Peptide inhibitors of MK2 show promise for inhibition of abdominal adhesions

Abstract

Background: Abdominal adhesions are a common side effect of surgical procedures with complications including infertility, chronic pain, and bowel obstruction, which may lead to the need for surgical lyses of the adhesions. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) has been implicated in several diseases, involving inflammation and fibrosis. Thus, the development of a cell-penetrating peptide (CPP) that modulates MK2 activity may confer therapeutic benefit after abdominal surgery in general and more specifically after bowel anastomosis.

Methods: This study evaluated the function of a CPP inhibitor of MK2 in human mesothelial cells and in a rat bowel anastomosis model. To determine IC50 and basic specificity, kinase inhibition was performed using a radiometric assay. Enzyme-linked immunoassay (ELISA) was used to evaluate interleukin-6 (IL-6) expression in response to IL-1β and tumor necrosis factor-α (TNF-α) stimulation in vitro to validate MK2 kinase inhibition. Following bowel anastomosis (10 rats for each control and treatment at 4 and 10 d), the rats were evaluated for weight loss, normal healing (colonic burst strength and hydroxyproline content at the anastomosis), and number and density of adhesions.

Results: The IC50 of the MK2 inhibitor peptide (22 μM) was similar to that of the nonspecific small molecule rottlerin (IC50 = 5 μM). The MK2 inhibitor peptide was effective at suppressing IL-1β and TNF-α stimulated IL-6 expression in mesothelial cells. In vivo, the MK2 inhibitor peptide was effective at suppressing both the density and number of adhesions formed as a result of bowel an anastamosis. Importantly, the peptide had no negative effect on normal healing.

Conclusions: In conclusion, the peptide inhibitor of MK2, MMI-0100, has the potential to significantly reduce inflammation through suppression of inflammatory cytokine expression and showed promise as a therapeutic for abdominal adhesions.

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Figure 1. IL-6 expression versus time and IL-1β or TNF-α dose in mesothelial cells

(A) IL-1β and (B) TNF-α increased IL-6 expression in a time and dose-dependent manner. Single factor ANOVAs were conducted within each time interval with p

Figure 1. IL-6 expression versus time and IL-1β or TNF-α dose in mesothelial cells

(A) IL-1β and (B) TNF-α increased IL-6 expression in a time and dose-dependent manner. Single factor ANOVAs were conducted within each time interval with p

Figure 2. IL-6 expression versus treatment with 1ng/ml IL-1β or 1ng/ml IL-1β + MMI-0100 in mesothelial cells

MMI-0100, at a concentration of 3,000 µM, significantly reduced IL-1β induced IL-6 expression at all time points.. Single factor ANOVAs were conducted within each time interval with p

Figure 3. IL-6 expression versus treatment with 10 ng/mL TNF-α or 10 ng/mL TNF-α + MMI-0100 in mesothelial cells

Both 3,000 and 1,000 µM MMI-0100 significantly reduced TNF-α induced IL-6 expression in mesothelial cells at all three time points.. Single factor ANOVAs were conducted within each time interval with p

Figure 4. IL-1β induced phosphorylation of hnRNPA0 is reduced with MMI-0100 treatment

(A) Western blot and (B) densimetric analysis showing MMI-0100 reduces IL-1β induced phosphorylation of hnRNPA0 after 6 hours. IL-1β induced phosphorylation disappears at 12 hours: 1) untreated, 2) IL-1β, 3) MMI-0100 (1 mM), 4) MMI-0100 (3 mM)

Figure 4. IL-1β induced phosphorylation of hnRNPA0 is reduced with MMI-0100 treatment

(A) Western blot and (B) densimetric analysis showing MMI-0100 reduces IL-1β induced phosphorylation of hnRNPA0 after 6 hours. IL-1β induced phosphorylation disappears at 12 hours: 1) untreated, 2) IL-1β, 3) MMI-0100 (1 mM), 4) MMI-0100 (3 mM)

Figure 5. IL-1β induced phosphorylation of HSPB1 with MMI -0100 treatment and AKT inhibition

(A) MMI-0100 treatment significantly decreases IL-1β induced HSPB1 phosphorylation at serine 15. (B) Treatment with an AKT inhibitor significantly reduced phosphorylation of HSPB1 at serine 82, which was not observed with peptide treatment. Data was normalized to total HSPB1 and cell number. Single factor ANOVAs were conducted with p

Figure 5. IL-1β induced phosphorylation of HSPB1 with MMI -0100 treatment and AKT inhibition

(A) MMI-0100 treatment significantly decreases IL-1β induced HSPB1 phosphorylation at serine 15. (B) Treatment with an AKT inhibitor significantly reduced phosphorylation of HSPB1 at serine 82, which was not observed with peptide treatment. Data was normalized to total HSPB1 and cell number. Single factor ANOVAs were conducted with p