Fermentation Supernatants of Pleurotus eryngii Mushroom Ameliorate Intestinal Epithelial Barrier Dysfunction in Lipopolysaccharide-Induced Caco-2 Cells via Upregulation of Tight Junctions

Georgia Saxami, Evangelia N Kerezoudi, Evdokia K Mitsou, Georgios Koutrotsios, Georgios I Zervakis, Vasiliki Pletsa, Adamantini Kyriacou, Georgia Saxami, Evangelia N Kerezoudi, Evdokia K Mitsou, Georgios Koutrotsios, Georgios I Zervakis, Vasiliki Pletsa, Adamantini Kyriacou

Abstract

In recent years, modulation of gut microbiota through prebiotics has garnered interest as a potential to ameliorate intestinal barrier dysfunction. The aim of the study was to examine the in vitro effect of fermentation supernatants (FSs) from rich in β-glucan Pleurotus eryngii mushrooms on the expression levels of tight junctions (TJs) genes in Caco-2 cells stimulated by bacterial lipopolysaccharides (LPS). Mushrooms were fermented using fecal inocula in an in vitro batch culture model. Caco-2 cells were subjected to LPS and FS treatment under three different conditions: pre-incubation with FS, co- and post-incubation. Reverse transcription PCR was applied to measure the expression levels of zonulin-1, occludin and claudin-1 genes. FSs from P. eryngii mushrooms led to a significant upregulation of the TJs gene expression in pre-incubation state, indicating potential preventive action. Down-regulation of all TJs gene expression levels was observed when the cells were challenged with LPS. The FS negative control (gut microbiota of each donor with no carbohydrate source) exhibited a significant upregulation of TJs expression levels compared to the cells that were challenged with LPS, for all three conditions. Overall, our data highlighted the positive and potential protective effects of P. eryngii mushrooms in upregulation of TJs' genes.

Keywords: Pleurotus eryngii; claudin-1; fermentation supernatants; gut barrier; occludin; zonulin 1.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effects of lipopolysaccharides (LPS) and FSs (FS-NC, FS-PEWS and FS-PEWSGM) on Caco-2 cell viability. Caco-2 cells were seeded in a 6-well plate and treated with (A) LPS at concentrations of 80, 100, 120, 150 and 200 ng/mL for 3, 24, 48 and 72 h, (B) FS-NC at concentrations of 1%, 2%, 5%, 8% and 10% for 3, 24, 48 and 72 h, (C) FS-PEWS at concentrations of 1%, 2%, 5%, 8% and 10% for 3, 24, 48 and 72 h, and (D) FS-PEWSGM at concentrations of 1%, 2%, 5%, 8% and 10% for 3, 24, 48 and 72 h. Values are mean ± SD of three independent experiments.
Figure 2
Figure 2
The effects of FSs from P. eryngii mushrooms (FS-PEWS and FS-PEWSGM) on zonulin-1 expression levels in LPS-induced Caco-2 cells. Caco-2 cells were subjected to LPS and FS treatment under three different conditions: pre incubation (FS/LPS), co-incubation (FS + LPS) and post-incubation (LPS/FS). Untreated: Caco-2 cells without any effect; FS-NC: FS of the negative control (NC: sample without additional carbon source); Cells + LPS: Caco-2 cells subjected to LPS (100 ng/mL). Data are expressed as mRNA expression (normalized with b-actin) relative to untreated cells as mean ± SD of two independent experiments. a statistically significant for FS-PEWS or FS-PEWSGM compared to untreated cells (p < 0.05), * statistically significant for FS-PEWS or FS-PEWSGM compared to Cells + LPS (p < 0.05) (Wilcoxon matched-pairs signed rank test or paired samples t-test).
Figure 3
Figure 3
The effects of FSs from P. eryngii mushrooms (FS-PEWS and FS-PEWSGM) on occludin expression levels in LPS-induced Caco-2 cells. Caco-2 cells were subjected to LPS and FS treatment under three different conditions: pre incubation (FS/LPS), co-incubation (FS + LPS) and post-incubation (LPS/FS). Untreated: Caco-2 cells without any effect; FS-NC: FS of the negative control (NC: sample without additional carbon source); Cells + LPS: Caco-2 cells subjected to LPS (100 ng/mL). Data are expressed as mRNA expression (normalized with b-actin) relative to untreated cells as mean ± SD of two independent experiments. a statistically significant for FS-PEWS or FS-PEWSGM compared to untreated cells (p < 0.05), † statistically significant for FS-PEWS or FS-PEWSGM compared to FS-NC (p < 0.05), * statistically significant for FS-PEWS or FS-PEWSGM compared to Cells + LPS (p < 0.05); ** statistically significant for FS-PEWS compared to FS-PEWSGM (p < 0.05) (Wilcoxon matched-pairs signed rank test or paired samples t-test).
Figure 4
Figure 4
The effects of FSs from P. eryngii mushrooms (FS-PEWS and FS-PEWSGM) on claudin-1 expression levels in LPS-induced Caco-2 cells. Caco-2 cells were subjected to LPS and FS treatment under three different conditions: pre incubation (FS/LPS), co-incubation (FS + LPS) and post-incubation (LPS/FS). Untreated: Caco-2 cells without any effect; FS-NC: FS of the negative control (NC: sample without additional carbon source); Cells + LPS: Caco-2 cells subjected to LPS (100 ng/mL). Data are expressed as mRNA expression (normalized with b-actin) relative to untreated cells as mean ± SD of two independent experiments. a statistically significant for FS-PEWS or FS-PEWSGM compared to untreated cells (p < 0.05), * statistically significant for FS-PEWS or FS-PEWSGM compared to Cells + LPS (p < 0.05) (Wilcoxon matched-pairs signed rank test or paired samples t-test).

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