathway.Cellular Physiology&Biochemistry,2017,42(2):685
[12]Zuo D,Duan Z,Jia Y,et al.Amphipathic silica nanoparticles induce cytotoxicity through oxidative stress mediated and p53dependent apoptosis pathway in human liver cell line HL-7702and rat liver cell line BRL-3A.Colloids&Surfaces B Biointerfaces,2016,145:232
[13]Yu C,Zhou Z,Wang J,et al.In depth analysis of apoptosis induced by silica coated manganese oxide nanoparticles in vitro.Journal of Hazardous Materials,2015,283:519
[14]Zheng L,Li Y,Li X,et al.Combination of hydroxyl acetylated curcumin and ultrasound induces macrophage autophagy with antiapoptotic and anti-lipid aggregation effects.Cellular Physiology Bio chemistry&Pharmacology,2016,39(5):1746-1760
[15]Zhao Y,Suo Y.MMPT as a reactive oxygen species generator induces apoptosis via the depletion of intracellular GSH contents in A549 cells.European Journal of Pharmacology,2012,688(1-3):6
[16]Singh K K.Mitochondria damage checkpoint,aging,and cancer.Annals of the New York Academy of Sciences,2006,1067(1):182-190
[17]Wong R S.Apoptosis in cancer:from pathogenesis to treatment.Journal of Experimental&Clinical Cancer Research,2011,30(1):87
[18]Zhang Y,Bao Y L,Wu Y,et al.Alantolactone induces apoptosis in RKO cells through the generation of reactive oxygen species and the mitochondrial pathway.Molecular Medicine Reports,2013,8(4):967
[19]张泰魏,李燕,许露,等.逍遥散加减方对HPRL模型大鼠卵巢颗粒细胞凋亡及相关蛋白的影响.中华中医药杂志,2018,33(9):3909-3912
[20]孙超,吴铭杰,江泽群,等.白花蛇舌草有效成分2-羟基-3-甲基蒽醌通过IL-6/STAT3信号通路诱导肝癌细胞凋亡作用机制.中华中医药杂志,2018,33(12):5346-5350