目的 基于网络药理学及体外实验探讨人参皂苷Rg₁调控铁死亡抑制慢性髓系白血病的作用机制。方法 运用CTD、GeneCards、HERB数据库筛选人参皂苷Rg₁和慢性髓系白血病靶点；采用FerrDb V2数据库获取铁死亡驱动与抑制靶点；通过Venn图获得交集靶点；使用STRING数据库和Cytoscape构建蛋白质相互作用（PPI）网络；利用DAVID数据库对交集靶点进行基因本体（GO）功能和京都基因与基因组百科全书（KEGG）通路富集分析；通过微生信在线作图平台进行可视化，并针对预测结果进行体外实验验证。采用CCK-8法和集落形成实验检测人参皂苷Rg₁作用后K562细胞增殖和分化能力影响；流式细胞术分析细胞周期分布；FerroOrange荧光探针检测细胞内Fe²⁺水平；BODIPY 581/591 C11荧光探针检测细胞脂质过氧化水平；透射电镜观察细胞线粒体形态变化。采用蛋白免疫印迹法和荧光定量PCR检测肿瘤蛋白53（p53）、亚精胺/精胺N1-乙酰转移酶1（SAT1）和花生四烯酸15-脂氧合酶（ALOX15）蛋白和mRNA的表达水平。结果 网络药理学结果表明，与铁死亡相关的人参皂苷Rg₁、慢性髓系白血病共同作用靶点26个，靶基因显著富集于癌症途径和p53信号通路等。实验结果表明，与对照组比较，人参皂苷Rg₁显著升高K562细胞增殖抑制率（P＜0.05）。人参皂苷Rg₁ 20 μmol/L作用48 h后，线粒体体积缩小、膜密度增加、线粒体嵴密度降低、融合甚至消失；K562细胞集落形成数减少（P＜0.01）；G₀/G₁期比例显著升高，S期比例降低（P＜0.001），G₂/M期比例升高（P＜0.01）；K562细胞内Fe²⁺、脂质ROS含量显著增高（P＜0.01、0.001）；K562细胞p53、SAT1、ALOX15蛋白和mRNA的表达显著上调（P＜0.01、0.001）。结论 人参皂苷Rg₁抑制慢性髓系白血病细胞增殖可能与调控p53/SAT1/ALOX15信号通路诱导铁死亡有关。

Objective To explore the mechanism by which ginsenoside Rg₁ regulates ferroptosis to inhibit chronic myeloid leukemia based on network pharmacology and in vitro experiments. Methods CTD, GeneCards and HERB databases were used to screen the targets of ginsenoside Rg₁ and chronic myeloid leukemia. FerrDb V2 database was adopted to obtain the genes driving and inhibiting ferroptosis. The intersection targets were obtained through Venn diagram. STRING database and Cytoscape were used to construct the PPI network. DAVID database was utilized to conduct GO functional and KEGG pathway enrichment analysis on the intersection targets. Visualization was performed through the online drawing platform of Microbiomics, and in vitro experiments were conducted to verify the prediction results. CCK-8 assay and colony formation assay were used to detect the effects of ginsenoside Rg₁ on the proliferation and differentiation ability of K562 cells. Flow cytometry was used to analyze the cell cycle distribution. FerroOrange fluorescent probe was used to detect the intracellular Fe²⁺ level. BODIPY 581/591 C11 fluorescent probe was used to detect the level of lipid peroxidation in cells. Transmission electron microscopy was used to observe the morphological changes of mitochondria. Western blotting and qPCR were used to detect the expression levels of p53, SAT1, and ALOX15 proteins and mRNAs. Results Results of network pharmacology indicate that there are 26 common target sites for the iron death-related ginsenoside Rg₁ and chronic myeloid leukemia, and the target genes are significantly enriched in cancer pathways and p53 signaling pathways, etc. The experimental results showed that compared with the control group, ginsenoside Rg₁ significantly increased the proliferation inhibition rate of K562 cells (P < 0.05). After 48 hours of treatment with ginsenoside Rg₁ 20 μmol/L, the mitochondrial volume decreased, the membrane density increased, the density of mitochondrial cristae decreased, fusion even disappeared; the number of K562 cell colonies decreased (P < 0.01); the proportion of G₀/G₁ phase significantly increased, the proportion of S phase decreased (P < 0.001), and the proportion of G₂/M phase increased (P < 0.01); the content of Fe²⁺ and lipid ROS in K562 cells significantly increased (P < 0.01, 0.001), the expression of p53, SAT1, ALOX15 protein and mRNA in K562 cells was significantly upregulated (P < 0.01, 0.001). Conclusion Ginsenoside Rg₁ inhibits the proliferation of leukemia K562 cells, which may be related to the induction of ferroptosis via regulation of the p53/SAT1/ALOX15 signaling pathway.

R286.3

国家自然科学基金资助项目（81860038）