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柴胡皂甙D诱导肝癌细胞同时发生凋亡及类凋亡性细胞死亡 被引量:10

Saikosaponin-d simultaneously induces apoptotic and paraptosis-like cell death in hepatoma cells
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摘要 目的探讨柴胡皂甙D(saikosaponin-d,SSD)诱导人肝癌Hep3B细胞死亡的作用及其可能机制。方法将肝癌Hep3B细胞分为空白对照组、DMSO(溶媒)对照组和3个SSD(5、10、15μmol/L)处理组,MTT法检测细胞活力;Annexin V-FITC/PI双标记后采用流式细胞仪定量检测死亡细胞的比例;Hoechst 33258染色后在形态学方面检测细胞凋亡;分光光度法检测半胱天冬酶-3(caspase-3)凋亡蛋白酶活性;倒置显微镜观察细胞形态改变;Western blotting和Real-time PCR技术分别用于检测CHOP(C/EBP homology protein)蛋白和mRNA表达。结果MTT结果显示,SSD浓度和时间依赖性抑制Hep3B细胞生长;流式细胞术结果表明SSD诱导Hep3B细胞死亡;Hoechst 33258染色后在荧光显微镜下观察,发现SSD处理组的细胞核出现凋亡细胞特征性的核染色体凝集、核固缩和碎裂;SSD可明显激活关键的凋亡执行分子caspase-3;通过倒置显微镜观察到SSD处理的细胞内出现大量空泡样改变——类凋亡的发生;总caspase抑制剂Z-VAD-FMK预处理能够有效抑制caspase-3活性,但仅能部分抑制SSD诱导的细胞死亡,且不能减少SSD诱导的细胞内空泡形成;SSD诱导应激诱导性分子CHOP蛋白和mRNA表达上调,该作用不能被Z-VAD-FMK抑制。结论SSD可诱导人肝癌Hep3B细胞同时发生caspase依赖性凋亡和非caspase依赖性类凋亡,其中CHOP表达升高可能是参与SSD诱导类凋亡的分子机制之一。 Objective To investigate the cell death-inducing effect of saikosaponin-d(SSD)on human hepatoma Hep3B cells and its potential mechanism.Methods Hepatoma Hep3B cells were divided into five groups:blank control group,DMSO(vehicle)group,and three SSD treatment groups treated with various doses of SSD(5,10,and 15μmol/L).MTT assay was used to evaluate cell viability.Flow cytometer was employed to quantitatively detect the percentage of dead cells after Annexin V-FITC/PI double staining.Apoptosis was detected morphologically after Hoechst 33258 staining.The activity of caspase-3 apoptotic protease was determined by spectrophotometry.Cell morphologic changes were observed with an inverted microscope.Western blotting and Real-time PCR were employed to evaluate the expression levels of C/EBP homology(CHOP)protein and mRNA,respectively.Results MTT assay showed that SSD inhibited the viability of human hepatoma Hep3B cells in concentration-and time-dependent manners.Sinomenine hydrochloride induced the death of Hep3B cells in a concentration-dependent manner indicated by flow cytometry.After staining with Hoechst 33258,the nuclei of SSD-treated cells showed nucleosomal agglutination,nucleosomal shrinkage and fragmentation under the fluorescence microscope,which are the characteristics of apoptotic cells.SSD significantly activated the key apoptotic executor caspase-3.The occurrence of paraptosis,characterized by extensive cytoplasmic vacuoles,was observed in SSD-treated cells under an inverted microscope.The pretreatment of a pancaspase inhibitor Z-VAD-FMK completely inhibited caspase-3 activity triggered by SSD,but only partially suppressed cell death and could not reduce the cytoplasmic vacuolation in SSD-treated cells.The protein and mRNA expressions of CHOP,a stress-inducible molecule,were upregulated by SSD,which could not be inhibited by Z-VAD-FMK.Conclusion SSD can simultaneously induce caspase-dependent apoptosis and caspase-independent paraptosis in human hepatoma Hep3B cells.The upregulated expression of CHOP may be the mechanism involved in SSD-induced paraptosis.
作者 卢新兰 卢朝辉 曹琰 张旭 王新 李雅睿 郭丹 和水祥 LU Xinlan;LU Chaohui;CAO Yan;ZHANG Xu;WANG Xin;LI Yarui;GUO Dan;HE Shuixiang(Department of Gastroenterology,The First Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710061;Department of Pathogen Biology and Immunology,Basic Medical College of Xi'an Jiaotong University,Xi'an 710061;Department of Gastroenterology,The First People's Hospital of Weinan,Weinan 714000,China)
出处 《西安交通大学学报(医学版)》 CAS CSCD 北大核心 2020年第6期966-971,共6页 Journal of Xi’an Jiaotong University(Medical Sciences)
基金 国家自然科学基金资助项目(No.81502095)。
关键词 柴胡皂甙D 细胞凋亡 类凋亡 肝癌 CHOP saikosaponin-d apoptosis paraptosis hepatoma C/EBP homology protein(CHOP)
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