(1.上海中医药大学基础医学院生物化学教研室(上海201203) ;2.上海中医药大学教学实验中心(上海201203))
20(S)-protopanaxadiol Activates ER Stress to Induce Cell Apoptosis in Human Umbilical Vein Endothelial Cells
ZHANG Xue-li1,HAN Zhi-fen2,XIA Hua-ying1,JIANG Jia-ye2,ZHANG Tong2
(1. College of Basic Medicine of Shanghai University of Traditional Chinese Medicine ;2. Experiment Center for Teaching and Learning of Shanghai University of Traditional Chinese Medicine)
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中文摘要:   目的:观察原人参二醇(protopanaxadiol,PPD)中20(S)-PPD对人脐静脉内皮细胞凋亡的作用并探讨其作用机制。  方法:20(S)-PPD处理人脐静脉内皮细胞,MTT法检测细胞增殖,DAPI染色检测细胞核形态变化,Calpain活性检测试剂盒检测胞质Calpain活性,内质网荧光探针3,3-二己基恶羰花青碘化物染色检测细胞内质网形态变化,实时定量 PCR检测拼接XBP-1以及CHOP mRNA水平,Western blot检测Caspase-3、Caspase-8、bax、bcl-2、ATF-6、磷酸化IRE1、磷酸化PERK、ATF-4以及CHOP蛋白水平。   结果:20(S)-PPD 10 μmol/L以上处理人脐静脉内皮细胞6 h或5 μmol/L 以上处理24 h均能显著抑制其细胞活力。20(S)-PPD 10 μmol/L 处理人脐静脉内皮细胞6 h后, DAPI 染色发现细胞出现明显染色质固缩及核碎片;内质网荧光探针3,3-二己基恶羰花青碘化物染色显示内质网形态发生显著变化,出现内质网碎片并且在核周聚集成颗粒;胞质Calpain活性未见显著改变;Western blot检测到凋亡相关蛋白Caspase-3活性片段,Caspase-8表达水平无显著变化且未检出其活性片段,bax 表达未见显著改变,而bcl-2表达显著下调, ATF-6表达未见显著改变,磷酸化IRE1显著增加;实时定量PCR结果显示拼接XBP-1s mRNA显著增加,磷酸化PERK及ATF-4显著增加,CHOP mRNA 及蛋白水平显著增加。  结论: 20(S)-PPD通过内质网应激激活IRE1与PERK途径,进一步下调bcl-2而导致人脐静脉内皮细胞凋亡。
Abstract:  Objective:To observe the effects of 20(S)-PPD on apoptosis and study the mechanism of 20(S)-PPD inducing cell apoptosis in human umbilical vein endothelial cells (HUVECs).  Methods:HUVECs were treated with 20(S)-PPD, and cell proliferation was detected by MTT assay. Cytoplasmic calpain activities were detected through Calpain Activity Assay Kit. Morphologies of endoplasmic reticulum were observed by ER fluorescent probe 3,3-dihexyloxacarbocyanine iodide staining. Splicing XBP-1 and CHOP mRNA levels were detected by Real-time quantitative PCR analysis. Caspase-3, Caspase-8, bax, bcl-2, ATF-6, phosphorylated IRE1, phosphorylated PERK and CHOP protein levels were detected by Western blot analysis.  Results:The cell growth of HUVECs was inhibited after treatment with 20(S)-PPD above 10 μmol/L for 6 h, or inhibited after treatment with 20(S)-PPD above 5 μmol/L for 24h. After 10 μmol/L 20(S)-PPD treatments, obvious condensed chromatins and fragmented nuclear bodies were observed in apoptotic cells by DAPI staining. The networks of ER appeared to be fragmented and aggregated to small patches distributed randomly around nucleus by 3,3-dihexyloxacarbocyanine iodide staining, while cytoplasmic calpain activities were not changed significantly. Cleaved Caspase-3 was detected in 10 μmol/L 20(S)-PPD treated HUVECs, Caspase-8, bax were not changed significantly, while bcl-2 protein levels decreased significantly after 10 μmol/L 20 (S)-PPD treatments. ATF6 was not changed significantly; phosphorylated IRE1 and splicing XBP-1 mRNA increased significantly; phosphorylated PERK and ATF-4 increased significantly. CHOP mRNA and protein levels increased significantly after 10 μmol/L 20(S)-PPD treatments.  Conclusion: 20(S)-PPD induces apoptosis in HUVECs by activating IRE1 and PERK signal pathway through ER stress to down-regulate bcl-2.
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