Volume 136, Issue 2, Pages 619-629 (February 2009) Inhibition of p38 Mitogen-Activated Protein Kinase Pathway as Prophylaxis of Postoperative Ileus in Mice  Sven Wehner, Stefan Straesser, Tim O. Vilz, Dimitrios Pantelis, Thais Sielecki, Vidal F. de la Cruz, Andreas Hirner, Joerg C. Kalff  Gastroenterology  Volume 136, Issue 2, Pages 619-629 (February 2009) DOI: 10.1053/j.gastro.2008.10.017 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Activation of p38-MAPK and JNK/SAPK after IM. (A) Phosphorylation (p) of p38-MAPK was detected in C57BL6/J mice ME lysates by immunoblotting, with maximum at 15 minutes after IM, decreasing within 45 minutes. JNK/SAPK phosphorylation was also observed after 15 minutes. However, phosphorylation remained unaffected for at least 60 minutes. (B) Preoperative semapimod treatment resulted in a diminished p38-MAPK phosphorylation, whereas pJNK/SAPK levels were not affected. Unphosphorylated proteins were blotted as loading controls. Control (CTL) ME specimens were from untreated animals. Gastroenterology 2009 136, 619-629DOI: (10.1053/j.gastro.2008.10.017) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Phosphorylation of p38-MAPK in op−/− and op+/− mice after IM. Levels of p38-MAPK phosphorylation (pp38) were determined from ME lysates by enzyme-linked immunosorbent assay 20 minutes postoperatively. pp38 levels were significantly increased (P < .001) in all groups after IM compared with unoperated op−/− controls (CTL). After IM, pp38 levels were significantly decreased in op−/− mice compared with the op+/− group (**P < .01), but did not differ from the semapimod (+S)-treated op−/− IM group (#) (*P < .05). CTL were unoperated op−/− mice. Values are expressed as mean ± SEM. Gastroenterology 2009 136, 619-629DOI: (10.1053/j.gastro.2008.10.017) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 mRNA expression of MIP-1α (A), IL-6 (B), MCP-1 (C), and ICAM-1 (D) in ME after IM. Three groups were investigated: placebo-treated, sham-operated mice, placebo-treated IM mice, and semapimod-treated IM animals. All genes were significantly up-regulated as early as 1 hour after IM and peaked 6 hours after IM, except ICAM-1, which peaked at 3 hours. Semapimod treatment resulted in a significant reduction of MIP-1α (3 and 6 hours), IL-6 (6 hours), MCP-1 (3, 6, and 24 hours), and ICAM-1 (1 and 3 hours) compared with the placebo IM group. Values are expressed as mean ± SEM. Asterisks indicate statistically significant differences (*P < .05, **P < .01, ***P < .001) between indicated samples (n = 4–5). Values are expressed as mean ± SEM. Gastroenterology 2009 136, 619-629DOI: (10.1053/j.gastro.2008.10.017) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Histogram showing neutrophil infiltration into ME. Myeloperoxidase (MPO) staining was performed 24 hours after IM or laparotomy (Sham) of placebo- or semapimod-treated (5 mg/kg) mice. Both, i.v. and i.p. applications of semapimod resulted in a significantly diminished neutrophil infiltration compared with placebo groups. Indicated probes differ significantly (**P < .01, ***P < .001) from each other. Semapimod IM i.v. versus IM i.p. group did not differ significantly (#). Values are expressed as mean ± SEM; n = 5–7. Gastroenterology 2009 136, 619-629DOI: (10.1053/j.gastro.2008.10.017) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 NO production in culture supernatants of ME. Muscle specimens from untreated and IM mice were taken 24 hours postoperatively and cultured for 24 hours. After IM, NO significantly increased (**P < .01) in the placebo group. NO was significantly decreased in the semapimod IM group compared with the placebo IM group (**P < .01). The semapimod IM group did not differ significantly from controls (CTL; #). Data are expressed as mean ± SEM; n = 4–5. Gastroenterology 2009 136, 619-629DOI: (10.1053/j.gastro.2008.10.017) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Measurement of jejunal circular smooth muscle contractility. Spontaneous and bethanechol-induced muscle contractility from controls and placebo or semapimod i.v.–treated IM mice. (A) Tracings of muscle contractility at 100 μmol/L bethanechol stimulation within the 3 different groups. In the placebo group, impaired muscle function was observed. Semapimod prevented the contractile impairment observed in vitro. (B) Bethanechol-induced smooth muscle contractility was significantly reduced (##P < .01, ###P < .001) in the placebo IM group at concentrations of 1–300 μmol/L compared with controls. The semapimod IM group did not differ significantly from unoperated controls, but did differ from the placebo IM group (*P < .05, ***P < .001). Data are expressed as the mean ± SD; n = 4 for controls and n = 8–9 for the IM groups. Gastroenterology 2009 136, 619-629DOI: (10.1053/j.gastro.2008.10.017) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Effect of semapimod on GIT (A–D) and colonic transit (E). GIT was measured as the percent of nonabsorbable fluorescein-labeled dextran in 15 gastrointestinal segments—stomach (Sto), small intestine (SI 1–9), cecum (Cec), and colon (Co)—90 minutes after oral ingestion. Placebo or semapimod was administered i.v (A and B) or i.p. (C and D). A and C show the intestinal distribution of fluorescein isothiocyanate–dextran after preoperative i.v. or i.p. drug administration, respectively. In semapimod-treated animals, most of the marker is located in distal jejunum compared with proximal jejunal location in placebo-treated animals. Calculation of the GC (B and D) demonstrated a significant delay of GIT in placebo-treated animals (***P < .001) after IM (P-IM) compared with semapimod-treated group, independent of their application routes. In the i.v. but not in the i.p. route, GIT from semapimod IM animals (S-IM) significantly differed (**P < .01) from semapimod sham groups (S-Sham). However, at both routes, it did not differ from placebo sham groups (P-Sham). (E) Colonic transit time showed a significant delay (***P < .001) in placebo-treated animals (P-IM) compared with unmanipulated controls (P-CTL). This delay was significantly improved (***P < .001) in the semapimod IM group (S-IM) and did not differ (#) from the respective control (S-CTL). GC for the 15 intestinal segments are displayed as mean (Sham, n = 5–6; IM, n = 9–10). Colonic transit times were displayed mean with all individual values (n = 5–6). Gastroenterology 2009 136, 619-629DOI: (10.1053/j.gastro.2008.10.017) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 Effect of semapimod on intestinal wound healing. Semapimod or placebo was administered i.v. 90 minutes before large bowel transection, followed by an anastomosis. (A) Hydroxyproline content was significantly increased at POD 5 (**P < .01) and 10 (***P < .001) in the semapimod group, and at POD 10 in the placebo group, compared with POD 2, respectively. Both groups did not differ from each other at any POD. (B) Anastomotic bursting strength increased in both groups significantly (***P < .001) at POD 5 and 10 compared with POD 2. However, both groups did not differ from each other at any POD. n = 8–11, data are expressed as single values with mean (bar). Gastroenterology 2009 136, 619-629DOI: (10.1053/j.gastro.2008.10.017) Copyright © 2009 AGA Institute Terms and Conditions