柴胡皂苷A诱导Huh7细胞凋亡并上调细胞自噬水平的研究
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摘要
目的:探讨柴胡皂苷A(SSA)对人肝癌Huh7细胞凋亡和自噬水平的影响。方法:通过MTT和流式细胞术检测不同浓度SSA对Huh7细胞增殖和凋亡的影响;构建重组质粒pEGFP-N1-LC3B并转染Huh7细胞,SSA培养基干预后共聚焦显微镜下观察细胞自噬水平。Western Blot检测凋亡蛋白Bax、Bcl-2、PCNA及自噬相关蛋白LC3B、Beclin1、Apg12-Apg5的表达变化。结果:SSA可明显抑制Huh7细胞增殖,促进细胞凋亡,增加胞质内自噬小体形成数量,上调Bax、LC3B-Ⅱ、Beclin1、Apg12-Apg5的表达,下调Bcl-2、PCNA的表达。结论:SSA体外诱导Huh7细胞凋亡的同时上调了细胞自噬水平。
Objective:To study whether saikosaponin A induces apoptosis of human hepatoma Huh7 cells is related to the change of autophagy level.Methods:The effects of different concentrations of SSA on proliferation and apoptosis of Huh7 cells were detected by MTT and flow cytometry,and then constructed recombinant plasmid pEGFP-N1-LC3 Band transfected into Huh7 cells.After intervened by SSA culture medium,the autophagy level was observed under confocal microscope.The expression of apoptosis proteins Bax,Bcl-2,PCNA and autophagy-related proteins LC3 B,Beclin1,and Apg12-Apg5 were detected by Western Blot.Results:SSA can significantly inhibit the proliferation of Huh7 cells,promote apoptosis,increase the number of autophagy bodies in the cytoplasm,up-regulate the expression of Bax,LC3 B-II,Beclin1,Apg12-Apg5 and downregulate the expression of Bcl-2,PCNA.Conclusion:SSA induced apoptosis of Huh7 cells in vitro and upregulated the autophagy level.
Objective:To study whether saikosaponin A induces apoptosis of human hepatoma Huh7 cells is related to the change of autophagy level.Methods:The effects of different concentrations of SSA on proliferation and apoptosis of Huh7 cells were detected by MTT and flow cytometry,and then constructed recombinant plasmid pEGFP-N1-LC3 Band transfected into Huh7 cells.After intervened by SSA culture medium,the autophagy level was observed under confocal microscope.The expression of apoptosis proteins Bax,Bcl-2,PCNA and autophagy-related proteins LC3 B,Beclin1,and Apg12-Apg5 were detected by Western Blot.Results:SSA can significantly inhibit the proliferation of Huh7 cells,promote apoptosis,increase the number of autophagy bodies in the cytoplasm,up-regulate the expression of Bax,LC3 B-II,Beclin1,Apg12-Apg5 and downregulate the expression of Bcl-2,PCNA.Conclusion:SSA induced apoptosis of Huh7 cells in vitro and upregulated the autophagy level.
引文
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17杨春燕.柴胡皂苷D诱导肝癌细胞株HepG2发生自噬的研究[J].齐齐哈尔医学院学报,2015,36(29):4373-74.
18 Xiao W,Ren MD,Lu GF,et al.The effects of saikosaponin-d on the expression of human hepatocellular carcinoma cell BECN1and autophagic function[J].Journal of Xi'an Jiaotong University(Medical Sciences),2017,38(01):127-130+150.
2 Mari1o G,Niso-Santano M,Baehrecke EH,et al.Selfconsumption:The interplay of autophagy and apoptosis[J].Nature Reviews Molecular Cell Biology,2014,15(2):81-94.
3 Kan YY,Wang YJ,Li Q,et al.Interaction between autophagy and apoptosis during tumor development[J].Chinese Journal of Comparative Medicine,2018,28(2):106-112.
4 Mizushima N,Levine B,Cuervo AM,et al.Autophagy fights disease through cellular self-digestion[J].Nature,2008,451(7182):1069-1075.
5 White E,Mehnert JM,Chan CS.Autophagy,metabolism,and cancer.[J].Cold Spring Harbor Symposia on Quantitative Biology,2015,21(22):5037.
6 Hu SC,Lee IT,Yen MH,et al.Anti-melanoma activity of Bupleurum chinense,Bupleurum kaoi and nanoparticle formulation of their major bioactive compound saikosaponin-d[J].Journal of ethnopharmacology,2016,179:432-442.
7 Xu KY,Li ZF,Gao J.Effect of Shu-yu capsule on liver-QI stagnation ratmodel of depressive disorder based on the analysisof gene expression microarray technique[J].Chinese Pharmacological Bulletin,2014,30(3):437-438.
8 Zheng TS,Li X.Research progress in mitochondrial apoptosis pathway[J].medical recapitulate,2013,19(18):3282-3285.
9 Andromachi K,Fabio F,Romilda C,et al.Autophagy and apoptosis-related genes in chronic liver disease and hepatocellular carcinoma[J].BMC Gastroenterology,2012,12(1):1-11.
10 Kang R,Zeh HJ,Lotze MT,et al.The Beclin 1network regulates autophagy and apoptosis[J].Cell Death&Differentiation,2011,18(4):571.
11 Yang Z,Klionsky DJ.Mammalian autophagy:Core molecular machinery and signaling regulation.[J].Current Opinion in Cell Biology,2010,22(2):124.
12 Hamasaki M,Furuta N,Matsuda A,et al.Autophagosomes form at ER-mitochondria contact sites[J].Nature,2013,495(7441):389-393.
13 Yang Z,Klionsky DJ.An overview of the molecular mechanism of autophagy[M].Springer Berlin Heidelberg,2009,335(1):1-32.
14 Klionsky DJ,Abdalla FC,Abeliovich H,et al.Guidelines for the use and interpretation of assays for monitoring autophagy[J].Autophagy,2012,8(4):445-544.
15 Huang R,Xu XJ,Liang YC,et al.Construction of prokaryotic expression vector with human autophagyrelated LC3Bgene and its activity detection[J].Military Medical Sciences,2014,(11):867-870.
16 Wu SL,Ding HL,Huang YY,et al.Autophagy in clinical therapy of tumor:research progress[J].Chinese Journal of Clinical Medicine,2017,24(5):797-801.
17杨春燕.柴胡皂苷D诱导肝癌细胞株HepG2发生自噬的研究[J].齐齐哈尔医学院学报,2015,36(29):4373-74.
18 Xiao W,Ren MD,Lu GF,et al.The effects of saikosaponin-d on the expression of human hepatocellular carcinoma cell BECN1and autophagic function[J].Journal of Xi'an Jiaotong University(Medical Sciences),2017,38(01):127-130+150.