Pleurotus eryngii Mushrooms Fermented with Human Fecal Microbiota Protect Intestinal Barrier Integrity: Immune Modulation and Signalling Pathways Counter Deoxycholic Acid-Induced Disruption in Healthy Colonic Tissue

Abstract

Background: This study explores the potential of the Pleurotus eryngii mushroom fermentation supernatant (FS-PEWS) as an intervention for mitigating sodium deoxycholate (SDC)-induced intestinal barrier dysfunction and inflammation. Methods: FS-PEWS was assessed for its protective effects against SDC-induced barrier dysfunction and inflammation using an in vitro Caco-2 cell model and ex vivo colonic biopsies from healthy adult donors, where barrier integrity, permeability, immunomodulation and receptor-mediated pathways were evaluated. Results: In Caco-2 cells, SDC exposure downregulated ZO-1, occludin, and claudin-1 expression, with FS-PEWS restoring ZO-1 and claudin-1 levels while maintaining cell viability. In colonic biopsies from healthy adults, FS-PEWS maintained tissue integrity and selectively mitigated transcellular permeability without affecting paracellular permeability when combined with the stressor. Additionally, FS-PEWS exhibited potent anti-inflammatory effects, reducing pro-inflammatory cytokines, e.g., TNF-α, IL-6, and IL-1β and modulating receptor-mediated pathways, i.e., TLR-4, dectin-1. Conclusions: These results demonstrate the potential of FS-PEWS to sustain intestinal barrier function and modulate immune responses under stress, highlighting its therapeutic potential for managing gut barrier dysfunction and inflammation associated with microbial metabolite-induced disruptions.

Keywords: Pleurotus eryngii mushrooms; Ussing chamber; cytokines; deoxycholic bile acid; gut barrier; signalling pathway.

Figure 1

Effects of sodium deoxycholate (SDC) on Caco-2 cell viability (MTT assay). Caco-2 cells were seeded in a 6-well plate and treated with SDC at concentrations of 0.2 mM, 1 mM, and 2 mM for 30, 60, 90, and 120 min. Cell viability is expressed as a percentage relative to the untreated control group, which was set at 100%. The results are presented as mean ± standard deviation of three independent experiments. ^a statistically significant compared to 0.2 mM; * statistically significant compared to 1 mM; p < 0.05 (Wilcoxon signed-rank test).

Figure 2

The relative expression of TJ genes in response to SDC-stimulated (1 mM) Caco-2 cells for 30, 60, 90, and 120 min. Data are expressed as mRNA expression (normalized to β-actin) relative to untreated cells as mean ± SD of two independent experiments. ^a statistically significant compared to untreated cells; p < 0.05 (Wilcoxon signed-rank test).

Figure 3

The relative expression of TJ (ZO-1, occludin, claudin-1) genes in response to incubation (2% v/v for 48 h) with FS-NC and FS-PEWS from a total of five faecal donors in the SDC-stimulated (1 mM for 90 min) Caco-2 cells. Data are expressed as mRNA expression (normalized to β-actin) relative to untreated cells as mean ± SD of two independent experiments. Untreated cells: culture cells without any effect, Cells + SDC: culture cells stimulated only with SDC, FS-NC/SDC: culture cells incubated with FSs of the negative control (basal medium with no carbohydrate source) and then stimulated with SDC; FS-PEWS/SDC: culture cells incubated with FSs of P. eryngii mushroom and then stimulated with SDC; ^a statistically significant compared to untreated cells; * statistically significant compared to SDC; p < 0.05 (Wilcoxon signed-rank test).

Figure 4

(A,B) Effects of FS-PEWS, SDC and their co-stimulation on colonic. (A) paracellular permeability; (B) transcellular permeability in biopsies mounted in Ussing chambers. Data (Δ90-0 min) are presented as a line intersecting the median. Dots represent the outlier values. Untreated biopsies: biopsies without any treatment (n = 10); biopsies + SDC: biopsies stimulated only with SDC (n = 10); biopsies + FS-PEWS: biopsies incubated only with FS of P. eryngii untreated mushroom from one universal faecaldonor (n = 10); biopsies + FS-PEWS/SDC: biopsies incubated with FS of P. eryngii untreated mushroom from one universal faecal donor and then stimulated with SDC (n = 9); ^a p < 0.05 statistically significant compared to untreated biopsies; * p < 0.05 statistically significant compared to SDC; † p < 0.05 statistically significant compared to FS-PEWS; Wilcoxon matched-pairs signed-rank test.

Figure 5

The relative expression of TJ (ZO-1, occludin, claudin-1) genes in response to incubation (2% v/v) with FS-PEWS with colonic biopsies obtained from a total of five high SDC responders mounted in the Ussing chamber ex vivo system. Data are expressed as mRNA expression (normalized to β-actin) relative to untreated biopsies as the mean ± SD of two independent experiments. Untreated biopsies: biopsies without any treatment; biopsies + SDC: biopsies stimulated only with SDC; biopsies + FS-PEWS: biopsies incubated only with fermented P. eryngii untreated mushroom (2% v/v) from one universal faecal donor; biopsies + FS-PEWS +SDC: biopsies incubated with fermented P. eryngii untreated mushroom (2% v/v) from one universal faecal donor and then stimulated with SDC; * statistically significant compared to SDC; † statistically significant compared to FS-PEWS; p < 0.05 (Wilcoxon signed-rank test).

Figure 6

The relative expression of cytokine (TNF-α, IL-1β, IFN-γ, IL-6, IL-8, IL-10) genes in response to incubation (2% v/v) with FS-PEWS with colonic biopsies obtained from a total of five high SDC responders mounted in the Ussing chamber ex vivo system. Data are expressed as mRNA expression (normalized to β-actin) relative to untreated biopsies as mean ± SD of two independent experiments. Untreated biopsies: biopsies without any treatment; biopsies + SDC: biopsies stimulated only with SDC; biopsies + FS-PEWS: biopsies incubated only with fermented P. eryngii untreated mushroom (2% v/v) from one universal faecal donor; biopsies + FS-PEWS + SDC: biopsies incubated with fermented P. eryngii untreated mushroom (2% v/v) from one universal faecal donor and then stimulated with SDC; ^a statistically significant compared to untreated cells; * statistically significant compared to SDC; † statistically significant compared to FS-PEWS; p < 0.05 (Wilcoxon signed-rank test).

Figure 7

The relative expression of receptors (TLR-2, TLR-4, NF-kB, mTOR, dectin-1) genes in response to incubation (2% v/v) with FS-PEWS with colonic biopsies obtained from a total of five high SDC responders mounted in the Ussing chamber ex vivo system. Data are expressed as mRNA expression (normalized to β-actin) relative to untreated biopsies as mean ± SD of two independent experiments. Untreated biopsies: biopsies without any treatment; biopsies + SDC: biopsies stimulated only with SDC; biopsies + FS-PEWS: biopsies incubated only with fermented P. eryngii untreated mushroom (2% v/v) from one universal faecal donor; biopsies + FS-PEWS + SDC: biopsies incubated with fermented P. eryngii untreated mushroom (2% v/v) from one universal faecal donor and then stimulated with SDC; ^a statistically significant compared to untreated cells; * statistically significant compared to SDC; † statistically significant compared to FS-PEWS; p < 0.05 (Wilcoxon signed-rank test).