The effect of Bacteroides fragilis and its postbiotics on the expression of genes involved in the endocannabinoid system and intestinal epithelial integrity in Caco-2 cells

Abstract

Purpose: Gut microbiota and its derivatives by constantly interacting with the host, regulate the host function. Intestinal epithelium integrity is under the control of various factors including the endocannabinoid system (ECS). Accordingly, we aimed at investigating the effect of Bacteroides fragilis and its postbiotics (i.e., heat-inactivated, cell-free supernatants (CFS) and outer membrane vesicles (OMVs)) on the expression of genes involved in ECS (cnr1, faah, pparg) and the epithelial barrier permeability (ocln, tjp1) in a Caco-2 cell line.

Methods: Caco-2 cell line was treated with live or heat-inactivated B. fragilis at MOIs of 50 and 100, or stimulated with 7% V/V CFS and B. fragilis OMVs at a dose of 50 and 100 µg/ml overnight. RT-qPCR was applied for expression analysis.

Results: Heat-inactivated B. fragilis induced cnr1, pparg, tjp1, and suppressed faah expression, while live B. fragilis had the opposite effect. OMVs increased pparg, and tjp1 expression by reducing the activity of ECS through an increase in faah and a reduction in cnr1 expression. Finally, an increase in the expression of pparg and ocln, and a reduction in the expression of cnr1 was detected in Caco-2 cells treated with CFS.

Conclusion: The live and heat-inactivated B. fragilis inversely affected cnr1, faah, pparg, and tjp1 expression in Caco-2 cells. Increased tjp1 mRNA levels by affecting the expression of ECS related genes is taken as an indication of the potential beneficial effects of B. fragilis postbiotics and making them potential candidates for improving permeability in the leaky gut syndrome.

Supplementary information: The online version contains supplementary material available at 10.1007/s40200-023-01264-8.

Keywords: Bacteroides fragilis; Endocannabinoid system; Epithelial barrier; Gut microbiota; Postbiotics.

Fig. 1

Effects of live (MOI 50 and MOI 100) and heat-inactivated (Heat-inactivated 50 and 100) forms of B. fragilis(a, b), its derived OMVs (50 and 100 µg/mL) (c, d), and CFS (7% V/V) (e, f) on the expression of ECS related genes (cnr1 and faah) in Caco-2 cells. Phosphate buffer solution (PBS), BHI and sucrose were treated as related controls. Expression data are normalized using gapdh as the internal control gene. (*) and (**) signify P-values of < 0.05 and < 0.001, respectively, obtained by one-way ANOVA and t-test statistical analyses. MOI: multiplicity of infection, OMV: outer membrane vesicle, CFS: cell free supernatant, ns: not significant

Fig. 2

Effects of live (MOI 50 and MOI 100) and heat-inactivated (Heat-inactivated 50 and 100) forms of B. fragilis(a), its derived OMVs (50 and 100 µg/mL) (b), and CFS (7% V/V) (c) on the expression of pparg gene in Caco-2 cells. Phosphate buffer solution (PBS), BHI and sucrose were treated as related controls. Expression data are normalized using gapdh as the internal control gene. (*) and (**) signify P-values of < 0.05 and < 0.001, respectively, obtained by one-way ANOVA and t-test statistical analyses. MOI: multiplicity of infection, OMV: outer membrane vesicle, CFS: cell free supernatant, ns: not significant

Fig. 3

Effects of live (MOI 50 and MOI 100) and heat-inactivated (Heat-inactivated 50 and 100) forms of B. fragilis(a, b), its derived OMVs (50 and 100 µg/mL) (c, d), and CFS (7% V/V) (e, f) on the expression of tight junction genes (tjp1 and ocln) in Caco-2 cells. Phosphate buffer solution (PBS), BHI and sucrose were treated as related controls. Expression data are normalized using gapdh as the internal control gene. (*) and (**) signify P-values of < 0.05 and < 0.001, respectively, obtained by one-way ANOVA and t-test statistical analyses. MOI: multiplicity of infection, OMV: outer membrane vesicle, CFS: cell free supernatant, ns: not significant