背景:本研究旨在利用网络药理学预测青黛草在治疗人类慢性粒细胞白血病(CML)中的活性成分和关键靶基因,并进行体外验证。
方法:通过公共数据库收集了靛蓝的活性成分和相应的靶标以及白血病相关基因。通过蛋白质-蛋白质相互作用(PPI)网络预测了靛蓝的核心靶标和途径,基因本体论(GO)功能,和京都基因和基因组百科全书(KEGG)途径富集分析。接下来,与白血病相关基因相交后,鉴定了青黛草活性成分的直接核心靶基因。随后,HL-60细胞用靛玉红(IND)刺激,然后使用CCK-8测定和细胞周期检查细胞增殖,细胞凋亡,和线粒体膜电位使用流式细胞术。使用Westernblot检测凋亡相关蛋白(裂解的Caspase9,裂解的Caspase7,裂解的Caspase3和裂解的parp)的含量,HSP90AA1蛋白,和PI3K/Akt信令(PI3K,p-PI3K,Akt,和p-Akt)在HL-60细胞内。
结果:共筛选出9种青黛草活性成分。前10名核心靶基因(TNF,PTGS2RELA,MAPK14,IFNG,PPARG,NOS2,IKBKB,鉴定了PPI网络中靛蓝活性成分的HSP90AA1和NOS3)。根据KEGG富集分析,这些靶点与白血病相关通路(如急性髓性白血病和CML)相关.与白血病相关基因相交后,发现IND参与了最多的目标信息对,并且处于目标网络的核心;HSP90AA1是IND的直接核心基因。此外,体外细胞实验证实IND可以抑制细胞增殖,引起G2/M期细胞周期停滞,增强HL-60细胞的凋亡,降低线粒体膜电位,并促进细胞凋亡相关蛋白水平。在IND治疗下,HSP90AA1过表达明显促进细胞增殖,抑制细胞凋亡。此外,IND通过抑制HSP90AA1表达对白血病细胞产生抑瘤作用。
结论:IND,靛蓝的活性成分,可以抑制CML进展,这可以通过抑制HSP90AA1和PI3K/Akt信号传导表达水平来实现。
BACKGROUND: This research intended to predict the active ingredients and key target genes of Indigo Naturalis in treating human chronic myeloid leukemia (CML) using network pharmacology and conduct the invitro verification.
METHODS: The active components of Indigo Naturalis and the corresponding targets and leukemia-associated genes were gathered through public databases. The core targets and pathways of Indigo Naturalis were predicted through protein-protein interaction (PPI) network, gene ontology (GO) function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Next, after intersecting with leukemia-related genes, the direct core target gene of Indigo Naturalis active components was identified. Subsequently, HL-60 cells were stimulated with indirubin (IND) and then examined for cell proliferation using CCK-8 assay and cell cycle, cell apoptosis, and mitochondrial membrane potential using flow cytometry. The content of apoptosis-associated proteins (Cleaved Caspase 9, Cleaved Caspase 7, Cleaved Caspase 3, and Cleaved parp) were detected using Western blot,
HSP90AA1 protein, and PI3K/Akt signaling (PI3K, p-PI3K, Akt, and p-Akt) within HL-60 cells.
RESULTS: A total of 9 active components of Indigo Naturalis were screened. The top 10 core target genes (TNF, PTGS2, RELA, MAPK14, IFNG, PPARG, NOS2, IKBKB,
HSP90AA1, and NOS3) of Indigo Naturalis active components within the PPI network were identified. According to the KEGG enrichment analysis, these targets were associated with leukemia-related pathways (such as acute myeloid leukemia and CML). After intersecting with leukemia-related genes, it was found that IND participated in the most pairs of target information and was at the core of the target network;
HSP90AA1 was the direct core gene of IND. Furthermore, the in-vitro cell experiments verified that IND could inhibit the proliferation, elicit G2/M-phase cell cycle arrest, enhance the apoptosis of HL-60 cells, reduce mitochondrial membrane potential, and promote apoptosis-related protein levels. Under IND treatment,
HSP90AA1 overexpression notably promoted cell proliferation and inhibited apoptosis. Additionally, IND exerted tumor suppressor effects on leukemia cells by inhibiting
HSP90AA1 expression.
CONCLUSIONS: IND, an active component of Indigo Naturalis, could inhibit CML progression, which may be achieved via inhibiting HSP90AA1 and PI3K/Akt signaling expression levels.