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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 10μmol/L以上处理人脐静脉内皮细胞6 h或5μmol/L以上处理24 h均能显著抑制其细胞活力。20(S)-PPD 10μmol/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。
Objective: To observe the effect of 20 (S) -PPD on protopanaxadiol (PPD) apoptosis in human umbilical vein endothelial cells and to explore its mechanism. Methods: Human umbilical vein endothelial cells were treated with 20 (S) -PPD, cell proliferation was detected by MTT assay, nuclear morphological changes were detected by DAPI staining, Calpain activity was detected by Calpain activity assay kit, endoplasmic reticulum fluorescent probe 3,3-bis Caspase-3, Caspase-8, bax, bcl-2, ATF-6 and Caspase-3 were detected by Western blot. Phosphorylated IRE1, phosphorylated PERK, ATF-4 and CHOP protein levels. Results: The viability of human umbilical vein endothelial cells treated with 20 (S) -PPD 10 μmol / L for 6 h or over 5 μmol / L for 24 h significantly inhibited cell viability. After treated with 20 (S) -PPD 10 μmol / L for 6 h, DAPI staining showed obvious chromatin condensation and nuclear fragmentation. Endoplasmic reticulum fluorescent probe 3,3-dihexyl-oxalicotin iodine The staining of the endoplasmic reticulum showed significant changes in the morphology of the endoplasmic reticulum, with fragments of endoplasmic reticulum and aggregated into granules in the nucleus. No significant changes were found in the activity of Calpain in the cytoplasm. The Caspase-3 active fragments, Caspase-3 The expression of bcl-2 was not significantly changed and the active fragment was not detected, bax expression was not significantly changed, but bcl-2 expression was significantly decreased, ATF-6 expression was not significantly changed, phosphorylation of IRE1 significantly increased; real-time quantitative PCR results showed splicing of XBP -1s mRNA was significantly increased, phosphorylated PERK and ATF-4 significantly increased, CHOP mRNA and protein levels increased significantly. CONCLUSION: 20 (S) -PPD can activate IRE1 and PERK through endoplasmic reticulum and further down-regulate bcl-2, leading to apoptosis of human umbilical vein endothelial cells.