青黄散组分配伍干预急性早幼粒细胞白血病增殖和凋亡的机制研究

Mechanism study of Qinghuang Powder components combination on proliferation and apoptosis intervention in acute promyelocytic leukemia

关键词:  青黄散组分  急性早幼粒细胞白血病  协同效应  多组学整合分析  PI3K/AKT信号通路  细胞凋亡

Key words:  Qinghuang Powder components  acute promyelocytic leukemia  synergistic effect  multi-omics integrated analysis  PI3K/AKT signaling pathway  apoptosis of cell

摘要:

[目的] 验证青黄散组分配伍对急性早幼粒细胞白血病（APL）的协同效应，通过多组学整合分析阐明其分子机制。[方法] 采用CCK-8法测定靛玉红（Indirubin）和二硫化二砷（As₂S₂）对NB4细胞的抑制活性，并用Chou-Talalay模型评估其协同效应。通过吉姆萨染色观察细胞形态变化，流式细胞术检测细胞凋亡率及周期分布。整合多数据库筛选药物-疾病靶点，进行GO和KEGG分析。采用转录组测序鉴定差异表达基因并进行生物信息学分析。蛋白免疫印迹法（Western blot）验证关键蛋白表达变化。[结果] Indirubin和As₂S₂对NB4细胞呈剂量依赖性抑制，Chou-Talalay分析证实Indirubin（80 μmol/L）与As₂S₂（16 μmol/L）配伍具显著协同效应（CI<1）。药物组合促进细胞凋亡并增加G0/G1期细胞比例（P＜0.05）。数据库整合分析获得44个关键靶点，转录组测序鉴定1 767个差异基因，均富集于磷脂酰肌醇-3激酶/蛋白激酶（PI3K/AKT）等信号通路。Western blot证实药物配伍抑制PI3K/AKT通路磷酸化（P＜0.05），增加BAX/BCL2比值，确认PI3K/AKT通路为青黄散组分配伍抗APL的关键靶点。[结论] 青黄散组分配伍通过协同抑制PI3K/AKT信号通路，调控细胞周期并促进凋亡，发挥抗APL作用，为临床应用提供分子药理学基础。

Abstract:

[Objective] To validate the synergistic effects of Qinghuang Powder components combination on acute promyelocytic leukemia（APL） and elucidate its molecular mechanisms through multi-omics integrated analysis. [Methods] CCK-8 assay was used to determine the inhibitory activity of Indirubin and Arsenic disulfide（As2S2） on NB4 cells，and their synergistic effects were evaluated using the Chou-Talalay model. Cell morphological changes were observed by Giemsa staining，while cell apoptosis rate and cycle distribution were detected by flow cytometry. Drug-disease targets were screened through multiple databases integration，followed by GO and KEGG analyses. Transcriptome sequencing was employed to identify differentially expressed genes with subsequent bioinformatics analysis. Western blot was used to verify key protein expression changes. [Results] Indirubin and As2S2 exhibited dose-dependent inhibition on NB4 cells. Chou-Talalay analysis confirmed significant synergistic effects（CI<1） between Indirubin（80 μmol/L） and As2S2（16 μmol/L）. The drug combination promoted cell apoptosis and increased the proportion of cells in G0/G1 phase（P＜0.05）. Integrated database analysis identified 44 key targets，while transcriptome sequencing revealed 1 767 differentially expressed genes，both enriched in pathways including PI3K/AKT signaling. Western blot confirmed that the drug combination inhibited PI3K/AKT phosphorylation（P＜0.05） and increased the BAX/BCL2 ratio，verifying PI3K/AKT pathway as a key target for Qinghuang Powder components against APL. [Conclusion] Qinghuang Powder components combination exerts anti-APL effects through synergistic inhibition of the PI3K/AKT signaling pathway，regulating cell cycle and promoting apoptosis，thus providing a molecular pharmacological basis for clinical application.