Exploration On The Molecular Mechanisms Of Daidzein Inhibiting Non-Small Cell Lung Cancer Based On Network Pharmacology And Metabolomics

Objective:Network pharmacology and metabolomics were used to analyze the effects of Daidzein（DD）on metabolites and their metabolic pathways in non-small cell lung cancer（NSCLC）tissue,which contributed to explaining the molecular mechanisms of DD inhibiting NSCLC and providing molecular theoretical basis for dietary guidance on soy food of NSCLC patients.Methods:（1）Network pharmacology was used to screen DD targets by Pharm Mapper and Swiss Target Prediction databases and NSCLC therapeutic targets by Gene Cards and OMIM databases.Network visualization and Protein-protein interaction（PPI）analysis of intersection targets between DD and NSCLC were performed by STRING database and Cytoscape 3.9.1.Furthermore,GO analysis and KEGG pathway analysis of the intersection targets were executed by R software.（2）NSCLC tissue of nude mice from our previous DD pharmacodynamic pre-experiment were pretreated.Subsequently,the effects of DD on metabolites in NSCLC tissue were analyzed using Widely targeted metabolomics based on Ultra performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）.VIP≥1,|log₂FC|≥1 and p<0.05 were regarded as the criteria to screen the differential metabolites between group normal saline（NS）and DD,and KEGG pathway enrichment analysis was executed for these differential metabolites.（3）KEGG analysis results of network pharmacology and metabolomics were integrated to identify the key metabolic pathway of DD inhibiting NSCLC.Subsequently,NSCLC tissue of nude mice from our previous formal experiment of DD pharmacodynamics were pretreated.The level of key metabolites and metabolic enzymes in this pathway were determined by Targeted metabolomics based on Multiple reaction monitoring（MRM）and Quantitative real-time PCR（qRT-PCR）,respectively,which contributed to further exploring the molecular mechanisms of DD inhibiting NSCLC.Results:（1）Network pharmacology showed that 280 targets from DD and 1655targets from NSCLC were retrieved,and a total of 120 targets were acquired after intersection.Core targets such as Akt1,Mapk14,Src,H-ras and Ppar-γwere obtained through PPI network and topological analysis.Moreover,intersection targets were mainly concentrated in KEGG pathways such as lipid and atherosclerosis,PI3K-Akt signaling pathway and inflammatory mediator regulation of transient receptor potential（TRP）channels.（2）Widely targeted metabolomics analysis demonstrated that there existed a significant separation and no overfitting between group NS and DD in the Orthogonal partial least squares-discrimination analysis（OPLS-DA）model.A total of 337 differential metabolites between groups were screened.Enrichment analysis and metabolic pathway analysis revealed that these differential metabolites were mainly enriched in several metabolic pathways such as inflammatory mediator regulation of TRP channels,linoleic acid metabolism and arachidonic acid metabolism.（3）KEGG analysis results of network pharmacology and metabolomics suggested that DD may inhibit NSCLC by regulating 12/15-lipoxygenase（12/15-LOX）metabolic pathway of inflammatory mediator regulation of TRP channels.The level of key metabolites in this pathway revealed that 15-hydroxyeicosatetraenoic acid（15-HETE）and13-hydroxyoctadecadienoic acid（13-HODE）increased in NSCLC tissue after DD treatment.qRT-PCR analysis showed that DD up-regulated the mRNA expression of12/15-LOX and Peroxisome proliferator-activated receptor-γ（PPAR-γ）in tissue.This suggested that the inhibition of DD on NSCLC may involve regulation of12/15-LOX and PPAR-γmRNA expression and 15-HETE and 13-HODE synthesis.Conclusions:The comprehensive application of network pharmacology,metabolomics and molecular biology techniques was performed to revealed that DD inhibited NSCLC by regulating 12/15-LOX metabolic pathway.The findings of this work are expected to provide clues for the molecular mechanisms of DD inhibiting NSCLC.