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Inhibition of ERK1/2 Worsens Intestinal Ischemia/Reperfusion Injury

PLoS ONE (2013) 8(9)
DOI: 10.1371/journal.pone.0076790
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Abstract

Background:The role of extracellular signal-regulated protein kinase (ERK) in intestinal ischemia/reperfusion (I/R) injury has not been well investigated. The aim of the current study was to examine the effect of inhibition of the ERK pathway in an in vitro and in vivo model of intestinal I/R injury.Methods:ERK1/2 activity was inhibited using the specific inhibitor, U0126, in intestinal epithelial cells under hypoxia/reoxygenation conditions and in mice subjected to 1 hour of intestinal ischemia followed by 6 hours reperfusion. In vitro, cell proliferation was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, apoptosis by DNA fragmentation, and migration using an in vitro model of intestinal wound healing. Cells were also transfected with a p70S6K plasmid and the effects of overexpression similarly analyzed. In vivo, the effects of U0126 on intestinal cell proliferation and apoptosis, intestinal permeability, lung and intestinal neutrophil infiltration and injury, and plasma cytokine levels were measured. Survival was also assessed after U0126. Activity of p70S6 kinase (p70S6K) was measured by Western blot.Results:In vitro, inhibition of ERK1/2 by U0126 significantly decreased cell proliferation and migration but enhanced cell apoptosis. Overexpression of p70S6K promoted cell proliferation and decreased cell apoptosis. In vivo, U0126 significantly increased cell apoptosis and decreased cell proliferation in the intestine, increased intestinal permeability, intestinal and lung neutrophil infiltration, and injury, as well as systemic pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β. Mortality was also significantly increased by U0126. Inhibition of ERK1/2 by U0126 also abolished activity of p70S6K both in vitro and in vivo models.Conclusion:Pharmacologic inhibition of ERK1/2 by U0126 worsens intestinal IR injury. The detrimental effects are mediated, at least in part, by inhibition of p70S6K, the major effector of mammalian target of rapamycin pathway. © 2013 Ban et al.

Figures

  • Figure 1. Activities of ERK1/2 and p70S6K were inhibited by U0126 in vitro and in vivo. A. IEC-6 cells were cultured in medium without FBS for 12 hours under hypoxic conditions followed treatment with U0126 or vehicle for 1 hour and then stimulated with 10% FBS under normoxic conditions for 20 minutes (n=3). Protein expression in cells was determined by Western blot analysis. B. IEC-6 cells were transfected with/without empty plasmid or p70S6K plasmid for 24 hours and cultured in medium without FBS for 12 hours under hypoxic conditions followed treatment with U0126 or vehicle for 1 hour and then stimulated with 10% FBS under normoxic conditions for 20 minutes (n=3). Protein expression was determined by Western blot analysis. C. Mice were pretreated with U0126 or vehicle and then subjected to one hour ischemia followed by 6 hours reperfusion in the intestine (n=5). Protein expression in the intestine was determined by Western blot analysis. U=U0126, Sham-U=Sham-U0126, IR=I/R, IR-U=I/R-U0126.
  • Figure 2. Inhibition of ERK1/2 and p70S6K by U0126 decreased cell proliferation and migration, and promoted cell apoptosis in vitro (n=3). A. After transfection with/without empty plasmid or p70S6K plasmid for 12 hours, IEC-6 cells were cultured in medium without FBS for 12 hours under hypoxic conditions followed by treatment with U0126 or vehicle for 1 hour and then stimulated with 10% FBS under normoxic conditions for 3 days. Cell proliferation was measured by MTT assay. B. After transfection with/without empty plasmid or p70S6K plasmid for 12 hours, IEC-6 cells were cultured in medium without FBS in a 12- well plate for 12 hours under hypoxic conditions. The confluent cell monolayer was then scraped with a 1-ml pipette tip. Cells were then treated with U0126 or vehicle for 1 hour followed by adding 10% FBS under normoxic conditions for 24 hours. The recovery area was measured. C. After transfection with/without empty plasmid or p70S6K plasmid for 12 hours, IEC-6 cells were cultured in FBS-free medium with U0126 or vehicle under hypoxic conditions for 24 hours followed by incubation in normoxic conditions for 6 hours. Cell apoptosis was determined by evaluation of DNA fragmentation. U=U0126.
  • Figure 3. Inhibition of ERK1/2 by U0126 increased intestinal cell apoptosis and decreased cell proliferation in vivo. Mice were pretreated with U0126 or vehicle and then subjected to one hour ischemia followed by 6 hours reperfusion in the intestine (n=5). A. Representative images of apoptotic cells in the intestinal tissue detected using an In Situ Cell Death Detection Kit. B. Quantitative measurement of apoptotic cells in the intestinal tissue. C. Representative images of immunohistochemical staining for Ki-67 in the intestinal tissue. Immunohistochemical staining was performed using the Dako-Cytomation EnVision + System-HRP (AEC) kit. C. Quantitative measurement of immunohistochemical staining for Ki-67 in the intestinal tissue. Sham-U=Sham-U0126, IR=I/R, IR-U=I/R-U0126.
  • Figure 4. Inhibition of ERK1/2 by U0126 increased intestinal inflammation, permeability and injury in vivo. Mice were pretreated with U0126 or vehicle and then subjected to one hour ischemia followed by 6 hours reperfusion in the intestine (n=5). The intestinal tissue was examined. A. MPO activity by a commercial Kit. B. Permeability by the ex-vivo isolated everted ileum sac method. C. Representative images by HE staining. D. Quantitative tissue damage by Park/Chiu scoring system. Sham-U=ShamU0126, IR=I/R, IR-U=I/R-U0126.
  • Figure 5. Inhibition of ERK1/2 by U0126 increased the levels of proinflammatory cytokines in plasma. Mice were pretreated with U0126 or vehicle and then subjected to one hour ischemia followed by 6 hours reperfusion in the intestine (n=5). The relatively levels of cytokines in plasma was determined using a commercial kit. A. The relatively level of cytokines TNF-α in plasma. B. The relatively level of cytokines IL-6 in plasma. C. The relatively level of cytokines IL-1β in plasma. Sham-U=Sham-U0126, IR=I/R, IRU=I/R-U0126.
  • Figure 6. Inhibition of ERK1/2 by U0126 increased lung inflammation and injury after intestinal ischemia/reperfusion. Mice were pretreated with U0126 or vehicle and then subjected to one hour ischemia followed by 6 hours reperfusion in the intestine (n=5). The lung tissue was examined. A. MPO activity by a commercial kit. B. Representative images by HE staining. C. Quantitative tissue damage. Sham-U=Sham-U0126, IR=I/R, IR-U=I/R-U0126.
  • Figure 7. Inhibition of ERK1/2 by U0126 increased animal mortality induced by intestinal ischemia/reperfusion. Mice were pretreated with U0126 or vehicle and then subjected to 75 minutes ischemia and the incision was closed. Animals were observed for 7 days for survival rates (n=10) (IR vs. IR-U0126, p<0.01). Sham-U=Sham-U0126, IR=I/R, IR-U=I/R-U0126.

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Ban, K., Peng, Z., & Kozar, R. A. (2013). Inhibition of ERK1/2 Worsens Intestinal Ischemia/Reperfusion Injury. PLoS ONE, 8(9). https://doi.org/10.1371/journal.pone.0076790

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