Ethanol impairs intestinal barrier function in humans through mitogen activated protein kinase signaling: a combined in vivo and in vitro approach

Abstract

Background: Ethanol-induced gut barrier disruption is associated with several gastrointestinal and liver disorders.

Aim: Since human data on effects of moderate ethanol consumption on intestinal barrier integrity and involved mechanisms are limited, the objectives of this study were to investigate effects of a single moderate ethanol dose on small and large intestinal permeability and to explore the role of mitogen activated protein kinase (MAPK) pathway as a primary signaling mechanism.

Methods: Intestinal permeability was assessed in 12 healthy volunteers after intraduodenal administration of either placebo or 20 g ethanol in a randomised cross-over trial. Localization of the tight junction (TJ) and gene expression, phosphorylation of the MAPK isoforms p38, ERK and JNK as indicative of activation were analyzed in duodenal biopsies. The role of MAPK was further examined in vitro using Caco-2 monolayers.

Results: Ethanol increased small and large intestinal permeability, paralleled by redistribution of ZO-1 and occludin, down-regulation of ZO-1 and up-regulation of myosin light chain kinase (MLCK) mRNA expression, and increased MAPK isoforms phosphorylation. In Caco-2 monolayers, ethanol increased permeability, induced redistribution of the junctional proteins and F-actin, and MAPK and MLCK activation, as indicated by phosphorylation of MAPK isoforms and myosin light chain (MLC), respectively, which could be reversed by pretreatment with either MAPK inhibitors or the anti-oxidant L-cysteine.

Conclusions: Administration of moderate ethanol dosage can increase both small and colon permeability. Furthermore, the data indicate a pivotal role for MAPK and its crosstalk with MLCK in ethanol-induced intestinal barrier disruption.

Trial registration: ClinicalTrials.gov NCT00928733.

Conflict of interest statement

Competing Interests: The authors hereby declare that the funder Top Institute Food and Nutrition had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors hereby declare that the affiliation to Top Institute Food and Nutrition had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Figures

Figure 1. Effect of placebo or single dose of 20 g of ethanol administration on blood ethanol and FAEE levels.

[A] Ethanol was analyzed in plasma of the individual volunteers after ethanol administration at the following time points: 0, 15, 30, 45, 60, 90, 120, and 180 min, and presented as mean ± SD. [B] Ethyl oleate and Ethyl palmitate were determined in plasma at 0, 30, and 180 min, and presented as mean ± SD (n = 12).

Figure 2. Effects of placebo and single dose of 20 g of ethanol on intestinal permeability, TJs protein and gene expression in the duodenum.

[A] Effects on gastroduodenal permeability (sucrose, 0–5 h urinary recovery). Data presented as boxplots displaying minimum, maximum, and the 25th, 50th and 75th percentiles (n = 12); P>0.05 vs. placebo. [B] Effects on small intestinal permeability (lactulose/rhamnose; L/R ratio, 0–5 h urine). Data presented as boxplots displaying minimum, maximum, and the 25th, 50th and 75th percentiles (n = 12); *P<0.01 vs. placebo. [C] Effects on large intestinal permeability (sucralose/erythritol; S/E ratio, 5–24 h urine). Data presented as boxplots displaying minimum, maximum, and the 25th, 50th and 75th percentiles (n = 11); *P<0.01 vs. placebo. [D] Representative images of immune-localization of ZO-1 (green), occludin (red), and nuclei (blue) at a 400x magnification are shown. Scale bar represents 10 µm. [E] Normalized intensity of immunofluorescent-labelled ZO-1 and occludin in duodenal mucosa. Z-stack images were at 40× objective power. Data are mean ± SD (n = 12). *P<0.05 for ZO-1, #P<0.05 for occludin. [F] Normalized mRNAs expression of occludin (occl), claudin 4 (clau4), and claudin 3 (clau3), MLCK and ZO-1 in duodenal biopsy specimens determined by qPCR. Each bar represents the mean± SD; *P<0.05 comparing ethanol with placebo.

Figure 3. Effect of placebo and single dose of 20 g of ethanol on phosphorylation of MAPK isoforms in intestinal epithelium.

Mucosal protein was isolated from tissue lysates, and the levels of phosphorylated MAPK isoforms were determined by ELISA using anti-phosphospecific antibodies against p-P38 [A], p-ERK1/2 [B], and [C] and p-JNK. Lysates of Hela cells treated with Anisomycin were used as positive control (+ve control) for phosphorylated P38 and JNK, whereas A431 cells treated with recombinant human epidermal growth factor were used for phosphorylated ERK1/2. (n = 12 separate sample preparations; *P<0.05 vs. placebo).

Figure 4. Effect of ethanol on phosphorylation of MAPK isoforms in Caco-2 cells.

[A] Phosphorylation of P38, [B] ERK1/2, and [C] JNK was determined by cell-based ELISA and expressed as the ratio of the phosphorylated to the total form of each protein. n = 3 and *P<0.0001 vs. control and #P<0.0001 vs. ethanol in the presence of SB2035809, PD98055, or SP600125, respectively. Effect of L-cysteine on ethanol-induced phosphorylation of P38 [D], ERK1/2 [E], and JNK [F], determined by cell-based ELISA, and expressed as the ratio of the phosphorylated to the total amount of each protein. H2O2 used as positive control. n = 3 and *P<0.0001 vs. control and #P<0.0001 vs. ethanol after pretreatment with L-cysteine.

Figure 5. Effects of MAPK inhibition on ethanol-induced barrier dysfunction in Caco-2 monolayers.

Confluent human intestinal epithelial cells (Caco-2) grown on inserts were assessed for trans-epithelial electrical resistance (TEER) and permeability to FITC-D4 after 3 h after treatment with P38 inhibitor [A] and [B], ERK1/2 inhibitor [C] and [D] and JNK inhibitor [E] and [F], respectively. Permeability is expressed as percentage of basal TEER and FITC-D4 permeation from apical to basal chamber. Some cells were treated only with medium or ethylene glycol tetra acetic acid (EGTA) as negative (-ve CTR) and positive control (+ve CTR), respectively. In addition, some monolayers were pretreated for 1 h with 100 µM of the P38-specific kinase inhibitor SB2035809, ERK1/2 PD98055 and JNK SP600125. Data are means ± SD of triplicate wells from three separate experiments. *P<0.0001 vs. control, #P<0.001, &P<0.0001 and $P<0.05 vs. ethanol after pretreatment with each MAPK inhibitor.

Figure 6. Effects of inhibition of MAPK isoforms on ethanol-induced redistribution of TJ and AJ proteins; activation of MLCK and reorganization of F-actin cytoskeleton in Caco-2 monolayers.

Monolayers subjected to aforementioned treatments were washed, fixed, permeabilized, and double-stained for ZO-1, occludin and E-cadherin, and ß-catenin. [A] Representative images of immunostaining of ZO-1 (green) and occludin (red) of three independent experiments. Scale bar  = 10 µm. [B] Representative images of immunostaining of E-cadherin (green) and β-catenin (red). Scale bar  = 10 µm. [C] Effects of MAPK inhibition on ethanol (EtOH)-induced myosin light chain (MLC) phosphorylation. Caco-2 cell monolayers were incubated with 40 mM ethanol (EtOH) for 3 h with or without MAPK inhibitors (as in Figure 6) or MLCK inhibitor (ML-7) pretreatment for 1 h, and analyzed by cell-based ELISA, using phosphospecific MLC antibodies (n = 3). Each bar represents the mean ± SD; *P<0.0001 vs. control and #P<0.0001 vs. ethanol. [D] Effects of MAPK inhibition on ethanol (EtOH)-induced F-actin reorganization. Caco-2 cell monolayers incubated with 40 mM ethanol for 3 h with or without MAPK inhibitors pretreatment for 1 h were fixed and stained for F-actin with Texas red-X phalloidin Scale bar  = 10 µm.