The Effects And Mechanism Of Qi-Zhen-Gui-Pi Decoction On Biological Behavior Of Lymphoma Cells

Objective To investigate the effects of Qi-Zhen-Gui-Pi decoction on proliferation,apoptosis,cell cycle distribution,migration and invasion of lymphoma cells,and to explore the mechanism through high throughput omics technology and network pharmacology.To illustrate the multi-component,multi-target and multi-pathway mode of action of traditional Chinese medicine formula.Methods The human lymphoma cell lines(SU-DHL-4,Jurkat and Raji)were treated with different concentrations of Qi-Zhen-Gui-Pi decoction.Cell proliferation was detected by CCK-8 method.Apoptosis and cell cycle distribution were detected by Flow cytometry(FCM).Migration and invasion abilities of lymphoma cells were detected by transwell chamber assay.Gene expression profiles of Jurkat were detected by Primeview human gene expression array.Ingenuity Pathway Analysis(IPA)was applied to identify enriched canonical pathways among differentially expressed genes.The m RNA and protein expression levels of selected genes were detected by real-time fluorescent quantitative PCR and Western Blot respectively.The main compounds and target proteins of Qi-Zhen-Gui-Pi decoction were analyzed by network pharmacology.KEGG pathway and gene ontology(GO)term enrichment analysis of the target genes were performed.And to investigate the main compounds involved in anti-lymphoma effect.Results(1)The result of CCK-8 method showed that treatment with different concentrations(5~25mg/m L)of Qi-Zhen-Gui-Pi decoction could significantly inhibit the proliferation of lymphoma cells in a dose dependent manner(P(27)0.01).The result of FCM showed that treatment with 20mg/m L and 25 mg/m L Qi-Zhen-Gui-Pi decoction for 24 h and 48 h could induce apoptosis of Jurkat and Raji cells(Raji cells in20mg/m L Qi-Zhen-Gui-Pi decoction treatment group: P<0.05;others: all P<0.01).The cell cycle was arrested in G2/M phase in all 3 lymphoma cell lines after they were treated with different concentrations of Qi-Zhen-Gui-Pi decoction(10、15、20mg/m L)for 48h(all P(27)0.01).The migration and invasion activity of Jurkat and Raji cells were significantly suppressed by Qi-Zhen-Gui-Pi decoction(15mg/m L,all P(27)0.01).(2)From the gene expression profiling data,a total of 2670 genes were dysregulated significantly,648 genes were significantly up-regulated and 1022 genes were significantly down-regulated after Jurkat cells were treated with Qi-Zhen-Gui-Pi(15mg/m L)decoction for 48h(|Fold Change|>1.5 and FDR<0.05).Based on IPA pathway analysis,PI3K/AKT signaling pathway and JAK/STAT signaling pathway were significantly inhibited among those significantly enriched pathways of differentially expressed genes.The m RNA and protein expression levels of six selected down-regulated genes(PIK3CA、NFKB1、IKBKB、Bcl-2、JAK2、STAT3)was analyzed using real-time fluorescent quantitative PCR and Western Blot respectively.The results show that Qi-Zhen-Gui-Pi decoction significantly down-regulated the expression of m RNA and protein levels of the above genes(real-time fluorescent quantitative PCR : STAT3: P<0.05,others: all P<0.01;Western Blot: all P<0.05).(3)The main compounds and their target proteins of Qi-Zhen-Gui-Pi decoction were analysed by Network pharmacological,and constructed the the herbal medicine-compound-target proteins network of Qi-Zhen-Gui-Pi decoction.The screening target genes and the lymphoma-related genes retrieved from the database were analyzed by Venn Diagram.The result showed that a total of 235 overlapping genes in the above two datasets,that may act as the potential anti-lymphoma target for Qi-Zhen-Gui-Pi decoction.For these potential target genes,KEGG pathway analysis showed that 117 pathways were significantly enriched,including PI3K/AKT signaling pathway,B cell receptor signaling pathway,apoptosis signaling pathway,MAPK signaling pathway,m TOR signaling pathway,NF-κB signaling pathway,JAK/STAT signaling pathway and p53 signaling pathway,etc.The most enriched GO terms were involved in response to drug,positive regulation of ERK1 and ERK2 cascade,enzyme binding,negative regulation of apoptotic process,positive regulation of transcription from RNA polymerase II promoter,etc.The results of bioinformatics and topology of network suggested that the anti-lymphoma compounds of Qi-Zhen-Gui-Pi decoction may be flavonoids and other compounds.Conclusion Qi-Zhen-Gui-Pi decoction can regulate the biological behavior of lymphoma cell lines.It may play an anti-lymphoma role through multiple genes and signaling pathways.The main anti-lymphoma compounds of Qi-Zhen-Gui-Pi decoction may be flavonoids and other compounds.The above experimental results proved that the anti-lymphoma effects of Qi-Zhen-Gui-Pi decoction have the characteristics of multi-component,multi-target and multi-pathway,and provided a preliminary theoretical basis for the clinical application of Qi-Zhen-Gui-Pi decoction in the treatment of lymphoma.