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. 2022 Nov 20;42(11):1739-1746.
doi: 10.12122/j.issn.1673-4254.2022.11.20.

[Effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells]

[Article in Chinese]
Y Hao  1 , J Ji  1 , C Liu  1 , N Zhang  1 , Y Gong  1
Affiliations

[Effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells]

[Article in Chinese]
Y Hao et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To investigate the effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells and explore the underlying mechanism.

Methods: The effects of different concentrations of matrine alone and in combination with LY294002 on the proliferation of K562 cells were examined with CCK-8 assay. The changes in morphology of K562 cells were observed following treatment for 48 h with 0.4 g/L matrine and 10 μmol/L Y294002, either alone or in combination, and cell apoptosis was detected using flow cytometry with annexin V-FITC/PI double labeling; the changes in cell cycle was detected with PI labeling. Western blotting was performed to examine the effect of matrine combined with LY294002 on expressions of p-mTOR, p-PI3K, Akt, p-Akt, cyclinD1, Bcl-2 and caspase-9 in the cells.

Results: Treatment with different concentrations of matrine, both alone and in combination with LY294002, inhibited the proliferation of K562 cells in a time- and concentration-dependent manner. Compared with matrine treatment alone, the combined treatment caused more obvious morphological changes of the cells, significantly increased cell apoptosis (P < 0.01), and induced cell cycle arrest in G0/G1 (P < 0.01). Western blotting showed that the protein expression levels of p-mTOR, cyclinD1, p-PI3K, p-Akt and Bcl-2 in K562 cells increased while the expression level of caspase-9 decreased significantly after the combined treatment (P < 0.01).

Conclusion: Matrine combined with LY294002 produces a synergistic inhibitory effect on K562 cells possibly by down-regulating the p-Akt expression in PI3K/Akt signaling pathway, reducing the expressions of p-mTOR, cyclinD1 and Bcl-2, and increasing the expression of caspase-9.

目的: 探究苦参碱（Mat）联合LY294002对人髓系白血病K562细胞增殖、凋亡和细胞周期的影响及可能机制。

方法: 采用CCK-8法检测不同浓度Mat单药和联合LY294002对K562细胞增殖的影响；将K562细胞分为对照组、Mat组、LY294002组和Mat+LY294002组，分别采用0.4 g/L Mat及10 μmol/L LY294002单独或联合干预48 h后，应用光学显微镜观察细胞形态变化，流式细胞术annexin Ⅴ-FITC/PI双标记检测细胞凋亡，PI单标记检测细胞周期变化，Western blot法检测Mat联合LY294002对K562细胞p-mTOR、p-PI3K、Akt、p-Akt、CyclinD1、Bcl-2、Caspase-9蛋白表达的影响。

结果: CCK-8结果显示，不同浓度Mat单药和联合LY294002均可抑制K562细胞的增殖，具有时间和浓度依赖性，且联合组增殖抑制率比单药组明显升高（P＜0.01）。形态学检查显示，联合用药组比单药组凋亡表现更明显。流式细胞结果显示，与对照组及单药组相比，联合组显著促进细胞凋亡（[42.50±2.63）%，P＜0.01]，显著增加了细胞周期G0/G1期细胞比率（[57.23±1.44）%，P＜0.01]。Western blot结果表明，两药联合后K562细胞p-mTOR、CyclinD1、p-PI3K、p-Akt、Bcl-2蛋白表达水平明显增高（P＜0.01），Caspase-9蛋白表达水平下降（P＜0.01）。

结论: Mat和LY294002联合应用可以增强对K562细胞生长抑制的协同作用，其机制可能是通过有效下调PI3K/Akt信号通路中的p-Akt表达，使p-mTOR、CyclinD1和Bcl-2蛋白表达下调，Caspase-9蛋白表达上调来实现的。

Keywords: Akt; LY294002; acute myeloid leukemia; apoptotic; cell cycle; matrine.

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Figures

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Mat和联合LY294002对K562细胞增殖抑制作用 EInhibitory effect of matrine alone and in combination with LY294002 on proliferation of K562 cells. **P < 0.01, compared with the single drug group, the combined medication group of the same time of action.
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Mat联用LY294002对K562细胞形态的影响 Morphological changes of K562 cells treated with matrine alone or in combination with LY294002. A: Cell morphology observed under inverted microscope (Original magnification: × 200). B: Cell morphology observed with Wright staining under optical microscope (× 1000). C: Cell morphology observed with acridine orange (AO) staining under fluorescence microscope (×200). 1: Control group; 2: Matrine group; 3: LY294002 group; 4: Matrine +LY294002 group.
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Mat联合LY294002对K562细胞凋亡的影响 Effects of matrine combined with LY294002 on apoptosis of K562 cells detected by flow cytometry. **P < 0.01, ***P < 0.0001. LY: LY294002
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流式细胞仪检测药物作用48 h后K562细胞的周期分布 Cell cycle distributions detected by flow cytometry after treatment for 48 h with matrine alone or in combination with LY294002.
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药物作用48 h后对K562细胞周期蛋白和细胞凋亡蛋白表达水平 Expression levels of cyclins and apoptotic proteins in K562 cells after treatment for 48 h with matrine alone or in combination with LY294002 detected by Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; #P < 0.01, ##P < 0.001 vs Mat group and LY294002 group. LY: LY294002.
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药物作用48 h后对K562细胞PI3K/AKT通路相关蛋白表达水平的影响 Expression level of proteins of PI3K/AKT pathway in K562 cells after treatment for 48 h with matrine alone or in combination with LY294002 detected by Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; #P < 0.01, ##P < 0.001 vs Mat group and LY294002 group.
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