The JNK Inhibitor XG-102 Protects against TNBS-Induced Colitis

The c-Jun N-terminal kinase (JNK)-inhibiting peptide D-JNKI-1, syn. XG-102 was tested for its therapeutic potential in acute inflammatory bowel disease (IBD) in mice. Rectal instillation of the chemical irritant trinitrobenzene sulfonic acid (TNBS) provoked a dramatic acute inflammation in the colon of 7–9 weeks old mice. Coincident subcutaneous application of 100 µg/kg XG-102 significantly reduced the loss of body weight, rectal bleeding and diarrhoea. After 72 h, the end of the study, the colon was removed and immuno-histochemically analysed. XG-102 significantly reduced (i) pathological changes such as ulceration or crypt deformation, (ii) immune cell pathology such as infiltration and presence of CD3- and CD68-positive cells, (iii) the production of tumor necrosis factor (TNF)-α in colon tissue cultures from TNBS-treated mice, (iv) expression of Bim, Bax, FasL, p53, and activation of caspase 3, (v) complexation of JNK2 and Bim, and (vi) expression and activation of the JNK substrate and transcription factor c-Jun. A single application of subcutaneous XG-102 was at least as effective or even better depending on the outcome parameter as the daily oral application of sulfasalazine used for treatment of IBD

c-Jun and phospho-c-Jun.

<p>Western blot analysis of c-Jun and phospho-c-Jun in colon homogenates from untreated controls (co) and 12 h or 24 h following trinitrobenzene sulfonic acid (TNBS) administration without or with XG-102 (100 µg/kg sc.). These blots are representative of 3 independent experiments.</p

Tissue damage scores.

<p>Tissue damage scores.</p

JNK2-Bim co-precipitation.

<p>JNK1 and JNK2 immunoprecipitates (IP) from colon tissue homogenates were analyzed by Western blotting with an anti-Bim antibody. Pounceau staining demonstrated equal loading (data not shown).</p

Representative CD3 and CD68 immunofluorescence.

<p>Representative CD3 (left) and CD68 (right) immunofluorescence of the distal colon from normal mice, trinitrobenzene sulfonic acid (TNBS) administration and treatment with sc. 100 µg/kg XG-102.</p

Hematoxylin and eosin (H&E) scores.

<p>Hematoxylin and eosin (H&E) scores from distal (A) and medial (B) colon. For the tissue damage score (hatched bars), the scores of ulcer, crypts and submucosa were summed-up for each individual animal, and the mean±SEM was calculated for each group. The mean±SEM of the infiltration score (grey bar) is separately shown. ***, ** = p<0.001 and p<0.01 for all groups compared with TNBS group.</p

Apoptosis.

<p>Western blot analysis of (A) caspase-3 and cleaved caspase-3, (B) Bax and Bim, and (C) FasL and p53 from colon extracts of untreated controls (co) 12 h, 24 h and 72 h following trinitrobenzene sulfonic acid (TNBS) administration without or with XG-102 (100 µg/kg sc.). These blots are representative of 3 independent experiments.</p

Production of TNF-α.

<p>TNF-α release (pg/ml) into the supernatant of organic colon culture from normal mice, following trinitrobenzene sulfonic acid (TNBS) only, and treatment with sc. 100 µg/kg XG-102. ** = p<0.01 for all groups compared with TNBS group.</p