Occludin deficiency promotes ethanol-induced disruption of colonic epithelial junctions, gut barrier dysfunction and liver damage in mice

Abstract

Background: Disruption of epithelial tight junctions (TJ), gut barrier dysfunction and endotoxemia play crucial role in the pathogenesis of alcoholic tissue injury. Occludin, a transmembrane protein of TJ, is depleted in colon by alcohol. However, it is unknown whether occludin depletion influences alcoholic gut and liver injury.

Methods: Wild type (WT) and occludin deficient (Ocln(-/-)) mice were fed 1-6% ethanol in Lieber-DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin. Junctional integrity was analyzed by confocal microscopy. Liver injury was assessed by plasma transaminase, histopathology and triglyceride analyses. The effect of occludin depletion on acetaldehyde-induced TJ disruption was confirmed in Caco-2 cell monolayers.

Results: Ethanol feeding significantly reduced body weight gain in Ocln(-/-) mice. Ethanol increased inulin permeability in colon of both WT and Ocln(-/-) mice, but the effect was 4-fold higher in Ocln(-/-) mice. The gross morphology of colonic mucosa was unaltered, but ethanol disrupted the actin cytoskeleton, induced redistribution of occludin, ZO-1, E-cadherin and β-catenin from the junctions and elevated TLR4, which was more severe in Ocln(-/-) mice. Occludin knockdown significantly enhanced acetaldehyde-induced TJ disruption and barrier dysfunction in Caco-2 cell monolayers. Ethanol significantly increased liver weight and plasma transaminase activity in Ocln(-/-) mice, but not in WT mice. Histological analysis indicated more severe lesions and fat deposition in the liver of ethanol-fed Ocln(-/-) mice. Ethanol-induced elevation of liver triglyceride was also higher in Ocln(-/-) mice.

Conclusion: This study indicates that occludin deficiency increases susceptibility to ethanol-induced colonic mucosal barrier dysfunction and liver damage in mice.

Keywords: Actin; Adherens junction; Alcohol; Claudin; Fatty liver; Tight junction.

Figure 1. Effect of chronic ethanol feeding on diet intake and body weight of occludin deficient mice

Adult wild type and occludin knockout (Ocln^−/−) mice were fed ethanol in Lieber DeCarli liquid diet (1–6%) (●) over 4-week period. Control mice were pair fed with isocaloric ethanol-free diet (○). Diet intake (A & B) and body weights (C & D) were recorded. Values are mean ± SEM (n = 11–15 as indicated in Methods section). Asterisks indicate the values for ethanol fed groups are significantly (p<0.05) different from the corresponding pair fed group values.

Figure 2. Chronic ethanol feeding induces colonic mucosal barrier dysfunction more severely in occludin deficient mice

Adult wild type (WT) and occludin knockout (Ocln^−/−) mice were fed ethanol (1–6%) (EF) in Lieber DeCarli liquid diet for 4 weeks. Control mice were pair fed with isocaloric ethanol-free diet (PF). Mucosal permeability of colon and ileum was evaluated by measuring vascular-to-luminal flux of FITC-inulin in vivo. Values for distal colon (A), proximal colon (B) and ileum (C) are mean ± SEM (n as indicated in the figure). Colonic sections were stained for TLR4 by immunofluorescence method (D). Fluorescence density was evaluated (E); values are mean ± SEM (n = 4). Asterisks in all graphs indicate the values that are significantly (p<0.05) different from the corresponding values for PF group. Hash tags indicate the values that are significantly different from corresponding values for WT group.

Figure 3. Chronic ethanol feeding disrupts colonic epithelial TJ more severely in occludin deficient mice

Adult wild type and occludin knockout (Ocln^−/−) mice were fed ethanol (1–6%) (EF) in Lieber DeCarli liquid diet for 4 weeks. Control mice were pair fed with isocaloric ethanol-free diet (PF). Absence of occludin in Ocln^−/− mice was confirmed by immunoblot analysis (A) and immunofluorescence staining (B). Gross mucosal morphology was examined by light microscopy of H & E stained colonic sections (C). Cryosections of distal colon was stained for occludin and ZO-1 by immunofluorescence method and confocal microscopy (D). The merged images represent overlay of occludin (green), ZO-1 (red) and nucleus (blue). ZO-1 fluorescence in the epithelial cells was evaluated by densitometry using Image J (E). Densitometric values are mean ± SEM (n = 4). Asterisks indicate the values for ethanol fed groups are significantly different from the corresponding values for pair fed group, and the pound symbol indicates the value that is significantly (p<0.05) different from corresponding value for wild type mice. Colonic mucosal extracts from pair fed and ethanol fed wild type mice were immunoblotted for occludin and β-actin (F). Occludin band density was measured and normalized to the density of corresponding band for β-actin (G). Values are mean ± SEM (n = 5). Asterisks indicate the value for ethanol fed group is significantly (p<0.05) different from the corresponding value for pair fed group.

Figure 4. Effect of ethanol feeding on Cldn-2 and Cldn-3 in colonic epithelium

Adult wild type and Ocln^−/− mice were fed ethanol (1–6%) (EF) in Lieber DeCarli liquid diet for 4 weeks. Control mice were pair fed with isocaloric ethanol-free diet (PF). Cryosections of distal colon were stained for Cldn-2 and Cldn-3 by immunofluorescence method and confocal images were captured (A). The merged images represent overlay of Cldn-2 (green), Cldn-3 (red) and nucleus (blue). Fluorescence for Cldn-2 and Cldn-3 was evaluated by densitometry using Image J (B). Densitometric values are mean ± SEM (n = 5). Asterisk indicates the value for ethanol fed group is significantly different from the corresponding value for pair fed group, and the pound symbol indicates the value that is significantly (p<0.05) different from corresponding value for wild type mice.

Figure 5. Ethanol-induced disruption of colonic epithelial adherens junction is more severe in occludin deficient mice

Adult wild type and Ocln^−/− mice were fed ethanol (1–6%) (EF) in Lieber DeCarli liquid diet for 4 weeks. Control mice were pair fed with isocaloric ethanol-free diet (PF). Cryosections of distal colon were stained for E-cadherin and β-catenin by immunofluorescence method and confocal images were captured (A). The merged images represent overlay of E-cadherin (green), β-catenin (red) and nucleus (blue). Fluorescence for β-catenin was evaluated by densitometry using Image J (B). Densitometric values are mean ± SEM (n = 5). Asterisk indicates the value for ethanol fed group that is significantly (p<0.05) different from the corresponding value for pair fed group.

Figure 6. Effect of ethanol feeding on colonic epithelial actin cytoskeleton

Adult wild type and Ocln^−/− mice were fed ethanol (1–6%) (EF) in Lieber DeCarli liquid diet for 4 weeks. Control mice were pair fed with isocaloric ethanol-free diet (PF). Cryosections of distal colon were stained for F-actin by immunofluorescence method and confocal images were captured (A). The merged images represent overlay of F-actin (green) and nucleus (blue). Fluorescence for F-actin was evaluated by densitometry using Image J (B). Densitometric values are mean ± SEM (n = 5). Asterisks indicate the values for ethanol fed group that are significantly (p<0.05) different from the corresponding values for pair fed group.

Figure 7. Effect of occludin knockdown on ethanol-induced TJ disruption in Caco-2 cells

Caco-2 cells were transfected with occludin-specific shRNA or control RNA. Control RNA and shRNA-transfected cells on transwells were treated with 200 μM acetaldehyde for 4 hours. Cell extracts were immunoblotted for occludin and β-actin (A). TER (B) and inulin permeability (C) were measured, and fixed cell monolayers were stained for occludin and ZO-1 (D). Values are mean ± SEM (n = 6). Asterisks indicate the values that are significantly (p<0.05) different from corresponding value for control RNA-transfected cell monolayers. Hash tags indicate the values that are significantly (p<0.05) different from corresponding values for sham-treated control cell monolayers.

Figure 8. Effect of chronic ethanol feeding on liver

Adult wild type and Ocln^−/− mice were fed ethanol (1–6%) (EF) in Lieber DeCarli liquid diet for 4 weeks. Control mice were pair fed with isocaloric ethanol-free diet (PF). Liver weights were recorded (A) and plasma ALT activity was measured (B). Values are mean ± SEM (n as indicated in the figure). Asterisks indicate the values for ethanol fed groups that are significantly (p<0.05) different from the corresponding values for pair fed group. Paraffin sections of liver were stained with H & E (C).

Figure 9. Ethanol-induced fat deposition in liver is more sever in occludin deficient mice

Adult wild type and Ocln^−/− mice were fed ethanol (1–6%) (EF) in Lieber DeCarli liquid diet for 4 weeks. Control mice were pair fed with isocaloric ethanol-free diet (PF). Cryo-sections of liver were stained with Oil Red-O (A), and triglyceride contents in liver extracts were measured by spectrophotometric assay (B). Values are mean ± SEM (n as indicated in the figure). Paraffin sections of liver were stained with Picro-Serius Red (C).