Objective To explore the effects of Bifidobacterium bifidum BD-1(BD-1)and its metabolites in inducing cy-tokine secretion in murine macrophage cell line RAW264.7 cells when using 2'-fucosyllactose(2'-FL)vs glucose as the sole carbon source,aiming to gain further insights into the immunomodulatory properties of BD-1 when using human milk oligosaccharides as a carbon source.Methods(1)BD-1 intervention experiment:live BD-1 bacteria were inoculated in modified MRS broth containing 2％2'-FL(2'-FL group)and 2％glucose as the sole carbon source(Glu group)respect-ively.Following anaerobic incubation for 24 hours,the viable BD-1 bacteria were separated from the cells and medium su-pernatants in each group.The cell components were co-cultured with RAW264.7 cells,with peptidoglycan(PGN)as the control for cellular composition.A negative control group without any intervention(Ctrl group)was established.(2)Inter-vention experiments with metabolites of BD-1:the experimental control consisted of medium containing 2％2'-FL or gluc-ose without BD-1 fermentation,while the negative control(Ctrl group)involved medium without any intervention with RAW264.7 cells.The co-culture supernatant of PGN and RAW264.7 cells was utilized as a positive control(PGN group).Following 24 hours of incubation,the total RNA from RAW264.7 cells was extracted and subjected to reverse transcrip-tion using quantitative real-time PCR.The mRNA expression levels of tumor necrosis factor(TNF)-α,interleukin(IL)-6,IL-10,IL-12 and Il-1β were assessed through RT-qPCR.Furthermore,the supernatants obtained from the co-cultures of RAW264.7 cells and the respective test materials in each group were collected to determine the secretion levels of these cy-tokines via enzyme-linked immunosorbent assay.Results(1)BD-1 intervention:compared to the PGN group,the secre-tion and gene expression of TNF-α,IL-6,and IL-10 were significantly elevated in both the 2'-FL group and Glu group(all P＜0.05).In comparison to the Glu group,there was a significant decrease in the secretion of IL-6 and IL-10 in the 2'-FL group(all P＜0.05),accompanied by a downward trend in their expression levels.(2)Metabolites of BD-1 intervention:both the secretion and gene expression of TNF-α,IL-6 and IL-10 were markedly higher in BD-1-2'-FL supernatant group and BD-1-Glu supernatant group compared to the 2'-FL group and Glu group(all P＜0.05).Additionally,there was a signi-ficantly higher secretion of IL-6 and IL-10 observed in the 2'-FL group than that in the Glu group(all P＜0.05),with a not-able increase seen specifically in IL-6 expression(P＜0.05)while an upward trend was noted in IL-10 expression level as well.Furthermore,when compared to the Glu group,it was found that organic acid production along with saccharide abundance were notably increased in the 2'-FL group.Conclusion There are variations in the characteristics of BD-1 cells cultured with human milk oligosaccharides or glucose as the sole carbon source,as well as in the cytokines secreted by macrophages.When BD-1 cells were nourished with human milk oligosaccharides as the sole carbon source,there was an observed inclination towards anti-inflammatory properties compared to glucose,evident through reduced secretion and gene expression of IL-6 in macrophages.Conversely,its metabolites exhibited a tendency to stimulate immune activation,resulting in increased secretion and gene expression of IL-6 and IL-10 in macrophages.
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