内质网应激调控重楼皂苷Ⅰ诱导肝癌HepG2细胞凋亡的研究  

The Study on Regulation of Endoplasmic Reticulum Stress in Polyphyllin Ⅰ-Induced Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells

作　　者：阳丹丹 齐琪 陈正礼[1,2] 陈兵[1] 黄超[1,2] 罗启慧[1,2] 刘文涛[1,2] 

机构地区：[1]四川农业大学动物医学院,实验动物疾病模型研究室,成都611130 [2]四川农业大学动物医学院,动物疾病与人类健康中心四川省重点实验室,成都611130

出　　处：《中国细胞生物学学报》2017年第11期1397-1406,共10页Chinese Journal of Cell Biology

基　　金：国家科技支撑计划课题(批准号:2014BAI03B01);国家重大科学仪器设备开发专项(批准号:2013YQ49085906)资助的课题~~

摘　　要：该文探讨了内质网应激在重楼皂苷Ⅰ(polyphyllin I,PPI)诱导人肝癌Hep G2细胞凋亡中的作用。采用CCK-8法检测不同浓度的重楼皂苷I处理Hep G2细胞6、12、24 h后细胞的增殖情况;采用Hoechst 33258染色检测细胞凋亡情况;应用q RT-PCR(Real-time quantitative PCR)检测内质网应激相关基因GRP78、ATF-6、PERK、IRE-1的m RNA水平;采用Western blot方法分析重楼皂苷I对Cleaved-caspase12、Caspase-12、Cleaved-caspase3、Bax、Bcl-2、IRE-1、XBP1、CHOP和P-JNK1蛋白质水平的影响;并用流式细胞术检测20 mmol/L内质网抑制剂4-苯基丁酸(4-phenyl butyric acid,4-PBA)对细胞凋亡率的影响。结果发现,重楼皂苷I以时间–浓度依赖的方式抑制Hep G2细胞的增殖;细胞出现典型的凋亡形态;ATF-6和PERK m RNA水平无明显变化,GRP78和IRE-1 mRNA水平较阴性对照组(0 h)均显著增加(P<0.05);重楼皂苷I通过增加Bax、减少Bcl-2蛋白质水平进而活化Caspase-3来诱导细胞凋亡。进一步研究发现,重楼皂苷I可上调内质网应激通路的IRE-1,下调P-JNK1和XBP1蛋白质水平及活化内质网应激的标志蛋白质Caspase-12,但CHOP蛋白质水平无明显变化。流式细胞术结果显示,4-PBA与2.5μmol/L重楼皂苷I联合作用组的细胞凋亡率显著增加(P<0.05)。因此,内质网应激参与了重楼皂苷I诱导人肝癌Hep G2细胞的凋亡,且在该过程中对细胞起到保护作用。This work was aimed to investigate the role of endoplasrnic reticulum stress in polyphyllin 1-induced apoptosis in human hepatocellular carcinoma HepG2 cells. HepG2 cells were treated with the different concentrations of polyphyllin I for 6, 12, 24 h, the inhibition of cell proliferation was detected by CCK-8; the qRT- PCR （Real-time quantitative PCR） was applied to detect GRP78, ATF-6, 1RE-1 and PERK mRNA levels; Westernblot was applied to determine the protein levels of Cleaved-caspasel2, Caspase-12, Cleaved-caspase3, Bax, Bcl-2, IRE-l, XBP1, CHOP and P-JNK1; the flow cytometry was applied to detect the effect of 20 mmol/L 4-PBA on apoptotic rate. The result showed that polyphyllin I could inhibit the proliferation and induce apoptosis of HepG2 cells in dose- and time-dependent manners; qRT-PCR results showed that the levels of ATF-6 and PERK mRNA had no obvious change, but the levels of GRP78 and IRE-I mRNA increased significantly, compared with the negative control group （P〈0.05）; Western blot results showed that polyphyllin I could up-regulate the levels of apoptotic proteins Bax and Cleaved-caspase3, and down-regulate the level of the anti-apoptotic protein Bcl-2. Our further study revealed that polyphyllin I could up-regulate the levels of endoplasmic reticulum stress proteins IRE-1 and Cleaved-caspasel 2, and down-regulate the levels of P-JNK1 and XBP 1 proteins, while the protein level of CHOP was no significant difference （P〉0.05）. Flow cytometry results showed that the apoptotic rate increased significantly in combination group of 4-PBA with 2.5 pmol/L polyphyllin 1 （P〈0.01）. In conclusion, endoplasmic reticulum stress was involved in polyphyllin I inducing HepG2 cells apoptosis, which played a protective role in this process.

关 键 词：重楼皂苷Ⅰ 细胞凋亡 内质网应激 HEPG2细胞 

分 类 号：R285[医药卫生—中药学]