Study On The Mechanisms Of Total Flavonoids From Sarcandrae Herba Inhibiting The Growth Of Human Leukemia Cells Based On Metabonomics And Transcriptome

[Objective]The purpose of this study was to prove that the Total Flavonoids from Sarcandrae Herba inhibit the activity of leukemia K562 cells.Using RNA-seq technology and cell metabolomics to study the changes of gene expression and metabolites in the intervention of K562 cells to explore the molecular mechanism of Total Flavonoids from Sarcandrae Herba to inhibit the growth of K562 cells,to provide a theoretical basis for the anti-leukemia of Total Flavonoids from Sarcandrae Herba and rheumatism,and to provide evidence and references for finding targets and pathways in the treatment of leukemia.[Methods]1.To explore the effect of Total Flavonoids from Sarcandrae Herba on human leukemia cells K562 cells were treated with serial concentrations of total flavonoids for 24,48,and 72h.Chemiluminescence was used to detect cell viability and calculate IC₅₀.Based on this,the optimal intervention time was determined.The cells were divided into a blank control group and Total Flavonoids from Sarcandrae Herba doses of 25,50,and 100μg/m L were tested by AO-EB and flow cytometry.2.Inhibition of Transcriptomics on Leukemia Cells by Total Flavonoids from Sarcandrae Herba K562 cells were divided into a blank control group and a total flavonoid treatment group for 48 hours,and total RNA was extracted for c DNA library construction,sequencing,and quality control.The transcriptome data was compared with reference genomes,gene expression levels and differences.Expression analysis,screening differentially expressed genes.Using differentially expressed genes as reference objects,further gene function annotation,signal pathway enrichment analysis,and protein network interaction analysis were performed.Finally,the key signal pathways were verified by WB experiments.3.The non-targeted metabolomics method based on LC-MS technology was used to analyze the changes of metabolites in the blank control group and the 100μg/m L Total Flavonoids from Sarcandrae Herba group after intervention for 48 h.Multivariate statistical analysis of metabolites and database comparisons are used to identify potential biomarkers.Finally,through the enrichment analysis of metabolic pathways and the combined analysis of transcriptome and metabolomics to elaborate the mechanism of K562 cells inhibition by Total Flavonoids from Sarcandrae Herba.[Results]1.Total Flavonoids from Sarcandrae Herba can significantly reduce the survival rate of K562 cells in a time-dependent and dose-dependent manner.The IC₅₀ at 24,48,and 72 h were 64.27,49.38,and 32.43μg/m L,respectively.According to K562 cell growth characteristics and IC₅₀ values,three dose groups of 25,50,and 100μg/m L were set for subsequent experiments.Compared with AO-EB staining,the total flavonoids of 50μg/m L and 100μg/m L Total Flavonoids from Sarcandrae Herba significantly promoted the apoptosis of K562 cells（P<0.01）,while the Total Flavonoids from Sarcandrae Herba of 25μg/m L did not promote K562 cell apoptosis.2.Analysis of RNA-seq sequencing data showed that the expression of 989 genes was significantly different between Total Flavonoids from Sarcandrae Herba intervention group and the blank control group（log₂（Fold change）>1,and P-adjust<0.05）.Among them,654genes were up-regulated and 335 genes were down-regulated.The GO enrichment analysis of differential genes showed that the number of GO terms significantly enriched in differentially expressed genes between the total flavonoid intervention group and the blank control group reached 1438,including 1,057 biological processes,145 cell components,and236 molecular functions.article.The results of KEGG analysis showed that the effect of total flavonoids in K562 cells on K562 cells was highly related to MAPK signaling pathway,neuroactive ligand-receptor interaction,complement and coagulation cascade.Analysis of the differential gene-protein interaction network showed that CXCL8,GNGT1,GNG12,MCHR1,FOS,CCR7,NMU,C3AR1,RPS2,and S1PR1 were at the core of the protein interaction network.WB validated the key targets of the MAPK pathway.The expression levels of p-JNK and CDC20 decreased,and the expression levels of P53 and CDKN1A increased（P<0.01）.There was no significant difference in the expression levels of p-ERK and p-P38.3.In the positive and negative ion modes,the blank control group and Total Flavonoids from Sarcandrae Herba can achieve good separation between QC samples both in positive and negative ion mode.After further screening and comparison with online databases such as HMDB and KEGG,98 metabolites were identified in LC-MS positive ion mode,60metabolites were identified in negative ion mode,and 158 metabolites were identified.Metabo Analyst 4.0 was introduced for pathway enrichment analysis,and it was significantly enriched in four metabolic pathways including phenylalanine,tyrosine and tryptophan biosynthesis,sphingolipid metabolism,glycerophospholipid metabolism,and phenylalanine metabolism.Then,the differential genes screened by transcriptomics and the differential metabolites screened by metabolomics were combined to find that sphingolipid metabolism is the most important metabolic pathway.[Conclusions]1.A certain concentration of Total Flavonoids from Sarcandrae Herba can inhibit the activity of leukemia K562 cells and promote their apoptosis.2.Total Flavonoids from Sarcandrae Herba inhibit the activity of leukemia cells and promote their apoptosis,which may be related to the decrease of JNK phosphorylation in the MAPK signaling pathway,which in turn increases the expression of P53 and CDKN1A genes and decreases the expression of CDC20.3.Total Flavonoids from Sarcandrae Herba inhibit the process of leukemia cells.By regulating amino acid metabolism,sphingolipid metabolism,and glycerophospholipid metabolism pathways,it inhibits the breakdown of amino acids,induces cell membrane damage,and interferes with the signal transmission of sphingosine between cells.