Chen Shan-Shan, Zhu Hua-Su, Dong Na, Zhang Ya-Ping, Wu Min, Liu Ling-Hong, Shi Qing, Li Dong, Ju Xiu-Li
Cheeloo College of Medicine, Shandong University Ji'nan 250012, China.
Research Center of Stem Cell and Regenerative Medicine, Shandong University Ji'nan 250012, China.
Zhongguo Zhong Yao Za Zhi. 2022 May;47(9):2541-2546. doi: 10.19540/j.cnki.cjcmm.20211206.701.
To investigate the toxicity and related mechanism of miltirone to human acute myeloid leukemia THP-1 cells. To be specific, the active components and targets of miltirone were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the target proteins were converted into standard gene names with UniProt. Acute leukemia-rela-ted target genes were screened from GeneCards and DisGeNET. Venn diagram was constructed with Venny 2.1 to yield the common targets of the disease and the drug. The protein-protein interaction(PPI) network was constructed by STRING and Cytoscape 3.8.2. THP-1 cells in the logarithmic growth phase were treated with dimethyl sulfoxide(DMSO), and 2.5, 5, 10, 15, and 20 μmol·L(-1) miltirone for 24 h, respectively. The proliferation rate of cells was analyzed by carboxyfluorescein diacetate succinimidyl ester(CFSE), apoptosis rate by flow cytometry with Annexin V-PE/7 AAD staining, and cell morphology by acridine orange staining. Real-time quantitative PCR(qPCR) was employed to detect the mRNA levels of nuclear receptor coactivator 2(NCOA2), poly(ADP-ribose) polymerase-1(PARP1), B-cell lymphoma-2(Bcl-2)-associated X protein(Bax), Bcl-2, and cysteine aspartyl protease-3(caspase-3). The effect of miltirone on apoptosis was detected in presence of caspase inhibitor Z-VAD-FMK. A total of 26 targets of miltirone, 1 046 genes related to acute leukemia, and 6 common targets of the two were screened out. Flow cytometry result showed miltirone at 10 μmol·L(-1) can inhibit proliferation and promote apoptosis of THP-1 cells. The typical manifestations of apoptosis, such as cell shrinkage, nuclear rupture, and chromatin agglomerate were displayed by acridine orange staining. The decreased mRNA levels of NCOA2 and PARP1 and increased Bax/Bcl-2 ratio and the activity of pro-apoptotic protein caspase-3 were observed. Z-VAD-FMK can attenuate the apoptosis-inducing effect of miltirone. This study indicates that miltirone can inhibit the proliferation and promote the apoptosis of THP-1 cells, by down-regulating NCOA2 and PARP1, raising Bax/Bcl-2 ratio, and activating caspase-3.
探讨米替龙对人急性髓系白血病THP-1细胞的毒性及相关机制。具体而言，从中药系统药理学数据库与分析平台（TCMSP）检索米替龙的活性成分及靶点，并通过UniProt将靶点蛋白转化为标准基因名称。从GeneCards和DisGeNET筛选急性白血病相关靶基因。用Venny 2.1构建维恩图以得出疾病与药物的共同靶点。通过STRING和Cytoscape 3.8.2构建蛋白质-蛋白质相互作用（PPI）网络。对数生长期的THP-1细胞分别用二甲基亚砜（DMSO）以及2.5、5、10、15和20 μmol·L⁻¹的米替龙处理24小时。通过羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）分析细胞增殖率，用膜联蛋白V-PE/7-AAD染色通过流式细胞术检测凋亡率，并用吖啶橙染色观察细胞形态。采用实时定量PCR（qPCR）检测核受体辅激活因子2（NCOA2）、聚（ADP-核糖）聚合酶-1（PARP1）、B细胞淋巴瘤-2（Bcl-2）相关X蛋白（Bax）、Bcl-2和半胱天冬酶-3（caspase-3）的mRNA水平。在存在半胱天冬酶抑制剂Z-VAD-FMK的情况下检测米替龙对凋亡的影响。共筛选出米替龙的26个靶点、1046个与急性白血病相关的基因以及二者的6个共同靶点。流式细胞术结果显示10 μmol·L⁻¹的米替龙可抑制THP-1细胞增殖并促进其凋亡。吖啶橙染色显示出凋亡的典型表现，如细胞皱缩、核破裂和染色质凝聚。观察到NCOA2和PARP1的mRNA水平降低，Bax/Bcl-2比值升高以及促凋亡蛋白caspase-3的活性增强。Z-VAD-FMK可减弱米替龙的诱导凋亡作用。本研究表明，米替龙可通过下调NCOA2和PARP1、提高Bax/Bcl-2比值以及激活caspase-3来抑制THP-1细胞增殖并促进其凋亡。
