PARK7 Diminishes Oxidative Stress-Induced Mucosal Damage in Celiac Disease

Abstract

Coeliac disease (CD) is a chronic, immune-mediated small intestinal enteropathy, accompanied with gluten-triggered oxidative damage of duodenal mucosa. Previously, our research group reported an increased mucosal level of the antioxidant protein Parkinson's disease 7 (PARK7) in children with CD. In the present study, we investigated the role of increased PARK7 level on the epithelial cell and mucosal integrity of the small intestine. The presence of PARK7 was investigated using immunofluorescent staining on duodenal mucosa of children with CD and on FHs74Int duodenal epithelial cells. To investigate the role of oxidative stress, FHs74Int cells were treated with H₂O₂ in the absence or presence of Comp23, a PARK7-binding compound. Intracellular accumulation of reactive oxygen species (ROS) was determined by DCFDA-based assay. Cell viability was measured by MTT, LDH, and Annexin V apoptosis assays. Disruption of cytoskeleton and cell adhesion was investigated by immunofluorescence staining and by real-time RT PCR. Effect of PARK7 on mucosal permeability was investigated ex vivo using intestinal sacs derived from control and Comp-23-pretreated mice. Comp23 treatment reduced the H₂O₂-induced intracellular accumulation of ROS, thus preserving the integrity of the cytoskeleton and also the viability of the FHs74Int cells. Accordingly, Comp23 treatment increased the expression of antioxidants (NRF2, TRX1, GCLC, HMOX1, NQO1), cell-cycle regulators (TP53, CDKN1A, PCNA, BCL2, BAX), and cell adhesion molecules (ZO1, CDH1, VCL, ITGB5) of H₂O₂-treated cells. Pretreatment with Comp23 considerably decreased the small intestinal permeability. In this study, we demonstrate that PARK7-binding Comp23 reduces the oxidative damage of duodenal epithelial cells, via increased expression of NRF2- and P53-regulated genes. Our results suggest that PARK7 plays a significant role in the maintenance of mucosal integrity in CD.

Figure 1

The presence of PARK7 in duodenal epithelial cells. Localization of PARK (red) was investigated by immunofluorescence staining in duodenal mucosa of children with celiac disease (CD) and controls (a) and in FHs74Int cells (b). Cell nuclei were counterstained with DAPI (blue). Scale bar: 200, 100, or 50 μm.

Figure 2

Effect of Comp23 on ROS accumulation inFHs71Int cells. FHs74Int duodenal epithelial cells were treated with different concentrations of H₂O₂ in the absence or presence of Comp23 (0.001 μM). Scatter plot (a) serves as a representative figure of the time-related fluorescence intensity after treatment with 1000 μM H₂O₂ with or without Comp23. Violin plot (b) indicates the slope distribution of time-related DCFDA fluorescence intensity change in the given group at different concentrations of H₂O₂. Relative mRNA expressions (c) at 1000 μM H₂O₂ concentration were determined by comparison with 18S ribosomal RNA as an internal control. Results are presented as mean + SD (n = 5). $p < 0.05 vs. Ø compound at the concerning H₂O₂ concentration (multiple t-test); ∗p < 0.05 vs. control+Ø compound (Mann-Whitney U test); #p < 0.05 vs. H₂O₂+Ø compound (Mann–Whitney U test).

Figure 3

Effect of Comp23 on the H₂O₂-induced cell death of FHs71Int cells. FHs74Int duodenal epithelial cells were treated with different concentrations of Comp23 in the absence or presence of H₂O₂ (1000 μM); then, oxidative stress-induced cytotoxicity was determined by MTT (a), LDH (b), and Annexin V apoptosis ((c, d); 0.001 μM Comp23) assays. Relative mRNA expressions (e) at 0.001 μM Comp23 concentration were determined by comparison with 18S ribosomal RNA as an internal control. Results are presented as mean + SD ((a, b, e): n = 5; (c): n = 9). ^∗p < 0.05 vs. control+Ø compound ((a, b, c): multiple t-test; (e): Mann-Whitney U test); #p < 0.05 vs. H₂O₂+Ø compound ((a, b, c): two-way ANOVA; (e): Mann-Whitney U test).

Figure 4

Cytoskeletal disruption of intestinal mucosa in celiac disease (CD) and of duodenal epithelial cells. Actin filament structure was investigated by phalloidin (orange) staining in duodenal mucosa of children with CD and controls (a) and in FHs74Int duodenal epithelial cells (b) after treatment with H₂O₂ (1000 μM) in the absence or presence of Comp23 (0.001 μM). Cell nuclei were counterstained with DAPI (blue). White arrows indicate burst cells. Scale bar: 500 μm, 100 μm, and 50 μm. The percent of intact and damaged cells (c) was determined by graphical analysis. Results are presented as the percentage of total cells in a field of view (n = 400‐500/treatment group). ^∗p < 0.05 vs. control+Ø compound; #p < 0.05 vs. H₂O₂+Ø compound (chi-square test).

Figure 5

Damage of cell adhesion in epithelial cells induced by oxidative stress. Colocalization of actin (red) and zonula occludens 1 (ZO-1, green) was investigated by immunofluorescence staining (a) in FHs74Int duodenal epithelial cells after treatment with H₂O₂ (1000 μM) in the absence or presence of Comp23 (0.001 μM). Cell nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Relative mRNA expressions (b) in FHs74Int cells were determined by comparison with 18S ribosomal RNA as an internal control. Results are presented as mean + SD (n = 5). ^∗p < 0.05 vs. control+Ø compound (Mann-Whitney U test); #p < 0.05 vs. H₂O₂+Ø compound (Mann-Whitney U test).

Figure 6

Mucosal permeability of small intestinal sacs of untreated and Comp23-treated mice. Permeability of small intestinal sacs derived from control and Comp23 pretreated mice, filled with 0.1% Evans blue diluted in DMEM were investigated in the absence or presence of H₂O₂ (1000 μM). The permeation of Evans blue was measured in every 20 minutes at 590 nm. The results are presented as mean ± SEM (n = 7‐9). ^∗p < 0.05 0 μM H₂O₂ vs. 1000 μM H₂O₂; #p < 0.05 0 μM H₂O₂ vs. 0 μM H₂O₂+Comp23; $p < 0.05 1000 μM H₂O₂ vs. 1000 μM H₂O₂+Comp23 at the concerning time (two-way ANOVA).

Figure 7

Schematic model of the proposed mechanism underlying the protective effect of PARK7 against oxidative damage of duodenal epithelial cells in celiac disease. Our results showed that treatment with Comp23, a PARK7-binding compound under oxidative stress conditions, results in enhanced transcriptional activity, inducing expression of stress-response elements, such as antioxidant or cell-cycle regulators. This effect of PARK7 leads to reduced cellular damage and improved cell adhesion, contributing to the maintenance of integrity in the inflamed mucosa. Comp23: PARK7-binding compound; ROS: reactive oxygen species; CAM: cell adhesion molecule.