Objective: To explore the effect of shikonin on autophagy and apoptosis of human promyelocytic leukemia cells and its possible mechanism.
Methods: Human promyelocytic leukemia cells NB4 in the logarithmic growth phase were divided into control group (untreated NB4 cells), shikonin group (0.3 µmol/L shikonin treatment), 740Y-P group (15 µmol/L PI3K/Akt/mTOR pathway activator 740Y-P treatment), shikonin+740Y-P group (0.3 µmol/L shikonin and 15 µmol/L 740Y-P co-treatment), after 24 hours of treatment, the cells were used for subsequent experiments. CCK-8 method was used to detect cell viability, monodansylcadaverine (MDC) staining to detect the aggregation of autophagic vesicles, flow cytometry to detect cell apoptosis, and Western blot to detect the expression of Beclin1, LC3, p62, Bax, cleaved caspase-3, Bcl-2 and PI3K/Akt/mTOR pathway related proteins.
Results: Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were increased in the shikonin group, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were decreased (all P < 0.05). Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the 740Y-P group (all P < 0.05). Compared with the shikonin group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the shikonin+740Y-P group (all P < 0.05). Compared with the control group, the expression of PI3K/Akt/mTOR pathway related proteins p-PI3K, p-Akt, and p-mTOR in NB4 cells were significantly decreased in the shikonin group, while those in the 740Y-P group were increased (all P < 0.05). Compared with the shikonin group, the expressions of p-PI3K, p-Akt, and p-mTOR proteins in NB4 cells were significantly increased in the shikonin+740Y-P group (all P < 0.05).
Conclusion: Shikonin may promote autophagy and apoptosis of NB4 cells by inhibiting PI3K/Akt/mTOR pathway.
题目: 紫草素对人早幼粒细胞白血病细胞自噬和凋亡的影响.
目的: 探讨紫草素对人早幼粒细胞白血病细胞自噬和凋亡的影响及可能的机制。.
方法: 取对数生长期的人早幼粒细胞白血病细胞NB4，将其分为对照组（未处理的NB4细胞）、紫草素组（0.3 μmol/L紫草素处理）、740Y-P组（15 μmol/L PI3K/Akt/mTOR通路激活剂740Y-P处理）、紫草素+740Y-P组（0.3 μmol/L紫草素与15 μmol/L 740Y-P共同处 理），处理24 h后，用于后续实验，CCK-8法检测各组细胞活力，单丹磺酰戊二酸染色检测细胞自噬囊泡的聚集情况，流式细胞术检测细胞凋亡，Western blot检测各组细胞中Beclin1、LC3、p62、Bax、cleaved caspase-3、Bcl-2及PI3K/Akt/mTOR通路相关蛋白的表达。.
结果: 与对照组比较，紫草素组NB4细胞内紫色点状荧光强度、细胞凋亡率及Beclin1、LC3-Ⅱ/LC3-Ⅰ、cleaved caspase-3、Bax蛋白相对表达量升高，OD450值（24、48 h）及Bcl-2、p62蛋白相对表达量降低（均P <0.05）；与对照组比较，740Y-P组NB4细胞内紫色点状荧光强度、细胞凋亡率及Beclin1、LC3-Ⅱ/LC3-Ⅰ、cleaved caspase-3、Bax蛋白相对表达量降低，OD450值（24、48 h）及Bcl-2、p62蛋白相对表达量升高（均P <0.05）；与紫草素组比较，紫草素+740Y-P组NB4细胞内紫色点状荧光强度、细胞凋亡率及Beclin1、LC3-Ⅱ/LC3-Ⅰ、cleaved caspase-3、Bax蛋白相对表达量降低，OD450值（24、48 h）及Bcl-2、p62蛋白相对表达量升高（均P <0.05）。与对照组比较，紫草素组NB4细胞中PI3K/Akt/mTOR通路相关蛋白p-PI3K、p-Akt、p-mTOR蛋白表达显著降低，而740Y-P组显著升高（均P <0.05）；与紫草素组比较，紫草素+740Y-P组NB4细胞中p-PI3K、p-Akt、p-mTOR蛋白表达显著升高（均P <0.05）。.
结论: 紫草素可能通过抑制PI3K/Akt/mTOR通路促进NB4细胞自噬与凋亡。.
Keywords: apoptosis; autophagy; phosphatidylinositol 3 kinases/protein kinase B/mammalian target of rapamycin pathway;; promyelocytic leukemia; shikonin.