| ObjectiveThe aims of this study are to confirm whether leonurine exhibits the immunomodulatory effect to promote the maturation and activity of monocyte derived dendritic cells(moDCs)from healthy donors(HDs)and multiple myeloma(MM)patients,and further explore the metabolic pathway of moDCs through the metabolomics analysis to preliminarily reveal the mechanism of leonurine on moDCs,which can provide evidence for the application of leonurine as a DC immune adjuvant in MM immunotherapy.Methods(1)Peripheral blood mononuclear cells(PBMCs)from peripheral blood of HDs and MM patients were isolated by density gradient centrifugation.PBMCs were cultured for 24 hours in the medium consisting of RPMI 1640 medium,2 mmol/L L-glutamine,5%human serum,100 U/mL penicillin and 0.1 mg/mL streptomycin.Then non-adherent cells were removed while adherent cells were kept for further incubation.Monocytes from adherent cells were incubated and generated into moDCs for 7 days in the medium consisting of RPMI 1640 medium,2 mmol/L L-glutamine,5%human serum,100 U/mL penicillin,0.1 mg/mL streptomycin,500 U/mL interleukin-4 and 800U/mL granulocyte-macrophage colony-stimulating factor.On the 1st day,the cells were evenly devided into 2 groups.One group was administrated with 1μM leonurine while the other group was administrated with dimethyl sulfoxide as the control group.On the8th day,the cells and incubation supernatant were harvested.The expression of maturation associated surface markers CD80,CD86,CD40,CD83 and HLA-DR on moDCs was analyzed by flow cytometry.The secretion of DC activity associated cytokines macrophage inflammatory protein 1-α(MIP-1α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in the supernatant was detected by enzyme-linked immunosorbent assay.(2)The moDCs administrated with or without 1μM leonurine were harvested,and the intracellular metabolites were extracted.The relative content of intracellular metabolites was detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The peak information in positive ion mode(POS)and negative ion mode(NEG)was combined.After pretreatment and metabolite annotation of the peak information,the metabolites and their relative content were obtained from the peak information.The differentially expressed metabolites between the leonurine group and the control group were ananlyzed by univariate analysis(UVA)and multivariate analysis(MVA).The differentially expressed metabolites were used for metabolic pathway analysis in order to search for the significantly affected pathway,which preliminarily revealed the mechanism of leonurine.Results(1)Leonurine significantly increased the proportion of moDCs(CD80~+CD86~+cells)in the harvested cells from MM patients(n=11,P=0.029),but had no significant effect on the proportion of moDCs in the harvested cells from HDs(n=14,P=0.300).Leonurine enhanced the mean fluorescence intensity(MFI)of CD40,CD83 and HLA-DR expression on HD-moDCs(n=14;P=0.084,P=0.042,P=0.013).Leonurine significantly enhanced the MFI of CD40,CD83 and HLA-DR expression on MM paitent-moDCs(n=11;P=0.025,P=0.020,P=0.006).Leonurine significantly increased the concentration of MIP-1α,IL-1βand IL-6 secreted by MM patient-moDCs(n=11;P=0.005,P=0.004,P=0.002),but had no significant effect on the concentration of MIP-1α,IL-1βand IL-6 secreted by HD-moDCs(n=14;P=0.748,P=0.373,P=0.140).(2)10606 peaks in POS and 8196 peaks in NEG were detected by LC-MS/MS,which were combined and then used for pretreatment and metabolite annotation.Finally,1124 metabolites were obtained.Differentially expressed metabolites between the leonurine group and the control group were analyzed by UVA and MVA,which should achieve the criteria of P<0.05 and variable importance in the projection(VIP)>1.184metabolites were significantly up-regulated while 71 metabolites were significantly down-regulated in the leonurine group compared to those in the control group.After annotation with kyoto encyclopedia of genes and genomes(KEGG)pathway database,42 differentially expressed metabolites were annotated to 15 KEGG pathways.After pathway analysis,the pathways which were significantly affected with the criteria of P<0.05 and Impact>0.2 were the arachidonic acid metabolism pathway and the linoleic acid metabolism pathway.ConclusionLeonurine promotes the maturation and activity of moDCs derived from both HDs and MM patients,the mechanism of which is related to the regulation on the arachidonic acid metabolism pathway and the linoleic acid metabolic pathway.Leonurine is potential to be applied as a DC immune adjuvant in MM immunotherapy. |
