ReFerm®: a postbiotic fermented oat gruel composition is reducing mast cell degranulation in the colon of patients with irritable bowel syndrome

Abstract

Background: Irritable bowel syndrome (IBS) is a highly prevalent gastrointestinal disorder that affects ~4% of the global population. ReFerm^® is a postbiotic product derived from oat gruel fermented with Lactobacillus plantarum 299v, and it has been shown to have beneficial effects on intestinal permeability in patients with IBS. In this study, we investigated the effects of ReFerm^® on regulators of intestinal permeability, namely mast cells and enteric glial cells.

Materials and methods: A total of 30 patients with moderate to severe IBS were treated with an enema containing ReFerm^® or a placebo twice daily. The patients underwent sigmoidoscopy with biopsies obtained from the distal colon at baseline and after 14 days of treatment. These biopsies were processed in two ways: some were fixed, embedded in paraffin, sectioned, and stained for mast cells and enteric glial cells; others were cryopreserved, lysed, and subjected to Western blotting to analyze the same markers.

Results: Treatment with ReFerm^®, but not the placebo, significantly reduced mast cell tryptase protein levels in the biopsy lysates. Although the number of mast cells remained unchanged in colonic biopsies, ReFerm^® treatment significantly reduced mast cell degranulation, a result not observed in the placebo group. Neither ReFerm^® or placebo treatment had an impact on total protein levels or the number of enteric glial cells in the biopsies.

Conclusion: ReFerm^® treatment significantly reduced both total mast cell tryptase levels and the degranulation of mast cells in colonic biopsies from patients with IBS, suggesting a decrease in mast cell activity as a potential mechanism underlying the beneficial effects of ReFerm^®. However, further research is required to assess the molecular mechanisms through which ReFerm^® operates in the colons of patients with IBS.

Clinical trial registration: https://clinicaltrials.gov, identifier: NCT05475314.

Keywords: enteric nervous system; functional gastrointestinal disorder; intestinal permeability; mucosal immunology; postbiotics.

Figure 1

Study design. Patients with moderate-to-severe irritable bowel syndrome were recruited based on inclusion and exclusion criteria. The patients underwent two sigmoidoscopies that were 2 weeks apart, and in the meantime, they were administered enemas with either ReFerm^® or placebo twice a day for 2 weeks. The collected biopsies were either fixed, embedded in paraffin, sectioned, and stained with markers for mast cells and enteric glial cells or lysed and analyzed by western blotting for mast cells and enteric glial cell protein markers.

Figure 2

The effect of ReFerm^® and placebo on mast cells in patients with irritable bowel syndrome. (A) Representative fluorescence images of colonic biopsies stained for mast cell tryptase (MCT) before treatment (BT) and post treatment (PT). Magenta = MCT (arrows), cyan = cell nuclei stained by DAPI. (B) The number of mast cells was similar in biopsies BT and PT with ReFerm^®. (C) The number of mast cells was similar in biopsies BT and PT with the placebo treatment. (D) Representative western blot membranes for MCT with β-actin as a loading control. (E) The quantification of western blotting for the ReFerm^® group showed significantly decreased MCT levels in PT compared to BT in the ReFerm^® group. (F) Quantification of western blots for the placebo group showed no differences between treatment groups. All data are given as a median and interquartile range, and comparisons are made using the Wilcoxon matched-pairs signed rank test. *p < 0.05. ns, non-significant; N, number of patients.

Figure 3

The effect of ReFerm^® and placebo on mast cell degranulation in patients with irritable bowel syndrome. (A) Representative images from single cells were double-stained for mast cell tryptase (MCT) and wheat germ agglutinin (WGA) to visualize the cell membrane. Magenta = MCT (arrows indicate extracellular granules), cyan = cell nuclei stained by DAPI. (B) The number of extracellular MCT granules was significantly decreased in the ReFerm^® group post treatment (PT) as compared to before treatment (BT). In total, 64 MCT+ cells were analyzed. (C) The size of MCT extracellular granules significantly decreased PT with ReFerm^® compared to BT. In total, 52 MCT+ cells were analyzed. (D) The number of extracellular MCT granules was not affected by the placebo treatment. In total, 62 MCT+ cells were analyzed. (E) The size of MCT extracellular granules was not affected by the placebo treatment. In total, 66 MCT+ cells were analyzed. All data are given as a median and interquartile range, and comparisons were made using the Wilcoxon matched-pairs signed rank test. ***p < 0.001, ****p < 0.0001, and ns, non-significant. Each point in the graphs represents one imaged and analyzed mast cell.

Figure 4

The effect of ReFerm^® and placebo on enteric glial cells identified by glial fibril acidic protein (GFAP) in patients with irritable bowel syndrome. (A) Representative fluorescence images of colonic biopsies stained for GFAP before treatment (BT) and post treatment (PT). Magenta = GFAP (arrows), cyan = cell nuclei stained by DAPI. (B) The number of GFAP⁺ cells was similar in biopsies BT and PT with ReFerm^®. (C) The number of GFAP+ cells was similar in biopsies BT and PT with placebo (D) Representative western blot membranes for GFAP with β-actin as a loading control. (E) The quantification of western blotting for the ReFerm^® group showed decreased levels of GFAP PT compared to BT in the ReFerm^® group; however, it did not reach statistical significance. (F) The quantification of western blots for the placebo group showed no difference in GFAP levels in PT compared to BT. All data are given as a median and interquartile range, and comparisons are made using the Wilcoxon matched-pairs signed rank test. ns, non-significant; N, number of patients.

Figure 5

The effect of ReFerm^® and placebo on enteric glial cells identified by calcium-binding protein β (S100β) in patients with irritable bowel syndrome. (A) Representative fluorescence images of colonic biopsies stained for S100β before treatment (BT) and post treatment (PT). Pink = S100β (arrows), blue = cell nuclei stained by DAPI. (B) The number of S100β+ cells was similar in biopsies BT and PT with ReFerm^®. (C) The number of S100β+ cells was similar in biopsies BT and PT with placebo (D) Representative western blot membranes for S100β+ with β-actin as a loading control. (E) Quantification of western blotting for the ReFerm^® group showed decreased levels of S100β PT compared to BT in the ReFerm^® group; however, it did not reach statistical significance. (F) Quantification of western blotting for the placebo group showed no difference in S100β levels in PT compared to BT. All data are given as a median and interquartile range, and comparisons are made using the Wilcoxon matched-pairs signed rank test. ns, non-significant; N, number of patients.