Metabolism Of Glaucocalyxin A In Vivo And In Vitro And The Mechanism Of Inhibiting Lung Cancer A549 Cell Proliferation

Objective:To establish the metabolism of glaucocalyxin A（glaucocalyxin A,GLA）in vivo and in vitro based on UHPLC-Q-TOF-MS technique and to explore the mechanism of GLA inhibiting the proliferation of lung cancer A549 cells.Methods:By using UHPLC-Q-TOF-MS,a practical method combined with DDA and DIA acquisitions was established and developed to identify metabolites of GLA in vivo and in vitro.The data acquired were mainly analyzed by Metabolite Pilot^TM2.0 software.According to the law of metabolic transformation and the cleavage pathway of GLA,the structure of GLA metabolites were deduced and the metabolic pathway was summarized.In the mechanism study,the prediction results of network pharmacology and the experimental results of metabolomics were used to make a comprehensive analysis.In the study of metabolomics,the A549 cells were treated with different concentrations of GLA for 24 h.Then the cytotoxicity was evaluated by Cell Counting Kit-8（CCK-8）assay.The determined IC₅₀value（4.0μg/m L）was used for the cells in the treat group,and the cells in the control group were normal A549.Metabolites were profiled on UHPLC-Q-TOF-MS platform with DIA data acquisition to analysis the metabolic profiles of GLA in A549 cells.Progenesis QI and SIMCA-P14.1software were used for data processing and analysis to select potential differential metabolites.The differential metabolites were identified via HMDB.Then the heat map analysis and metabolic pathway screening were carried out through the platform of Metabo Analyst 4.0.In the research of network pharmacology,the target genes of drug（GLA）and disease（NSCLC）were identified by some online websites,and then the core target genes were screened by Cytoscape software.Afterwards,the core genes were introduced into Metascape to predict the biological processes of GLA affecting A549.Results:A total of 32 phaseⅠmetabolites and 6 phaseⅠmetabolites were identified in rats in vivo and in vitro.Among them,25,18,17 and 7metabolites were identified in urine,feces,bile and plasma,respectively.Seven metabolites were identified in rat intestinal flora（RIF）.It mainly involved oxidation,demethylation,hydrogenation,water addition,dehydrogenation,ketone formation,methylation conjugation,sulfate binding,and glucuronic acid binding.There were 5 metabolites both in vivo and in vitro,which were dioxygenated metabolites,hydrated metabolites,dioxygenated and hydrated metabolites,metabolites oxidized to carboxylic acids,and methylated metabolites.Among them,methylated metabolites only appearred in vitro.In metabolomics experiment,46 differential metabolites were screened and identified.The main metabolic pathways interfered by GLA included D-Glutamine and D-glutamate metabolism,taurine and hypotaurine metabolism,cysteine and methionine metabolism.The results of network pharmacological prediction showed that the core target genes were closely related to signal transduction diseases,cellular,cellular responses to stress,pathways in cancer and other biological processes.Conclusions:In this study,UHPLC-Q-TOF-MS data acquisition technique was used for the metabolites of GLA in rats in vivo and in vitro firstly,which laid the foundation for further elucidation of its pharmacokinetics.The metabolic effects of GLA on A549 were analyzed by means of cell metabolomics and network pharmacology,and the metabolic pathways and other biological processes which were greatly affected by GLA were found.These results provided a reference for explaining the molecular mechanism of GLA inhibiting the proliferation of A549 and provided a new research strategy for pharmacodynamics.