NMR METABOLOMICS AND PROTEOMICS ON CELL MONOLAYER STUDYING THE EFFECT OF SYMBIOTICS

Introduction:  The gut microbiota has shown to be involved in changes of the body fat mass in humans. The aim of this study was to investigate if symbiotics may enhance the gut health benefit of pre- and probiotics. By growing a Caco-2 cell monolayer on permeable membranes in-vitro, the cell model can be used to mimic the small intestine epithelium. Stimulations were performed by adding selected fecal water samples from a large clinical trial on symbiotics to the apical side of the Caco-2 cell monolayer. To explore the mechanistic effects, NMR metabolomics was used to detect indirect effects of the Caco-2 cell monolayer and fecal water on the metabolic profiles of the cell media. Proteomics on the cells were carried out to determine if proteins were regulated by symbiotic treatment. Methods & Results:  Stimulations were performed by adding fecal samples diluted in PBS and sterilized by filtration through a 0.22-micron filter from 26 subjects. The samples belong to 5 different groups: Placebo, A_responder, A_non-responder, A+B_responder, A+B_non-responder, where A+B is the symbiotic treatment. Caco-2 cells were seeded onto filter inserts and incubated in 24-well plates for 18 days. Stimulations were performed for 24 hours with 4 replicates. A hundred μL sterile fecal water and 300 μL cell media was added to the apical side. Four replicates of control samples on each plate were obtained by the addition of 400 μL cell media to the apical side. Cell media from the apical and basolateral sides are collected at the end of the intervention. CaCo-2 cells were harvested, lysed and digested. 1 H NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer equipped with a Prodigy CryoProbe using standard 1D NOESY experiments. The 1H NMR spectra were automatically aligned, phased and binned prior to being analyzed by multivariate statistics. Proteomics were performed by using a nanoflow-LC connected to an Orbitrap Fusion. Proteins were identified using SEQUES HT + Percolator and quantified label-free in GeneData Expressionist. Regulated proteins were analyzed by Ingenuity Pathway Analysis (IPA) software. Results in a total of 7057 proteins were identified using 1% false discovery rate (FDR). Statistical filtering of the preliminary quantitative results showed that 145 proteins were significantly regulated. Pathway analysis of regulated proteins in IPA, showed that several pathways were affected including inflammatory responses, immunological disease, and lipid metabolism. PCA analysis using the 145 proteins or the binned NMR spectra showed a weak tendency for subjects to separate between responders and non-responders in the group who received symbiotics. Conclusion:  TEER measurements on the Caco-2 cell monolayer model indicate a stronger gut barrier integrity as an effect of the symbiotic group. Differences in the metabolic profiles was found on the apical side between symbiotic responder vs. non-responder groups. No clear metabolic effect of the caco-2 cell monolayer could be found. Proteomics suggests that the effect from the symbiotic treatment effects pathways involved in cell proliferation of gut epithelium cells and ion transport across the cell layer. Regulated pathways observed in this study needs to be further investigated for their relationship to a symbiotic diet.