Inhibition of ERK1/2 worsens intestinal ischemia/reperfusion injury.
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.


CONCLUSIONS
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.
Department of Surgery, University of Texas Health Science Center at Houston, Houston, Texas, United States of America.
Kechen
K
Ban
Zhanglong
Z
Peng
Rosemary A
RA
Kozar
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D018836:Inflammation Mediators / Q000378:metabolism; D007410:Intestinal Diseases / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology*; D008171:Lung Diseases / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D015427:Reperfusion Injury / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology*
D002070:Butadienes; D016207:Cytokines; D004791:Enzyme Inhibitors; D018836:Inflammation Mediators; D009570:Nitriles; C113580:U 0126; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3