Decoding The Mechanism Of Shen Qi Sha Bai Decoction In Treating Acute Myeloid Leukemia Based On Network Pharmacology And Molecular Docking

BackgroundAcute Myeloid Leukemia(AML)is a serious blood disorder that accounts for 10-20% of childhood leukemia and 70-80% of adult leukemia.Although modern medical advances have significantly improved the prognosis of AML patients,the average 5-year survival rate is only30-50%,making it crucial to investigate the molecular mechanisms regulating AML and new therapeutic measures.Traditional Chinese medicine(TCM)treating AML has a history of more than 2000 years in China.For example,TCM like arsenic trioxide exhibits therapeutic effects on acute promyelocytic leukaemia.The other TCM like tripterygium wilfordii has a therapeutic effect on AML.The Traditional Chinese Medicine formula of Shen Qi Sha Bai Decoction(SQSBD),which is composed of 12 kinds of herbal medicines including Hedyotis Diffusae Herba,Scutellariae Barbatae Herba,Lobeliae Chinensis Herba,Codonopsis Radix,licorice,Hedysarum Multijugum Maxim,Scutellariae Radix,Dioscoreaebulbiferae,Indigo Naturalis,Glehniae Radix,Asparagi Radix,and Agrimonia Eupatoria.A clinical study revealed that SQSBD combined with chemotherapy is more effective that chemotherapy alone in the treatment of AML.The total effective rate of the combination group and the chemotherapy group was 91.1 and 73.9%,respectively.However,the pharmacological mechanism of SQSBD treating AML remains largely unclear.Recently,network pharmacology has been developed based on system biology,network biology and polypharmacology.This new area of research involves identifying the synergies and potential mechanisms of multi-component and multi-target drugs by analyzing various networks of complex and multi-level interactions based on omics and systems biology technologies.Since TCM prescriptions are considered as multi-component,multi-target therapies,which may meet the need to treat multiple complex diseases in an integrated manner,network pharmacological approaches are suitable for investigation of TCM on treatment of AML.We explored the mechanism of SQSBD in the treatment of AML through network pharmacology and molecular docking studies,with a view to obtaining the key components of SQSBD and the core targets regulating AML,and providing more theoretical basis for the treatment of AML by Chinese medicine.ObjectivesThe mechanism of SQSBD in the treatment of AML was studied through network pharmacology and molecular docking,with a view to obtaining the key components of SQSBD and the core targets regulating AML,and providing more theoretical basis for the treatment of AML by Chinese medicine.MethodsDifferential expression analysis was used to identify AML differential genes;network topology analysis to screen core network and targets;functional enrichment analysis to determine the biological function of targets;molecular docking technique to simulate the affinity of active ingredient-target interaction;MTT assay to detect cell proliferation;flow cytometry to detect cell cycle and cell differentiation indicators;Western Blot assay to detect target protein expression;and Wright-Giemsa staining to observe cell morphological changes.Results1.Predicting the candidate targets of Shen Qi Sha Bai Decoction in treating acute myeloid leukemia based multi-database integration analysisDifferential analysis of the GSE37307 dataset yielded a total of 1505 AML differential genes(|log(FC)|>1 and adj.P value<0.05),of which 687 were highly expressed and 818 were lowly expressed in the AML group.SQSBD identified 268 active ingredients(DL values≥0.18 and OB values≥30%)and 264 drug targets.The number of overlapping targets was46 and the number of active ingredients involved in the interactions was 153.2.Identifying the core targets of Shen Qi Sha Bai Decoction in treating acute myeloid leukemia based bioinformatics and network topology analysisFunctional enrichment analysis of the candidate targets showed that 109 KEGG pathways and 880 GO annotation entries were significantly enriched(count>2 and adj.P value<0.05).11 pathways clearly associated with AML were reported in the literature,with26 targets involved in enrichment.The core network obtained from topological analysis of the candidate target protein interaction network consisted of 96 nodes and 1764 edges,with 8candidate targets involved in interactions.Combining the results of functional enrichment analysis and protein network analysis,CDK1,MAPK1,PCNA,JUN,HSPB1 and STAT3 were identified as core targets.3.Studing the the mechanisms of core targets regulating acute myeloid leukemia based bioinformatics and molecular dockingR2 database,GEO database and combined GTEX and TCGA database analysis of core target differences showed significant differences in core target expression in both AML and normal groups(P < 0.05).The results of the core target single gene GSEA analysis were mainly enriched to the cell cycle,apoptosis and other KEGG pathways that regulate AML.Core target and gene set correlation analysis showed that STAT3 and MAPK1 were positively associated with cell differentiation and apoptosis(P < 0.05),and CDK1 and PCNA were positively associated with the cell cycle(P<0.05).The molecular docking results showed that the absolute values of the binding energies of all active ingredients and corresponding targets were greater than 5 kcal/mol,with Licochalcone A-MAPK1 and Quercetin-CDK1 showing the strongest affinity.4.Cellular assays to verify the effect of active ingredients on AML cellsMTT results revealed that Quercetin and Licochalcone A significantly inhibited the proliferation of both AML cells U937 and HL60(P<0.05),and flow cytometry and Western Blot assays showed that Quercetin induced G2/M phase block in U937 and HL60 cells(P<0.05),downregulating CDK1(Tyr15)phosphorylation levels(P < 0.05);Licochalcone A promoted increased CD11 b expression on the surface of U937 and HL60 cells(P<0.05)and upregulated ERK2(Tyr204)phosphorylation levels(P < 0.05),and Wright-Giemsa staining assays showed that Licochalcone A induced downward differentiation of AML morphology.ConclusionThe mechanism of SQSBD for AML treatment involves multiple components,multiple targets and multiple pathways,of which CDK1,MAPK1,PCNA,JUN,HSPB1 and STAT3 are the core targets.The active components inhibit the proliferation of AML cells by blocking the cell cycle and inducing cell differentiation through regulating the phosphorylation levels of the core targets.