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Study on the mechanism of diterpenoid DP from
Euphorbia fischeriana against leukemia- ZHONGGUO YAOFANG Vol. 34, Issue 7, Pages: 825-831(2023)
- 作者机构：
1.齐齐哈尔医学院医药科学研究院（博士后工作站），黑龙江 齐齐哈尔;161006
2.黑龙江中医药大学博士后流动站，哈尔滨 150040
3.齐齐哈尔医学院公共卫生学院，黑龙江 齐齐哈尔 161006
4.齐齐哈尔医学院药学院，黑龙江 齐齐哈尔 161006
5.齐齐哈尔医学院附属第三医院妇产科，黑龙江 齐齐哈尔 161099
- 作者简介：
- 基金信息：
DOI：10.6039/j.issn.1001-0408.2023.07.11
CLC： R966;R285Published：15 April 2023，
Received：09 October 2022，
Revised：07 March 2023，
扫 描 看 全 文
马立威,陈哲,王文豹等.狼毒大戟中二萜类化合物DP抗白血病的作用机制研究 Δ[J].中国药房,2023,34(07):825-831.
MA Liwei,CHEN Zhe,WANG Wenbao,et al.Study on the mechanism of diterpenoid DP from Euphorbia fischeriana against leukemia[J].ZHONGGUO YAOFANG,2023,34(07):825-831.
马立威,陈哲,王文豹等.狼毒大戟中二萜类化合物DP抗白血病的作用机制研究 Δ[J].中国药房,2023,34(07):825-831. DOI： 10.6039/j.issn.1001-0408.2023.07.11.
MA Liwei,CHEN Zhe,WANG Wenbao,et al.Study on the mechanism of diterpenoid DP from Euphorbia fischeriana against leukemia[J].ZHONGGUO YAOFANG,2023,34(07):825-831. DOI： 10.6039/j.issn.1001-0408.2023.07.11.
摘要
目的
2
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
2
以LY294002（PI3K特异性抑制剂）为工具药，采用MTT、Annexin Ⅴ-FITC/PI、AO-EB染色法观察并测定DP作用24 h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24 h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24 h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Western blot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
2
10 μmol/L DP和10 μmol/L DP+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、20 μmol/L DP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；10 μmol/L DP和10 μmol/L DP+LY294002作用后细胞中caspase-3、caspase-9和Bim mRNA转录水平均显著升高（
P
＜0.05或
P
＜0.01），FoxO3a mRNA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）；10 μmol/L DP+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
2
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
2
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
2
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
2
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
2
DP can inhibit the proliferation and induce the apoptosis of HL60 cells via inhibiting PI3K/Akt signaling pathway.
关键词
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
12-deoxyphorbol-13-palmitatehuman myeloid leukemia HL60 cellsphosphoinositide 3-kinase/protein kinase B signaling pathway
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