(-)-Epicatechin and NADPH oxidase inhibitors prevent bile acid-induced Caco-2 monolayer permeabilization through ERK1/2 modulation

Abstract

Secondary bile acids promote gastrointestinal (GI) tract permeabilization both in vivo and in vitro. Consumption of high fat diets increases bile acid levels in the GI tract which can contribute to intestinal permeabilization and consequent local and systemic inflammation. This work investigated the mechanisms involved in bile acid (deoxycholic acid (DCA))-induced intestinal epithelial cell monolayer permeabilization and the preventive capacity of (-)-epicatechin (EC). While EC prevented high fat diet-induced intestinal permeabilization in mice, it did not mitigate the associated increase in fecal/cecal total and individual bile acids. In vitro, using differentiated Caco-2 cells as a model of epithelial barrier, EC and other NADPH oxidase inhibitors (VAS-2870 and apocynin) mitigated DCA-induced Caco-2 monolayer permeabilization. While EC inhibited DCA-mediated increase in cell oxidants, it did not prevent DCA-induced mitochondrial oxidant production. Prevention of DCA-induced ERK1/2 activation with EC, VAS-2870, apocynin and the MEK inhibitor U0126, also prevented monolayer permeabilization, stressing the key involvement of ERK1/2 in this process and its redox regulation. Downstream, DCA promoted myosin light chain (MLC) phosphorylation which was related to MLC phosphatase (MLCP) inhibition by ERK1/2. DCA also decreased the levels of the tight junction proteins ZO-1 and occludin, which can be related to MMP-2 activation and consequent ZO-1 and occludin degradation. Both events were prevented by EC, NADPH oxidase and ERK1/2 inhibitors. Thus, DCA-induced Caco-2 monolayer permeabilization occurs mainly secondary to a redox-regulated ERK1/2 activation and downstream disruption of TJ structure and dynamic. EC's capacity to mitigate in vivo the gastrointestinal permeabilization caused by consumption of high-fat diets can be in part related to its capacity to inhibit bile-induced NADPH oxidase and ERK1/2 activation.

Keywords: Bile acids; Deoxycholic acid; Epicatechin; High fat; Intestinal permeability.

Graphical abstract

Fig. 1

Effects of a high-fat diet and of EC supplementation on fecal/cecal bile acid profiles in mice. Mice were fed a control diet (empty bars), a high-fat diet (HF) (black bars), or the high-fat diet supplemented with 20 mg EC/kg body weight (HFE) (blue bars). A- Total fecal bile acids and B-E- individual cecum bile acids were measured as described in methods. Results are shown as means ± SEM and are the average of 5–7 animals/group. Values having different superscripts are significantly different (p < 0.05, One-way ANOVA) MCA: muricholic acid, CA: cholic acid, CDCA: chenodeoxycholic acid, DCA, deoxycholic acid; LCA: lithocholic acid, HDCA: hyodeoxycholic acid, UDCA: ursodeoxycholic acid, T-α-MCA: tauro-α-muricholic acid, T-β-MCA: tauro-β-muricholic acid, TCA: taurocholic acid, TCDCA: taurochenodeoxycholic acid, THCA: taurohyocholic acid, GCA: glycholic acid, GCDCA: glycochenodeoxycholic acid, TDCA: taurodeoxycholic acid, TLC: taurolithocholic acid, THDCA: taurohyodeoxycholic acid, TUDCA: tauroursodeoxycholic acid, GDCA: glycohyodeoxycholic acid and GHDCA: glycohyodeoxycholic acid. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

DCA causes an increase in Caco-2 cell monolayer paracellular permeability that is prevented by EC. Caco-2 cell monolayers were treated with 100 μM DCA in the absence or the presence of 0–10 μM EC added to the upper chamber and cells incubated for 0–4 h. Caco-2 cell monolayer permeability was evaluated by measuring FITC-dextran paracellular transport. A- Cell viability of Caco-2 cells treated with DCA and in the absence or the presence of 1–10 μM EC for 6 h. B- Kinetics of FITC-dextran paracellular transport in Caco-2 monolayers in the absence of additions (empty circles), and in the presence of 100 μM DCA without (black triangles) or with (blue triangles) simultaneous addition of 5 μM EC. *Significantly different from controls at the corresponding time point. C,D- Dose-dependent inhibition by EC of DCA-induced FITC-dextran paracellular transport after 2 h (C) or 4 h (D) incubation. Results are shown as mean ± SEM of 5 independent experiments. A,C,D- Data were normalized to control values. Values having different superscripts are significantly different (p < 0.05, One-way ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

DCA causes an increase in Caco-2 cell oxidant: effects of EC. Caco-2 cell monolayers were treated with 100 μM DCA in the absence or the presence of 5 μM EC for 0–4 h. A,B- mRNA levels of NOX1 and NOX4 were measured by RT-PCR and referred to β-actin mRNA content. mRNA levels were measured A-between 0-4 h for cells incubated without or with DCA, or B- for 2 h without or with addition of DCA and in the absence or the presence of 5 μM EC. C- Kinetics (0–4 h) of oxidant production evaluated using the probes Amplex® Red (red circles), DCFDA (grey triangles), and DHE (blue triangles) as described in methods. D- Effects of DCA and 5 μM EC on oxidant production as measured with Amplex® Red (AR) after 1 h incubation, or with DCFDA and DHE after 4 h incubation. E-G- Mitochondrial oxidant production was evaluated with MitoSOX. E- typical FACS scan profiles for cells incubated in the absence (C) or the presence of 100 μM DCA (DCA). NC: negative control of cells not added with MitoSOX. F- -Kinetics of MitoSOX fluorescence of cells incubated in the presence of DCA. G- MitoSOX fluorescence in cells incubated without or with DCA and in the absence or presence of 5 μM EC for 2 h. Results are shown as mean ± SEM of 4–5 independent experiments. Data were normalized to control values. Kinetic graphs: control values are shown as a dashed grey line. *Significantly different from controls at the corresponding time point. Bars: values having different superscripts are significantly different (p < 0.05, One-way ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

ERK1/2 activation is involved in DCA-induced Caco-2 cell monolayer permeabilization: inhibitory actions of EC and other NADPH oxidase inhibitors. Caco-2 cell monolayers were incubated for 0–4 h with or without 100 μM DCA in the absence or the presence of 5 μM EC, 1 μM apocynin, 1 μM VAS-2870 or 10 μM U0126. A- The kinetics of ERK1/2 and NF-κB activation in the presence of 100 μM DCA was evaluated by Western blot by measuring the phosphorylation of ERK1/2 (T202/Y204) and p65 (Ser536), respectively. B- Cells were incubated with or without 100 μM DCA and in the absence or the presence of 1 μM EC for either 0.5 h to assess EGFR phosphorylation (Tyr1068) or 2 h for ERK1/2 phosphorylation. C,D- Cells were incubated with or without 100 μM DCA and in the absence or the presence of apocynin, VAS-2870 or U0126 for 2 h. C,D- Effects of the NADPH oxidase and ERK1/2 inhibitors on (C) DCA-induced ERK1/2 phosphorylation measured by Western blot, and (D) Caco-2 cell monolayer permeabilization evaluated as the FITC-dextran paracellular transport. Western blot bands were quantified and values for phosphorylated proteins were referred to the respective total protein content. Results are shown as mean ± SEM of 4–6 independent experiments. A- *Significantly different from controls at the corresponding time point. B-D- Values having different superscripts are significantly different (p < 0.05, One-way ANOVA).

Fig. 5

DCA promotes an increase in MLC phosphorylation independently from MLCK but dependent on MLC phosphatase inhibition: effects of EC, and NADPH oxidase and ERK1/2 inhibitors. Caco-2 cell monolayers were incubated for 2 h with or without 100 μM DCA and in the absence or the presence of 5 μM EC, 1 μM apocynin, 1 μM VAS-2870 or 10 μM U0126. (A) MLC (Ser19) and (D) MYPT1 (Thr696) phosphorylation levels were measured by Western blot. After quantification values for phosphorylated proteins were referred to the respective total protein content. β-actin was measured as loading control. B,C- MLCK mRNA levels were measured by RT-PCR and values referred to the β-actin mRNA content. B- Kinetics of DCA-mediated effects on MLCK mRNA levels, C- Effects of the inhibitors on the MLCK mRNA content after 2 h incubation with DCA. Results are shown as mean ± SEM of 4–6 independent experiments. Values having different superscripts are significantly different (p < 0.05, One-way ANOVA).

Fig. 6

DCA decreases the expression of tight junction proteins: effects of EC, NADPH oxidase and ERK1/2 inhibitors. Caco-2 cell monolayers were incubated for 0–4 h with or without 100 μM DCA and in the absence or the presence of 5 μM EC, 1 μM apocynin, 1 μM VAS-2870 or 10 μM U0126. The expression of TJ proteins (ZO-1, occludin, claudin-1 and claudin-2) was measured by Western blot. Bands were quantified and values referred to β-actin levels. A-Kinetics of DCA-mediated effects on ZO-1 (full circles), occludin (black triangles), claudin-1 (empty circles) and claudin-2 (red triangles) protein profiles. B-D- Effects of EC, apocynin, VAS-2870 and U0126 on occludin (B) and ZO-1 (C) protein levels and (D) MMP-2 activity measured by zymography after 2 h incubation with 100 μM DCA. Results are shown as mean ± SE of 4–6 independent experiments. Values having different superscripts are significantly different (p < 0.05, One-way ANOVA test). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)