The Role of Claudins in the Pathogenesis of Dextran Sulfate Sodium-Induced Experimental Colitis: The Effects of Nobiletin

Abstract

Background: The pathogenesis of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease is not well understood. This study investigated the roles and regulation of the claudin-1, -2, -3, and -4 isoforms in the pathogenesis of ulcerative colitis, and the potential therapeutic effects of nobiletin.

Methods: Colitis was induced in rats by administering dextran sulfate sodium [DSS] in drinking water for seven days. Animals were treated daily with nobiletin [oral, 60 mg/Kg body weight] and studied in four groups, C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. On day seven, the animals were sacrificed, and colonic tissues were collected and analyzed.

Results: Both macroscopic and microscopic findings suggest the progression of colitis. In the inflamed colon, claudin-1 and -4 proteins were decreased, claudin-2 increased, while the claudin-3 protein remained unchanged. Except for claudin-1, these changes were not paralleled by mRNA expression, indicating a complex regulatory mechanism. Uniform β-actin expression along with consistent quality and yield of total RNA indicated selectivity of these changes. Nobiletin treatment reversed these changes.

Conclusions: Altered expression of the claudin isoforms -1, -2, and -4 disrupts tight junctions, exposing the lamina propria to microflora, leading to electrolyte disturbance and the development of ulcerative colitis. Nobiletin with its anti-inflammatory properties may be useful in IBD.

Keywords: claudins; colitis; dextran sulfate sodium; inflammation; myeloperoxidase; tight junctions.

Figure 1

(A) Body weight (Wt) change [percentage of day 0 body weight] and (B) the food efficiency ratios of the animals from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The data are the mean ± SE [n = 10]. *, ^# indicate significance at p < 0.05 between the indicated pair of groups.

Figure 2

(A) Colon weights [g/cm colon length], (B) the disease activity score, (C) a representative histology of hematoxylin and eosin-stained [left panel] and alcian blue-stained [right panel] colonic tissue sections, and (D) the histological score of the animals from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The data are the mean ± SE [n = 10]. *, ^# indicate significance at p < 0.05 between the indicated pair of groups. Magnification: 10×. Scalebar = 100 μm.

Figure 3

A bar diagram showing MPO activity units/min-mg in the colonic segments from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The data are the mean ± SE [n = 10]. *, ^# indicate significance at p < 0.05 between the indicated groups.

Figure 4

(A) Representative photomicrographs showing immunofluorescence staining, and (B–E) a bar diagram showing the expression levels of the indicated claudin isoforms measured as mean fluorescence intensity [MFI, arbitrary units] using a specific primary antibody and an FITC-labeled secondary antibody in the tissue sections of colon from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The tissue sections were counter-stained with DAPI. Claudins are expressed in the surface lining epithelium [arrows] and in the epithelial cells [arrow heads] lining the glands [G]. L—lumen of the colon. Magnification = 20×, Scalebars = 100 μm. The data are the mean ± SE [n = 10]. *, ^# indicate significance at p < 0.05 between the indicated pair of groups.

Figure 5

(A) A bar diagram showing the protein expression levels relative to β-actin [ratios] of claudin-1, (B) claudin-2, (C) claudin-3, and (D) claudin-4 isoforms in the colon from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The data are the mean ± SE [n = 10]. *, ^# indicate significance at p < 0.05 between the indicated pair of groups. p < 0.05 with respect to C. The insets show the representative ECL Western blot analysis of the indicated claudin isoform and β-actin.

Figure 6

A bar diagram showing the mRNA expression levels as ratios relative to β-actin mRNA of the indicated claudin mRNA isoforms -1, -2, and -4 using (A) the endpoint RT-PCR method and (B) SYBR green RT-PCR-based C_T calculations for claudin isoforms -1 and -2 in the LiCl-purified colonic RNA samples from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The data are the mean ± SE [n = 10]. *, ^# indicate significance at p < 0.05 between the indicated pair of groups.

Figure 7

A proposed mechanism of the pathogenesis of DSS-induced ulcerative colitis and its reversal by nobiletin treatment in colitis rats. The figure was created using BioRender with permission.