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狼毒大戟中二萜类化合物DP抗白血病的作用机制研究
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| 篇名： | 狼毒大戟中二萜类化合物DP抗白血病的作用机制研究 |
| TITLE： | Study on the mechanism of diterpenoid DP from Euphorbia fischeriana against leukemia |
| 摘要： | 目的 探究狼毒大戟中二萜类化合物12-去氧佛波醇-13-棕榈酸酯（DP）能否通过磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）信号通路发挥抗白血病的作用，为将其开发成抗白血病新药提供实验依据。方法以LY294002（PI3K特异性抑制剂）为工具药，采用MTT、AnnexinⅤ-FITC/PI、AO-EB染色法观察并测定DP作用24h后对人原髓细胞白血病细胞HL60增殖、凋亡的影响；采用ELISA法测定DP作用24h后细胞培养液中乳酸脱氢酶（LDH）的释放量及细胞中胱天蛋白酶3（caspase-3）、caspase-9的活性。采用实时荧光定量-PCR法检测DP作用24h后细胞中caspase-3、caspase-9、叉头框O3a（FoxO3a）、B细胞淋巴瘤2细胞死亡的相互作用介质（Bim）的mRNA转录水平；采用Westernblot法检测细胞中磷酸化FoxO3a（p-FoxO3a）、磷酸化Akt（p-Akt）的蛋白表达水平，并采用免疫染色法在激光共聚焦显微镜下观察细胞中FoxO3a蛋白核转位情况。结果10μmol/LDP和10μmol/LDP+LY294002对HL60细胞有明显的增殖抑制及凋亡诱导作用（P＜0.01）；5、10、20μmol/LDP作用后的细胞均呈典型的凋亡形态学特征，细胞培养液中LDH释放量和细胞中caspase-3、caspase-9活性均显著升高（P＜0.05或P＜0.01），其作用具有一定的浓度依赖性趋势；10μmol/LDP和10μmol/LDP+LY294002作用后细胞中caspase-3、caspase-9和BimmRNA转录水平均显著升高（P＜0.05或P＜0.01），FoxO3amRNA转录水平和p-FoxO3a、p-Akt蛋白表达水平均显著降低（P＜0.05或P＜0.01）；10μmol/LDP+LY294002组细胞可见FoxO3a蛋白的核转位变化，且该变化较LY294002组明显。结论DP可通过抑制PI3K/Akt信号通路抑制人白血病HL60细胞增殖并诱导其凋亡。 |
| ABSTRACT： | OBJECTIVE To explore whether diterpenoid 12-deoxyphorbol-13-palmitate （DP） from Euphorbia fischeriana can exert anti-leukemia effects through the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signal pathway， and to provide experimental evidence for developing it into a new anti-leukemia drug. METHODS Using LY294002 （PI3K specific inhibitor） as tool drug， the effects of 24 h DP treatment on the proliferation and apoptosis of human myeloid leukemia HL60 cells were detected by MTT method， Annexin Ⅴ-FITC/PI staining and AO-EB staining. ELISA method was used to detect lactic dehydrogenase （LDH） release and the activities of cysteinyl aspartate specific proteinase 3 （caspase-3） and caspase-9. The transcriptional level of caspase-3， caspase-9， forkhead box O3a （FoxO3a） and B cell lymphoma 2 interacting mediator of cell death （Bim） mRNA were detected by real-time quantitative polymerase chain reaction （qRT-PCR）. The protein expression of phosphorylated FoxO3a （p- FoxO3a） and phosphorylated Akt （p-Akt） were detected by Western blot method. The nuclear translocation of FoxO3a protein was detected by immunostaining combined with laser confocal microscopy. RESULTS 10 μmol/L DP and 10 μmol/L DP+LY294002 could inhibit the proliferation and induce the apoptosis of HL60 cells （P＜0.01）. After treatment of 5， 10， 20 μmol/L DP， HL60 cells showed typical morphological characteristics of apoptosis； DP could significantly increase the levels of LDH release and the activities of caspase-3 and caspase-9 （P＜0.05 or P＜0.01）， in dose-dependent manner. After treatment of 10 μmol/L DP and 10 μmol/L DP+LY294002， the transcriptional levels of caspase-3， caspase-9 and Bim mRNA were increased significantly （P＜0.05 or P＜0.01）， and transcriptional level of FoxO3a mRNA and protein expressions of p-FoxO3a and p-Akt were decreased significantly （P＜0.05 or P＜0.01）. Nuclear translocation changes were observed in FoxO3a protein in 10 μmol/L DP+LY294002 group， and the change was more significant than that of LY294002 group. CONCLUSIONS DP can inhibit the proliferation and induce the apoptosis of HL60 cells via inhibiting PI3K/Akt signaling pathway. |
| 期刊： | 2023年第34卷第07期 |
| 作者： | 马立威；陈哲；王文豹；张金玲；张洪涛；葛鹏玲；刘吉成 |
| AUTHORS： | MA Liwei，CHEN Zhe，WANG Wenbao，ZHANG Jinling，ZHANG Hongtao，GE Pengling，LIU Jicheng |
| 关键字： | 狼毒大戟；12-去氧佛波醇-13-棕榈酸酯；人原髓细胞白血病细胞HL60；磷脂酰肌醇3激酶/蛋白激酶B信号通路 |
| KEYWORDS： | Euphorbia fischeriana； 12-deoxyphorbol-13-palmitate； human myeloid leukemia HL60 cells； phosphoinositide 3- |
| 阅读数： | 96 次 |
| 本月下载数： | 3 次 |
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