Figure 4.

A. muciniphila-derived EVs reduce LPS-induced intestinal permeability through AMPK phosphorylation. (a) Trans-epithelial electrical resistance in Caco-2 cells 4 h after treatment with LPS (5 μg ml⁻¹), LPS (5 μg ml⁻¹)+EcEVs (1 μg ml⁻¹) and LPS (5 μg ml⁻¹)+AmEVs (1 μg ml⁻¹). (b) In vitro permeability assay in Caco-2 cells 4 h after LPS (5 μg ml⁻¹), LPS (5 μg ml⁻¹)+EcEV (1 μg ml⁻¹) and LPS (5 μg ml⁻¹)+AmEV (1 μg ml⁻¹) treatment. FITC-dextran was added to the upper chamber in the Caco-2 transwell chamber assay, and the transport of FITC-labeled dextran across the Caco-2 monolayer culture was measured by fluorescence spectrofluorometry. (c) Expression of tight junction proteins in Caco-2 cells after LPS, LPS+EcEV and LPS+AmEV treatments. A graph showing the relative occludin expression (normalized to actin) is also shown (*P<0.05). (d) AmEV treatment increased occludin expression and phosphorylation of AMPKα2 thr-172 in a dose-dependent manner. Caco-2 cells were treated with 0.1, 1 and 10 μg ml⁻¹ AmEVs, and the expression of occludin, p-AMPK, total AMPK and actin was analyzed after 4 h by immunoblotting. A graph showing the relative protein expression normalized to actin is shown (occludin expression in NT vs the specified treatment group ^##P<0.01, ^###P<0.001; p-AMPK expression in NT vs the specified treatment group **P<0.01). (e) Time course of AmEV-induced occludin and p-AMPK expression in Caco-2 cells. Caco-2 cells were treated with 1 μg ml⁻¹ AmEVs, and the expression of occludin, p-AMPK, total AMPK and actin was analyzed after 1, 2, 4 and 8 h by immunoblotting. The graph shows the relative protein expression after normalization to actin (occludin expression in NT vs the specified time point ^#P<0.05; p-AMPK expression in NT vs the specified time point *P<0.05, **P<0.01). (f) Western blot showing the effect of the AMPK inhibitor on AmEV-treated Caco-2 cells. Caco-2 cells were treated with the AMPK-specific inhibitor compound C for 30 min, after which 1 μg ml⁻¹ AmEVs was applied. Protein expression of occludin and p-AMPK was observed after 4 h. The graph shows the relative occludin expression after normalization to actin (occludin expression in NT vs the specified time point ^#P<0.05). All data are presented as the mean±s.e.m. of three experiments performed in triplicate. NS, not significant.