Kechen Ban

Glutamine protects against apoptosis via downregulation of Sp3 in intestinal epithelial cells.

Glutamine plays a key role in intestinal growth and maintenance of gut function, and as we have shown protects the postischemic gut (Kozar RA, Scultz SG, Bick RJ, Poindexter BJ, Desoigne R, Weisbrodt NW, Haber MM, Moore FA. Shock 21: 433-437, 2004). However, the precise mechanisms of the gut protective effects of glutamine have not been well elucidated. In the present study, RNA microarray was performed to obtain differentially expressed genes in intestinal epithelial IEC-6 cells following either 2 mM or 10 mM glutamine. The result demonstrated that specificity protein 3 (Sp3) mRNA expression was downregulated 3.1-fold. PCR and Western blot confirmed that Sp3 expression was decreased by glutamine in a time- and dose-dependent fashion. To investigate the role of Sp3, Sp3 gene siRNA silencing was performed and apoptosis was assessed. Silencing of Sp3 demonstrated a significant increase in Bcl-2 and decrease in Bax protein expression, as well as a decrease in caspase-3, -8, and -9 protein expression and activity. The protein expression of apoptosis-related proteins after hypoxia/reoxygenation was similar to that of normoxia and correlated with a decrease in DNA fragmentation. Importantly, the addition of glutamine to Sp3-silenced cells did not further lessen apoptosis, suggesting that Sp3 plays a major role in the inhibitory effect of glutamine on apoptosis. This novel finding may explain in part the gut-protective effects of glutamine.

Enteral glutamine: a novel mediator of PPARγ in the postischemic gut

Early enteral nutrition supplemented with glutamine, arginine, omega‐3 fatty acids, and nucleotides has been shown to decrease infection complications in critically injured patients. Concern has been raised, however, that under conditions of hyperinflammation, these diets may be injurious through the induction of inducible NO synthase by enteral arginine. In a rodent model of gut ischemia/reperfusion, inflammation and injury are intensified by enteral arginine and abrogated by glutamine. These findings correlate with the degree of metabolic stress imposed upon the gut by hypoperfusion. Glutamine is metabolized by the gut and therefore, can contribute back energy in the form of ATP, whereas arginine is a nonmetabolizable nutrient, using but not contributing energy. Recent data suggest that one of the molecular mechanisms responsible for the gut‐protective effects of enteral glutamine is the activation of peroxisome proliferator‐activated receptor γ. This anti‐inflammatory transcription factor belongs to the family of nuclear receptors, plays a key role in adipocyte development and glucose homeostasis, and has been recognized as an endogenous regulator of intestinal inflammation. Preliminary clinical studies support the use of enteral glutamine in patients with gut hypoperfusion.

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. 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.

Plasma-Mediated Gut Protection After Hemorrhagic Shock is Lessened in Syndecan-1-/- Mice.

ABSTRACT We have shown in a rodent model of hemorrhagic shock (HS) that fresh frozen plasma (FFP) reduces lung inflammation and injury that are correlated with restitution of syndecan-1. As the gut is believed to contribute to distant organ injury and inflammation after shock, the current study sought to determine if the protective effects of plasma would extend to the gut and to elucidate the contribution of syndecan-1 to this protective effect. We also examined the potential role of TNF&agr;, and a disintegrin and metalloproteinase (ADAM)-17, both intestinal sheddases of syndecan-1. Wild-type (WT) and syndecan-1−/− (KO) mice were subjected to HS followed by resuscitation with lactated Ringers (LR) or FFP and compared with shock alone and shams. Small bowel and blood were obtained after 3 h for analysis of mucosal injury and inflammation and TNF&agr; and ADAM-17 protein expression and activity. After HS, gut injury and inflammation were significantly increased compared with shams. Resuscitation with LR decreased both injury and inflammation that were further lessened by FFP. KO mice displayed worsened gut injury and inflammation after HS compared with WT mice, and LR and FFP equivalently inhibited injury and inflammation. Both systemic and intestinal TNF&agr; and ADAM-17 followed similar trends, with increases after HS, reduction by LR, and a further decrease by FFP in WT but not KO mice. In conclusion, FFP decreased gut injury and inflammation after hemorrhagic shock, an effect that was abrogated in syndecan-1−/− mice. Plasma also decreased TNF&agr; and ADAM-17, representing a potential mechanistic link to its protection via syndecan-1.

Glutamine activates peroxisome proliferator-activated receptor-γ in intestinal epithelial cells via 15-S-HETE and 13-OXO-ODE: a novel mechanism

Glutamine possesses gut-protective effects both clinically and in the laboratory. We have shown in a rodent model of mesenteric ischemia-reperfusion that enteral glutamine increased peroxisome proliferator-activated receptor-γ (PPAR-γ) and was associated with a reduction in mucosal injury and inflammation. The mechanism by which glutamine activates PPAR-γ is unknown, and we hypothesized that it was via a ligand-dependent mechanism. Intestinal epithelial cells, IEC-6, were co-transfected with PPAR-γ response element-luciferase promoter/reporter construct. Cells were pretreated with increasing concentrations of glutamine ± GW9662 (a specific antagonist of PPAR-γ) and analyzed for PPAR-γ response element luciferase activity as an indicator of PPAR-γ activation. PPAR-γ nuclear activity was assessed by electrophoretic mobility shift assay. Cell lysates were subjected to tandem mass spectroscopy for measurement of prostaglandin and lipoxygenase metabolites. A time- and concentration-dependent increase in PPAR-γ transcriptional activity, but not mRNA or protein, was demonstrated. Activity was abrogated by the PPAR-γ inhibitor, GW9662, and changes in activity correlated with PPAR-γ nuclear binding. Glutamine, via degradation to glutamate, activated the metabolic by-products of the lipoxygenase and linoleic acid pathways, 15-S-hydroxyeicosatetraenoic acid and dehydrogenated 13-hydroxyoctaolecadienoic acid, known endogenous PPAR-γ ligands in the small bowel. This novel mechanism may explain the gut-protective effects of enteral glutamine.

Syndecan 1 plays a novel role in enteral glutamine's gut-protective effects of the postischemic gut.

ABSTRACT Syndecan 1 is the predominant heparan sulfate proteoglycan found on the surface of epithelial cells and, like glutamine, is essential in maintaining the intestinal epithelial barrier. We therefore hypothesized that loss of epithelial syndecan 1 would abrogate the gut-protective effects of enteral glutamine. Both an in vitro and in vivo model of gut ischemia-reperfusion (IR) was utilized. In vitro, intestinal epithelial cells underwent hypoxia-reoxygenation to mimic gut IR with 2 mM (physiologic) or 10 mM glutamine supplementation. Permeability, caspase activity, cell growth, and cell surface and shed syndecan 1 were assessed. In vivo, wild-type and syndecan 1 knockout (KO) mice received ± enteral glutamine followed by gut IR. Intestinal injury was assessed by fluorescent dye clearance and histopathology, permeability as mucosal-to-serosal clearance ex vivo in everted sacs, and inflammation by myeloperoxidase (MPO) activity. In an in vitro model of gut IR, glutamine supplementation reduced epithelial cell permeability and apoptosis and enhanced cell growth. Shed syndecan 1 was reduced by glutamine without an increase in syndecan 1 mRNA. In vivo, intestinal permeability, inflammation, and injury were increased after gut IR in wild-type mice and further increased in syndecan 1 KO mice. Glutamine’s attenuation of IR-induced intestinal hyperpermeability, inflammation, and injury was abolished in syndecan 1 KO mice. These results suggest that syndecan 1 plays a novel role in the protective effects of enteral glutamine in the postischemic gut.

Protective Role of p70S6K in Intestinal Ischemia/Reperfusion Injury in Mice

The mTOR signaling pathway plays a crucial role in the regulation of cell growth, proliferation, survival and in directing immune responses. As the intestinal epithelium displays rapid cell growth and differentiation and is an important immune regulatory organ, we hypothesized that mTOR may play an important role in the protection against intestinal ischemia reperfusion (I/R)-induced injury. To better understand the molecular mechanisms by which the mTOR pathway is altered by intestinal I/R, p70S6K, the major effector of the mTOR pathway, was investigated along with the effects of rapamycin, a specific inhibitor of mTOR and an immunosuppressant agent used clinically in transplant patients. In vitro experiments using an intestinal epithelial cell line and hypoxia/reoxygenation demonstrated that overexpression of p70S6K promoted cell growth and migration, and decreased cell apoptosis. Inhibition of p70S6K by rapamycin reversed these protective effects. In a mouse model of gut I/R, an increase of p70S6K activity was found by 5 min and remained elevated after 6 h of reperfusion. Inhibition of p70S6K by rapamycin worsened gut injury, promoted inflammation, and enhanced intestinal permeability. Importantly, rapamycin treated animals had a significantly increased mortality. These novel results demonstrate a key role of p70S6K in protection against I/R injury in the intestine and suggest a potential danger in using mTOR inhibitors in patients at risk for gut hypoperfusion.

Protection by enteral glutamine is mediated by intestinal epithelial cell peroxisome proliferator-activated receptor-γ during intestinal ischemia/reperfusion.

ABSTRACT We have demonstrated that enteral glutamine provides protection to the postischemic gut, and that peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) plays a role in this protection. Using Cre/lox technology to generate an intestinal epithelial cell (IEC)–specific PPAR&ggr; null mouse model, we now investigated the contribution of IEC PPAR&ggr; to glutamine’s local and distant organ–protective effects. These mice exhibited absence of expression of PPAR&ggr; in the intestine but normal PPAR&ggr; expression in other tissues. After 1 h of intestinal ischemia under isoflurane anesthesia, wild-type and null mice received enteral glutamine (60 mM) or vehicle followed by 6 h of reperfusion or 7 days in survival experiments and compared with shams. Small intestine, liver, and lungs were analyzed for injury and inflammatory parameters. Glutamine provided significant protection against gut injury and inflammation, with similar protection in the lung and liver. Changes in systemic tumor necrosis factor-&agr; reflected those seen in the injured organs. Importantly, mice lacking IEC PPAR&ggr; had worsened injury and inflammation, and glutamine lost its protective effects in the gut and lung. The survival benefit found in glutamine-treated wild-type mice was not observed in null mice. Using an IEC-targeted loss-of-function approach, these studies provide the first in vivo confirmation in native small intestine and lung that PPAR&ggr; is responsible for the protective effects of enteral glutamine in reducing intestinal and lung injury and inflammation and improving survival. These data suggest that early enteral glutamine may be a potential therapeutic modality to reduce shock-induced gut dysfunction and subsequent distant organ injury.

Intraluminal tranexamic acid inhibits intestinal sheddases and mitigates gut and lung injury and inflammation in a rodent model of hemorrhagic shock.

BACKGROUND Intravenous tranexamic acid (TXA) is an effective adjunct after hemorrhagic shock (HS) because of its antifibrinolytic properties. TXA is also a serine protease inhibitor, and recent laboratory data demonstrated that intraluminal TXA into the small bowel inhibited digestive proteases and protected the gut. A Disintegrin And Metalloproteinase 17 (ADAM-17) and tumor necrosis factor &agr; (TNF-&agr;) are effective sheddases of intestinal syndecan-1, which when shed, exposes the underlying intestinal epithelium to digestive proteases and subsequent systemic insult. We therefore hypothesized that intraluminal TXA as a serine protease inhibitor would reduce intestinal sheddases and syndecan-1 shedding, mitigating gut and distant organ (lung) damage. METHODS Mice underwent 90 minutes of HS to a mean arterial pressure of 35 ± 5 mm Hg followed by the intraluminal administration of TXA or vehicle. After 3 hours, the small intestine, lung, and blood were collected for analysis. RESULTS Intraluminal TXA significantly reduced gut and lung histopathologic injury and inflammation compared with HS alone. Gut, lung, and systemic ADAM-17 and TNF-&agr; were significantly increased by HS but lessened by TXA. In addition, gut and lung syndecan-1 immunostaining were preserved and systemic shedding lessened after TXA. TXA reduced ADAM-17 and TNF-&agr;, but not syndecan-1, in TXA-sham animals compared with sham vehicles. CONCLUSION Results of the present study demonstrate a beneficial effect of intraluminal TXA in the gut and lung after experimental HS in part because of the inhibition of the syndecan-1 shedding by ADAM-17 and TNF-&agr;. Further studies are needed to determine if orally administered TXA could provide similar intestinal protection and thus be of potential benefit to patients with survivable hemorrhage at risk for organ injury. This is particularly relevant in patients or soldiers who may not have access to timely medical care.

Enteral arginine modulates inhibition of AP-1/c-Jun by SP600125 in the postischemic gut.

We previously demonstrated that enteral arginine increased c-Jun/activator protein-1 (AP-1) DNA-binding activity and iNOS expression in a rodent model of mesenteric ischemia/reperfusion (I/R). The objective of this study was to specifically investigate the role of AP-1 in arginine’s deleterious effect on the postischemic gut. We hypothesized that AP-1 inhibition would mitigate the effects of arginine. Using a rodent model of mesenteric I/R we demonstrated that gut neutrophil infiltration, activity of c-Jun/AP-1, as well as iNOS expression were increased by I/R and further increased by arginine while lessened by inhibition of c-Jun using the pharmacologic c-Jun N-terminal kinase inhibitor, SP600125. Similar results were demonstrated using a cell culture model of oxidant stress in IEC-6 cells. Importantly, effects of SP600125 were comparable to those of c-Jun silencing. Lastly, the specific iNOS inhibitor, 1400W, had no effect on either AP-1 or c-Jun. In conclusion, SP600125 attenuated the activity of c-Jun/AP-1, iNOS expression, and neutrophil infiltration induced by arginine following mesenteric I/R. Our data suggest that AP-1 inhibition mitigates the injurious inflammatory effects of arginine in the postischemic gut. Further investigation into the pathologic role of enteral argninine in the postischemic gut is warranted.