MC-12, an annexin A1-based peptide, is effective in the treatment of experimental colitis

Abstract

Annexin A1 (ANXA1) inhibits NF-κB, a key regulator of inflammation, the common pathophysiological mechanism of inflammatory bowel diseases (IBD). MC-12, an ANXA1-based tripeptide, suppresses NF-κB activation. Here, we determined the efficacy of MC-12 in the control of IBD. Mice with colitis induced by dextran sodium sulfate (DSS) or 2,4,6-trinitro benzene sulfonic acid (TNBS) were treated with various doses of MC-12 administered intraperitoneally, orally or intrarectally. We determined colon length and the histological score of colitis, and assayed: in colon tissue the levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10 by RT-PCR; prostaglandin E(2) (PGE(2)), cytoplasmic phospholipase A(2) (cPLA(2)) and myeloperoxidase by immunoassay; and COX-2 and NF- κB by immunohistochemistry; and in serum the levels of various cytokines by immunoassay. In both models MC-12: reversed dose-dependently colonic inflammation; inhibited by up to 47% myeloperoxidase activity; had a minimal effect on cytoplasmic phospholipase A(2); reduced significantly the induced levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10, returning them to baseline. DSS and TNBS markedly activated NF-κB in colonic epithelial cells and MC-12 decreased this effect by 85.8% and 72.5%, respectively. MC-12 had a similar effect in cultured NCM460 normal colon epithelial cells. Finally, MC-12 suppressed the induction of COX-2 expression, the level of PGE(2) in the colon and PGE(2) metabolite in serum. In conclusion, MC-12, representing a novel class of short peptide inhibitors of NF-κB, has a strong effect against colitis in two preclinical models recapitulating features of human IBD. Its mechanism of action is complex and includes pronounced inhibition of NF-κB. MC-12 merits further development as an agent for the control of IBD.

Figure 1. The effect of MC-12 on DSS- and TNBS-induced colitis in mice.

Mice (7/group) received 2% DSS in drinking water or 2.5% TNBS intra-colonically to induce colitis and were treated with vehicle or MC-12 given IP, PO or IR. A: The body weight of mice of DSS model during treatment by IP, PO or IR, expressed as percentage of baseline (day 0). B: The colon length of DSS model with MC-12 treatments by IP, PO or IR. C: The body weight of mice of TNBS model during treatment, expressed as percentage of baseline (day 0). D: The colonic length of mice of TNBS model in three treatment groups. These studies were repeated at least once giving similar results. Values are mean ± SEM. *, statistically significant difference from the vehicle-treated group.

Figure 2. MC-12 ameliorates colitis induced by both DSS and TNBS.

Paraffin sections of colonic tissues were stained with hematoxylin & eosin and their histological scores were determined as in Methods. A: Normal mucosa of the C57BL/6 mouse. B: Severe inflammation including infiltration by inflammatory cells, edema, loss of crypts and ulcerations are seen in a DSS + vehicle-treated mouse. C, D: MC-12, 5 or 25 mg/kg, IP, significantly decreased DSS-induced colonic inflammation. E: Treatment with MC-12, 25 mg/kg PO, decreased DSS-induced colonic inflammation. F: Treatment with MC-12, 25 mg/kg, IR, markedly reduced DSS-induced colitis. G: Colonic mucosa from a TNBS vehicle-treated mouse, showing severe crypt loss and inflammatory cell infiltration (arrow). Inset: normal mucosa of a healthy SJL/J mouse. H: Treatment with MC-12 25 mg/kg for 2 days decreased the inflammatory cell infiltration and crypt loss (arrow indicates remaining crypts). I, J: The histological score of the various study groups of both DSS- and TNBS-induced colitis. Values are mean ± SEM. H&E staining; magnification 100x.

Figure 3. The effect of MC-12 on parameters of inflammation.

MPO (A, B), cPLA₂ (C, D) activity and the mRNA levels of proinflammatory and anti-inflammatory cytokines (E, F) were measured in colon tissue samples of both DSS- and TNBS-induced colitis mouse models. In both models MC-12 significantly inhibited MPO activity and the mRNA levels of all cytokines but not cPLA₂ activity. Values are mean ± SEM. *p<0.05 compared to vehicle-treated group, **p<0.01 compared to vehicle-treated group.

Figure 4. MC-12 reduces the serum levels of cytokines.

The serum levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10 were measured by ELISA as in Methods. Both DSS and TNBS induced their levels and treatments with MC-12, 25 mg/kg administered PO, IP or IR reduced these levels 1.6- to 5.1-fold. *p<0.01, #p<0.05 compared to vehicle-treated controls.

Figure 5. MC-12 inhibits NF-κB activation induced by DSS and TNBS in vivo and in vitro.

Colon tissue sections were immunohistochemically stained with anti-phospho-NF-κB p65 antibody. Normal colon mucosa from C57BL/6 mice (A) or from SJL/J mice (F) shows a few cells with p-p65 nuclear positive staining. Colon mucosa with DSS (B) or TNBS (G) shows increased NF-κB nuclear-positive cells, most of which are crypt epithelial cells. Markedly less p-p65 nuclear translocation was shown in colon mucosa from DSS model treated with MC-12, 5 mg/kg ip (C), 25 mg/kg ip (D) or 25 mg/kg po (E) or from TNBS model treated with MC-12 25 mg/kg ip (H). Changes in p-p65 nuclear positive, evident in the photos are quantified (I, J). DSS increased NF-κB-DNA binding in NCM460 cells determined by EMSA, and MC-12 30 and 300 µM significantly blocked this effect (K). Values are mean ± SEM. **p<0.01 compared to vehicle-treated group. IHC staining; magnification 200x.

Figure 6. MC-12 inhibits COX-2 induction and decreases PGE₂ levels in DSS- and TNBS-induced colitis. A:

Representative photomicrographs of tissue sections with COX-2 immunohistochemical staining on colon tissue from normal (no treatment), vehicle- or MC-12-treated mice from the DSS (upper panels) or TNBS (lower panel) groups. MC-12 treatment: 25 mg/kg ip. B: The COX-2 expression scores in the various groups of animals (n = 8/group). Differences were evaluated by Pearson’s χ² method. C, D: MC-12 decreased the levels of PGE₂ in colonic mucosa and of PGE₂ metabolite in serum. Values are mean ± SEM. **p<0.01 compared to vehicle-treated group, *p<0.05 compared to vehicle-treated group.