Résumé
ObjectiveTo predict the therapeutic target genes and related signaling pathways of Qinghuangsan （QHP） in the treatment of acute myeloid leukemia （AML） by network pharmacology，molecular docking，and further clarify its mechanisms through in vitro cell experiment. MethodThe active components and targets of QHP were retrieved from traditional Chinese medicine systems pharmacology database and analysis platform （TCMSP），traditional Chinese medicine integrated database （TCMID），TargetNet and SwissTargetPrediction databases，and AML-related target genes were obtained by GeneCards and online mendelian inheritance in man （OMIM） databases. After screening the common targets of QHP and AML，the protein-protein interaction （PPI） network of the common targets was constructed with STRING，followed by gene ontology （GO） term and Kyoto encyclopedia of genes and genomes （KEGG） pathway enrichment analysis based on RStudio software and clusterProfiler，Bioconductor packages. At the same time，Cytoscape software is used to construct the network of "disease-component-target" and "compound-target-pathway". Select the active ingredients of QHP for molecular docking with the top 8 targets in the "compound-target-pathway" network. In vitro cell experiment and Western blot were used to further verify the anti-AML effect of QHP. ResultThe prediction results show that there are 11 main active components of QHP，and 22 common targets of QHP and AML are collected. KEGG pathway analysis results show that phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） and mitogen-activated protein kinase （MAPK） signaling pathways may play a key role in the treatment of AML disease by QHP. "Compound-target-pathway" network analysis showed that the top 8 targets include Akt1，phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit alpha （PIK3CA），mitogen-activated protein kinase kinase 1 （MAP2K1），TP53，serine/threonine kinase （RAF1），B cell lymphoma（Bcl）-2，cysteine aspartic acid specific protease（Caspase）-9 and JUN. Molecular docking results showed that 3-indolyl-β-D-glucopyranoside was optimally docked with MAP2K1，isovitexin docked with PIK3CA，and indirubin docked with Bcl-2. Cell experiments show that 3-indolyl-β-D-glucopyranoside，isovitexin and indirubin can effectively inhibit the proliferation of AML cells，regulate the MAPK/PI3K signaling pathway，and inhibit the expression of Bcl-2 protein. ConclusionQHP can treat AML through "multi-component，multi-target，multi-pathway" synergistic treatment，and its mechanism of pharmacology may be related to the regulation of MAPK signaling pathway and PI3K/Akt signaling pathway.
Résumé
Objective:To investigate the effect of different dose of Realgar compatible with Indigo Naturalis on the transitional constituents of Indigo Naturalis in rat serum based on the compatibility of Qinghuangsan.Method:Indigo Naturalis test solution, the drug-containing serum of three different proportions of Qinghuangsan (10 g of Indigo Naturalis compatible with 52.5, 105, 210 mg of Realgar for group A, B and C, respectively) and blank serum were detected by UPLC-Q-TOF-MS/MS, in combination with the chemical components identified in Indigo Naturalis test solution, the differences of transitional constituents of Indigo Naturalis in rat serum from the group A, B and C were analyzed. HL-60 cells (human leukemia cells) were treated with the three groups of Qinghuangsan drug-containing serum and the effect of drug-containing serum on the activity of HL-60 cells was detected by cell counting kit-8 (CCK-8) assay.Result:A total of 19, 22, 25 of transitional constituents were detected in Qinghuangsan drug-containing serum from group A, B and C, respectively. The three groups of drug-containing serum all contained 5 prototype components from Indigo Naturalis test solution, including tryptanthrin, indigo, indirubin, 2-aminobenzoic acid and N-phenyl-2-naphthylamine, respectively. The results of CCK-8 assay showed that Qinghuangsan drug-containing serum of group C had the strongest inhibitory effect on HL-60 cells.Conclusion:After fixed Indigo Naturalis dose, with the increase of Realgar dose, the transitional constituents in rat serum increase and the inhibitory effect on HL-60 cells also gradually enhances, which indicates that Realgar may promote the absorption of active components in Indigo Naturali in vivo, thus enhance the efficacy, further explains the compatibility law and pharmacodynamic material basis of different proportions of Realgar and Indigo Naturalis.