Effect Of Dichloromethane Extraction Phase Of Patrinia Scabiosaefolia Fisch.Stem On Proliferation And Differentiation Of K562

Objective: To investigate the effects of Dichloromethane extraction phase of ethanolic extract of Patrinia Scabioscaefolia Fisch.(DPSS)on the proliferation inhibition,apoptosis promotion and differentiation induction of K562 cells,and to investigate the mechanism of DPSS on K562 cells at the cellular and molecular levels.Methods: K562 cells were routinely cultured in vitro.The stem of Patrinia scabiosaefolia Fisch.was concentrated by 70% ethanol and extracted with petroleum ether,ethyl acetate,dichloromethane and n-butanol.MTT assay was used to detect the effects of DPSS at 0,25,50,100 and 200 μg/m L on the proliferation of K562 cells at24,48 and 72 hours.Flow cytometry was used to analyze the changes of cell cycle and apoptosis at 24 and 48 hours.Wright-Gimesa staining was used to observe the morphological changes of K562 cells.The cell surface antigens CD33 and CD11 b were detected by flow cytometry.Illumina sequencing technology(based on RNA-seq)was used for transcriptome sequencing analysis of K562 cells and K562 cells that had been subjected to 100 μg/m L DPSS for 24 h.The sequencing data were processed by Fast QC and Hisat2 software,and the "limma" package was used to Differential analysis was performed to screen the group of differentially expressed genes between the two groups,followed by GO enrichment,KEGG enrichment and protein interaction network construction for differential genes using bioinformatics technology to analyze the effects of up-and down-regulated genes on cell growth,and finally some genes were selected for validation using q PCR technology.Results: The proliferation of K562 cells treated with different concentrations of DPSS was inhibited in a time-dose dependent manner(r = 0.96).Cell cycle analysis showed that with the increase of DPSS concentration,G0/G1 phase cells decreased(r=-0.70)while G2/M phase cells increased(r= 0.88).The results of apoptosis showed that200 μg/m L DPSS had the highest apoptosis rate after 48 h.Morphological observation showed that the cell body increased,the amount of cytoplasm increased,the ratio of nucleus to cytoplasm decreased,and the nuclear chromatin was rough.Cell differentiation antigen,CD11 b and CD117,were positively expressed treated with DPSS.The analysis of differential expression genes showed that there were 1844 upregulated genes and 2227 down-regulated genes between the control and 100 μg/m L DPSS groups.Functional annotation of GO genes showed that the differential genes were mainly localized in organelles,membrane fraction and macromolecular complex fraction;most of the molecular functions had catalytic activity,molecular function regulator and nucleic acid binding transcription factor activity;involved in cellular processes,metabolic processes and biological regulation.KEGG enrichment analysis revealed that DEGs were involved in 97 significant KEGG pathways,including metabolic pathway,apoptotic pathway,autophagic lysosomal pathway,MAPK signaling pathway,pyrimidine metabolism,PI3K-Akt signaling pathway,P53 signaling pathway and NF-kappa B signaling pathway.etc.Conclusion: DPSS can inhibit the proliferation of K562 cells by inducing apoptosis through cycle block,and can induce the differentiation of K562 cells to monocytes,thus having a potential anti-leukemic effect.