Departments of 1 Critical Care Medicine, 2 Surgery, and 4 Pathology, and 3 Center for Biologic Imaging, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Administration of pyruvate, an effective
scavenger of reactive oxygen species, has been shown to be salutary in
numerous models of redox-mediated tissue or organ injury. Pyruvate,
however, is unstable in solution and, hence, is not attractive for
development as a therapeutic agent. Herein, ethyl pyruvate, which is
thought to be more stable than the parent compound, was formulated in a
calcium-containing balanced salt solution [Ringer ethyl pyruvate solution (REPS)] and evaluated in a murine model of hemorrhagic shock
and resuscitation (HS/R). Resuscitation with REPS instead of Ringer
lactate solution (RLS) significantly improved survival at 24 h and
abrogated bacterial translocation to mesenteric lymph nodes and the
development of increased ileal mucosal permeability to FITC-labeled
dextran (4,000 Da) at 4 h. Mice treated with REPS instead of RLS
also had lower circulating levels of alanine aminotransferase at 4 h. Treatment with REPS instead of RLS decreased activation of nuclear
factor-B in liver and colonic mucosa after HS/R and also decreased
the expression of inducible nitric oxide synthase, tumor necrosis
factor, cyclooxygenase-2, and interleukin-6 mRNA in liver, ileal
mucosa, and/or colonic mucosa. These data support the view that
resuscitation with REPS modulates the inflammatory response and
decreases hepatocellular and gut mucosal injury in mice subjected to
HS/R.
translocation; bacterial; permeability; mucosal; tumor necrosis factor; cyclooxygenase-2; inducible nitric oxide synthase
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INTRODUCTION |
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REACTIVE SPECIES
OF OXYGEN (ROS) have been implicated as being important mediators
in a variety of pathological conditions, including burns
(20), various forms of
ischemia-reperfusion (I/R) injury (8, 15, 16,
26), and hemorrhagic shock (9, 10, 17, 32).
Examples of ROS of biological importance include superoxide radical
anion (O). Although O
. In addition, in a series of reactions catalyzed by
ionized iron, O
Pyruvate, a key intermediate in cellular metabolism, is an effective
scavenger of ROS. In a reaction characteristic of -keto carboxylic
acids in general, pyruvic acid (the simplest member of this class of
compounds) rapidly undergoes nonenzymatic decarboxylation in the
presence of H2O2 to form acetate, carbon
dioxide, and water (5, 33). Recently, pyruvate also has
been shown to be capable of scavenging OH · (11).
Administration of exogenous pyruvate has been shown to be salutary in
numerous models of redox-mediated tissue or organ injury
(6-8, 39).
Despite these promising findings, the usefulness of pyruvate as a therapeutic agent is limited by its poor stability in solution (54). When dissolved in an aqueous solvent, pyruvate spontaneously undergoes condensation and cyclization reactions to form a variety of products, some of which may be toxic (31). To circumvent this issue, Sims et al. (48) formulated a derivative of pyruvic acid, namely ethyl pyruvate, in a calcium- and potassium-containing balanced salt solution. These investigators showed that treatment with this fluid, called Ringer ethyl pyruvate solution (REPS), ameliorates structural and functional damage to the intestinal mucosa caused by mesenteric I/R in rats. Subsequently, Tawadrous et al. (51) showed that resuscitation with REPS instead of Ringer lactate solution (RLS) prolongs survival and decreases intestinal mucosal injury in rats subjected to hemorrhagic shock. In this study, Tawadrous et al. (51) also obtained biochemical evidence that resuscitation with REPS instead of RLS ameliorates hepatic and intestinal mucosal lipid peroxidation. These findings support the view that ethyl pyruvate is an effective ROS scavenger.
ROS have been implicated in the activation or modulation of a number of
important intracellular signal transduction pathways, including
signaling mediated by the transcription factor nuclear factor (NF)-B
(52). Activation of the NF-
B pathway is important in
regulating the expression of a number of genes involved in the
inflammatory response, including inducible nitric oxide synthase (iNOS)
(36), cyclooxygenase (COX)-2 (19), tumor
necrosis factor (TNF) (27), and interleukin (IL)-6
(1, 23, 40, 45). Recent work by several groups has shown
that many of these genes are activated in mice subjected to hemorrhagic
shock and resuscitation (HS/R) (13, 46, 47, 50).
In view of the foregoing, the goal of the present investigation was to
gain additional information about the effects of infusing REPS instead
of RLS to resuscitate experimental animals from hemorrhagic shock.
Specifically, we sought to test the hypothesis that treatment with REPS
would blunt activation of NF-B signaling and the activation of
several inflammatory genes in three organs: the liver, the ileum, and
the colon. In these studies, we show that treatment with REPS
ameliorated HS/R-induced hepatocellular injury and gut barrier
dysfunction and also downregulated the inflammatory response associated
with resuscitation from hemorrhagic shock.
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MATERIALS AND METHODS |
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This research protocol complied with the regulations regarding animal care as published by the National Institutes of Health and was approved by the Institutional Animal Use and Care Committee of the University of Pittsburgh Medical School. Male C57BL/6 mice weighing 20-25 g (Jackson Laboratories, Bar Harbor, ME) were used in this study. The animals were maintained at the University of Pittsburgh Animal Research Center with a 12:12-h light-dark cycle and free access to standard laboratory feed and water. Animals were not fasted before the experiments. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
Experimental designs for animal experiments.
The shock model employed has been described previously
(17). Briefly, mice were anesthetized by intramuscular
pentobarbital sodium (90 mg/kg). Both femoral arteries were surgically
prepared and cannulated. The left artery was used for continuous blood pressure monitoring. The right artery was used for blood withdrawal and
blood and fluid administration. The mice were subjected to hemorrhagic
shock by withdrawal of blood (2.25 ml/100 g body wt) over 10 min to
achieve a mean arterial pressure (MAP) of 30 mmHg. MAP was maintained
at 30 mmHg for 2.0 h with continuous monitoring of blood pressure
and withdrawal and return of blood as needed. Cannulas, syringes, and
tubing were flushed with heparin sodium (1,000 U/ml) before all
procedures. The animals were resuscitated to an initial MAP 80 mmHg by administration of all remaining shed blood plus intra-arterial
injection of 2× shed blood volume of either RLS or REPS over 30 min.
Sham controls were subjected to the same anesthetic and cannulation
procedures but were not subjected to hemorrhagic shock. The composition
of RLS was as follows (in mM): 109 NaCl, 4.0 KCl, 2.7 CaCl2, and 28 sodium lactate. The composition of REPS was
as follows (in mM): 130 NaCl, 4 KCl, 2.7 CaCl2, and 28 ethyl pyruvate.
Intestinal mucosal permeability. Intestinal mucosal permeability to the fluorescent tracer fluorescein isothiocyanate- dextran with a molecular mass of 4,000 Da (FD-4) was determined using an everted gut sac method, as previously described by Wattanasirichaigoon et al. (55). Everted gut sacs were prepared in ice-cold modified Krebs-Henseleit bicarbonate buffer (KHBB, pH 7.4). One end of the gut segment was ligated with a 4-0 silk suture. The segment was then everted onto a thin plastic rod, and the resulting gut sac was secured with a 4-0 silk suture to the grooved tip of a 3-ml plastic syringe containing KHBB. The sac was gently distended by injecting 1.5 ml of KHBB. The sac was suspended in a 50-ml beaker containing 40 ml of KHBB plus FD-4 (40 mg/ml). The solution in the beaker was temperature jacketed at 37°C and was continuously bubbled with a gas mixture containing 95% O2-5% CO2. We took a 1.0-ml sample from the beaker before putting in the gut sac to determine the initial external (i.e., mucosal surface) FD-4 concentration. The sac was incubated for 30 min in the KHBB solution containing FD-4. The length of the gut sac was measured. Fluid from the inside of the sac was aspirated for the determination of FD-4 concentration. The serosal and mucosal samples were centrifuged for 10 min at 1,000 g. The supernatant (300 µl) was diluted with phosphate-buffered saline (PBS, 2.7 ml). Fluorescence was measured using a Perkin-Elmer LS-50 fluorescence spectrophotometer (Palo Alto, CA) at an excitation wavelength of 492 nm (slit width, 2.5 nm) and an emission wavelength of 515 nm (slit width, 10 nm). Permeability was expressed as the mucosal-to-serosal clearance of FD-4 as previously described (55).
Bacterial translocation. The skin was cleaned with 10% povidone iodine. Using sterile technique, we opened the abdominal cavity and exposed the viscera. The MLN complex was removed, weighed, and placed in a grinding tube containing 0.5 ml of ice-cold PBS. The MLN were homogenized with glass grinders, and a 250-µl aliquot of the homogenate was plated onto brain-heart infusion and MacConkey agar (Becton Dickinson, Franklin Lakes, NJ). The plates were examined 24 h later after being aerobically incubated at 37°C. The colonies were counted and results expressed as colony-forming units (CFU) per gram of tissue.
Serum ALT measurement. Blood (200 µl) was obtained by cardiac puncture and placed in a 0.5-ml centrifugation tube on ice. The samples were then centrifuged at 5,000 g for 3 min. The serum was collected and assayed for ALT using an automated assay system.
Immunohistochemistry for iNOS expression. Tissues were fixed in 2% paraformaldehyde for 1 h, then treated with 30% sucrose overnight. Fixed sections were washed three times in PBS containing 0.5% BSA and 0.15% glycine, pH 7.4 (buffer A). The fixed and washed sections were incubated for 30 min with purified goat IgG (50 mg/ml) at 25°C and then washed three more times with buffer A. All the preceding steps were designed to ensure minimal nonspecific reaction to the antibodies used. Sections were then incubated for 60 min with a primary antibody to iNOS (1 µg/ml; Transduction Laboratories, Newcastle, UK). This step was followed by three washes in buffer A and a 60-min incubation with a fluorescently labeled second antibody (Alexa 488, 1-2 mg/ml; Molecular Probes, Eugene, OR) mixed with buffer A. The sections were then washed six times (5 min/wash) in buffer A, washed for 1 min in PBS containing 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), an ultraviolet-excited DNA stain to delineate nuclei, and then mounted in gelvatol and coverslipped for light microscopy. Observation was with an Olympus Provis microscope equipped with a cooled charge-coupled device camera.
Assessment of NF-B activation.
To prepare nuclear extracts, we homogenized murine tissue samples with
T-PER (Pierce, Rockford, IL), using a 1:20 ratio of tissue-to-sample
preparation reagent, as directed by the manufacturer's instructions.
The samples were centrifuged at 10,000 g for 5 min to pellet
tissue debris. The supernatant was collected and frozen at
80°C.
Nuclear protein concentration was determined using a commercially
available Bradford assay (Bio-Rad, Hercules, CA).
RT-PCR. Total RNA was extracted from harvested tissues with chloroform and TRI reagent (Molecular Research Center, Cincinnati, OH) exactly as directed by the manufacturer. We treated the total RNA with DNAFree (Ambion, Houston, TX) as instructed by the manufacturer, using 10 U DNase I/10 µg RNA. Two micrograms of total RNA were reverse transcribed in a 40-µl reaction volume containing 0.5 µg oligo(dT)15 (Promega), 1 mM of each dNTP, 15 units avian myeloblastosis virus RT (Promega), and 1 U/µl recombinant RNasin ribonuclease inhibitor (Promega) in 5 mM MgCl2, 10 mM Tris · HCl, 50 mM KCL, and 0.1% Triton X-100 (pH 8.0). The reaction mixtures were preincubated at 21°C for 10 min before DNA synthesis. The RT reactions were carried out for 50 min at 42°C and were heated to 95°C for 5 min to terminate the reaction. Reaction mixtures (50 µl) for PCR were assembled using 5 µl cDNA template, 10 units AdvanTaq Plus DNA polymerase (Clontech, Palo Alto, CA), 200 µM of each dNTP, 1.5 mM MgCl2, and 1.0 µM of each primer in 1× AdvanTaq Plus PCR buffer. PCR reactions were performed using a model 480 thermocycler (Perkin-Elmer, Norwalk, CT). Amplication was initiated with 5 min of denaturation at 94°C. The PCR conditions for amplifying cDNA for TNF, IL-6, and COX-2 were as follows: denaturation at 94°C for 45 s, annealing at 61°C for 45 s, and polymerization at 72°C for 45 s. To ensure amplification was in the linear range, we empirically identified the optimal number of cycles as 33, 35, and 35 for TNF, IL-6, and COX-2, respectively. Amplification of cDNA for iNOS was carried out by denaturing at 94°C for 45 s, annealing at 58°C for 1 min, and polymerizing at 72°C for 45 s for 35 cycles. This number of PCR cycles was empirically determined to ensure that amplification was in the linear range. After the last cycle of amplification, the samples were incubated in 72°C for 10 min and then held at 4°C. The 5' and 3' primers for iNOS were CAC CAC AAG GCC ACA TCG GAT T and CCG ACC TGA TGT TGC CAT TGT T, respectively (Invitrogen, Carlsbad, CA); the expected product length was 426 bp. The 5' and 3' primers for TNF were GGC AGG TCT ACT TTG GAG TCA TTG C and ACA TTC GAG GCT CCA GTG AAT TCG G, respectively; the expected product length was 307 bp. The 5' and 3' primers for IL-6 were CTG GTG ACA ACC ACG GCC TCC CCT and ATG CTT AGG CAT AAC GCA CTA GGT, respectively; the expected product length was 600 bp. The 5' and 3' primers for COX-2 were GTC TGA TGA TGT ATG CCA CAA TCT G and GAT GCC AGT GAT AGA GGG TGT TGA A, respectively; the expected product length was 276 bp. 18S ribosomal RNA was amplified to verify equal loading. For this reaction, the 5' and 3' primers were CCC GGG GAG GTA GTG ACG AAA AAT and CGC CCG CTC CCA AGA TCC AAC TAC, respectively; the expected product length was 200 bp. Ten microliters of each PCR reaction were electrophoresed on a 2% agarose gel, scanned at a NucleoVision imaging workstation (NucleoTech, San Mateo, CA), and quantified using GelExpert release 3.5.
Statistical methods.
Results are presented as means ± SE. Differences in CFU between
groups were analyzed using the Wilcoxon U-test. Other
continuous data were analyzed using Student's t-test or
analysis of variance followed by Fisher's least significant
differences test as appropriate. Survival data were analyzed using the
2 test. P values < 0.05 were considered
significant. Summary statistics are presented for RT-PCR data, but
these results were not subjected to statistical analyses, since the
method employed was only semiquantitative and the samples sizes
(n = 3-4) were small.
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RESULTS |
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Survival.
Experiment A was not designed as a survival study. The mice
were intentionally killed 4 h after the end of resuscitation to assess intestinal barrier function and to obtain blood and tissue samples for determinations of plasma ALT concentration, NF-B activation, and steady-state mRNA levels for several genes.
Nevertheless, in experiment A, all of the mice in the Sham
and REPS groups survived until they were killed at 4 h after
resuscitation (Table 1). In contrast,
only 12 of 15 mice in the RLS group survived to the 4-h
postresuscitation time point. Experiment B was performed as a survival study. In this experiment, all of the mice in the Sham group
and 11 of 12 mice in the REPS group survived for at least 24 h,
whereas 5 of 10 mice in the RLS group died before the 24-h postresuscitation time point. At 24 h, all of the surviving mice in the Sham and REPS groups were active and eating and drinking normally.
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Intestinal barrier dysfunction.
In the RLS group, ileal mucosal permeability to FD-4 was approximately
twofold greater than in the Sham group (Fig.
1A). However, resuscitation
with REPS instead of RLS significantly ameliorated the increase in
mucosal permeability induced by the HS/R protocol. Bacterial
translocation to MLN was minimal in sham-operated controls but was
extensive in the RLS group (Fig. 1B). Resuscitation with REPS virtually abrogated bacterial translocation after HS/R.
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Hepatocellular damage.
The mean plasma ALT concentration was significantly greater in the RLS
group than in the sham-operated control group (Fig. 2). However, the mean circulating level
of this marker of hepatocellular injury was significantly lower in the
REPS group than in the RLS group.
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NF-B activation.
We used EMSA to detect the transcription factor NF-
B in nuclear
extracts prepared from samples of liver and ileal and colonic mucosa
obtained from mice 4 h after resuscitation from hemorrhagic shock
with either RLS or REPS. We also assayed nuclear extracts prepared from
samples obtained from control animals (Sham group) that were
anesthetized and cannulated but not subjected to shock. There was
evidence of basal DNA binding of NF-
B in all the tissues examined,
but especially in samples of ileal mucosa (Fig.
3A). After resuscitation with
RLS, NF-
B DNA binding increased in liver and colonic mucosa but was
unchanged in ileal mucosa. To confirm the identity of the activated
protein-DNA complex, we carried out binding assays with samples that
were preincubated with specific antibodies directed against p50 and
p65. Although we failed to observe a supershift with the anti-p50
antibody, we observed both a supershifted band and decreased intensity
of the NF-
B band with the p65 antibody. Moreover, binding of the
protein to labeled NF-
B binding element was completely inhibited by
a 100-fold excess of unlabeled NF-
B duplex oligonucleotide but not
by a similar molar excess of unlabeled HIF-1 duplex oligonucleotide.
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Expression of stress-related genes.
Hepatic iNOS, TNF, and COX-2 mRNA expression clearly increased after
HS/R in the RLS group (Fig. 4,
A-C, respectively). Resuscitation with REPS instead of
RLS decreased post-HS/R expression of all three genes.
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Immunohistochemistry.
HS/R was associated with an apparent increase in hepatic iNOS
expression in the RLS group but not the REPS group (Fig.
6). These findings were consistent with
the results obtained by semiquantitative RT-PCR.
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DISCUSSION |
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In 1904, Holleman (14) reported that pyruvate and
related -keto acids with the general structure R
CO
COOH
reduce H2O2 nonenzymatically in a reaction that
yields carbon dioxide and water. In the case of pyruvic acid, this
oxidative decarboxylation reaction can be written as follows:
CH3COCOO
+ H2O2
CH3COO
+ H2O + CO2. Subsequent studies verified that this reaction is rapid and stoichiometric (5, 28). In addition to
scavenging H2O2, pyruvate is also capable of
scavenging OH · (11). In 1991, Salahudeen et al.
(39) reported that intravenous infusion of sodium pyruvate
protects rats from renal parenchymal injury induced by injecting
H2O2 into the renal artery. After the
publication of that paper, a number of other investigators reported
that treatment with pyruvate could protect animals from the deleterious
effects of a variety of conditions thought to be mediated by ROS,
including myocardial I/R (4, 7, 8, 11, 35), intestinal I/R (6), and HS/R (30).
Despite these findings, pyruvate has not been developed as a therapeutic agent, probably because it is relatively unstable in solution (54) and is capable of forming potentially toxic byproducts (31). Ethyl pyruvate, a simple derivative of pyruvate, is more stable than the parent compound (Alfred Ajami, Xanthus, personal communication). Nevertheless, this compound has not been extensively evaluated as a therapeutic agent. One reason for the paucity of prior work with ethyl pyruvate may relate to its poor solubility (0.25% wt/vol in saline). Sims et al. (48), however, discovered that the use of a balanced, calcium-containing salt solution (analogous to RLS) markedly increases the solubility of ethyl pyruvate to 1.5% (wt/vol) or 130 mM. The basis for the increased solubility of ethyl pyruvate in a Ringer-type solution is thought to be stabilization of the enolate form of ethyl pyruvate by Ca2+ (48).
A formulation of ethyl pyruvate in a Ringer-type balanced salt solution, i.e., REPS, has been evaluated in two prior studies. In the first, Sims et al. (48) showed that pre- and posttreatment of rats subjected to mesenteric I/R largely prevented structural damage to the intestinal mucosal caused by this stress and also significantly ameliorated the development of gut mucosal hyperpermeability after reperfusion. Subsequently, in a study using a rat model of HS/R, Tawadrous et al. (51) showed that resuscitating rats with REPS instead of RLS prevented the development of mucosal hyperpermeability and ameliorated lipid peroxidation, a marker of ROS-mediated stress, in liver and gut.
In the present study, we have both confirmed and substantially extended the observations made in the prior study of REPS as a resuscitation fluid for HS/R. The differences between our laboratory's earlier study and the present one can be summarized as follows. First, we used a rat model of hemorrhagic shock in the study by Tawadrous et al. (51), whereas for the studies described herein we used a model of hemorrhagic shock in mice. Second, in the study by Tawadrous et al. (51), we showed that resuscitation with REPS prolongs survival during the first 4 h after resuscitation from shock, whereas we are now able to report that resuscitation with REPS instead of RLS improves permanent survival in a murine model of HS/R. Third, as in the previous study by Tawadrous et al. (51), we showed that resuscitation from hemorrhagic shock with REPS instead of RLS significantly ameliorated ileal mucosal hyperpermeability to FD-4. Hyperpermeability to hydrophilic macromolecules, however, is only one manifestation of gut barrier dysfunction. However, another important feature of gut barrier dysfunction associated with HS/R is increased bacterial translocation to MLN (2). It is noteworthy, therefore, that in the present study, we found that resuscitation with REPS instead of RLS abrogated bacterial translocation. This finding is consistent with data from previous studies (9, 10) showing that various ROS scavengers are capable of ameliorating bacterial translocation caused by hemorrhagic shock. Fourth, in the present study, we also showed that resuscitation with REPS ameliorated HS/R-induced hepatocellular damage. This observation is consistent with data obtained in two other recent studies, wherein treatment before resuscitation with the ROS scavengers tempol (32) and N-2-mercaptopropionyl glycine (17) ameliorated hepatocellular injury associated with HS/R in rodents. Fifth, in the present study, we carried out experiments to examine the effects of REPS on various aspects of the inflammatory response after HS/R.
Previous investigators have shown that HS/R is associated with
activation of the transcription factor NF-B in various organs and
tissues, such as liver (1, 13), lung (13),
pulmonary mononuclear cells (45), heart (27),
and kidney (25). Our results, showing HS/R-induced
activation of NF-
B in liver and colonic mucosa, are consistent with
these observations. Interestingly, we found that NF-
B was
constitutively activated in ileal mucosa, and HS/R caused little or no
change in the degree of NF-
B DNA binding in this tissue. A high
basal level of ileal mucosal NF-
B activation was previously
described by Pritts et al. (36). To confirm the
specificity of the EMSA for NF-
B, we carried out both cold
competition and supershift assays. As expected, competition with an
excess of unlabeled NF-
B probe eliminated binding by the
32P-labeled NF-
B consensus duplex oligonucleotide,
whereas addition of an excess quantity of an unlabeled irrelevant
duplex oligonucleotide had no effect on binding by the hot NF-
B
probe. This observation supports the specificity of the EMSA for
NF-
B DNA binding. We observed clear evidence of a supershift when
extracts were preincubated with a commercially available antibody
against p65. This finding is in agreement with previously reported data
reported by Pritts et al. (36) and suggests that the
detected NF-
B dimers contained p65. However, we were unable to
demonstrate clear evidence of a supershift when extracts were
preincubated with an antibody against p50. Because this finding is
inconsistent with reports from several other laboratories, we carried
out similar studies using a different anti-p50 antibody from another
supplier. Similar results, however, were obtained (data not shown). We
are unable to determine whether this observation represents a problem
with the antibodies we used or actual evidence that we detected DNA binding by NF-
B-like constructs without a p50 subunit.
The transcriptionally active form of NF-B is a homo- or
heterodimer made up of various proteins belonging to the NF-
B
family. These proteins include p50, p65 (RelA), c-Rel, p52, and RelB
(3). In resting cells, however, these homo- or
heterodimeric forms of NF-
B exist in the cytoplasm in an inactive
form due to binding by a third inhibitory protein called I
B
(3). Upon stimulation of the cell by a proinflammatory
trigger, I
B is phosphorylated on two key serine residues, targeting
I
B for ubiquination and subsequent proteosomal degradation.
Phosphorylation of I
B is thought be mediated by various I
B
kinases (IKKs) (18). Phosphorylation and degradation of
I
B permit translocation of the transcriptionally active (dimeric)
form of NF-
B into the nucleus and subsequent binding of the
transcription factor to cis-acting elements in the promoter
regions of various NF-
B-responsive genes.
Although the upstream events that lead to IKK activation are unclear
(and probably differ depending on the inciting proinflammatory stimulus), it has been proposed that ROS are important in this process.
Several lines of evidence support this view. First, numerous studies
have shown that providing an exogenous source of ROS (e.g., by adding
H2O2 to the medium for cultured cells) can
trigger activation of NF-B (23, 38, 40). Second,
stimulating cells with various proinflammatory substances (e.g., TNF)
leads to endogenous production of ROS (41). Third, various
compounds with known antioxidant activity, such as
N-acetylcysteine and pyrrolidine dithiocarbamate (PDTC),
have been shown to block activation of NF-
B in cultured cells, not
only by exogenous ROS but also by other proinflammatory stimuli
(34, 41). Fourth, cytokine-stimulated activation of NF-
B tends to be exaggerated when cells are pretreated with an agent, such as buthionine sulfoximine, that depletes intracellular levels of glutathione, an important endogenous ROS scavenger
(43). Finally, certain ROS scavengers, such as PDTC and
dimethylthiourea, have been shown to block NF-
B activation in vivo
as well (22, 49). Indeed, previous studies have shown that
HS/R-induced NF-
B activation is downregulated by agents that either
scavenge ROS or block their synthesis, including allopurinol
(45-47) and IRFI-042 (a vitamin E analog)
(1). Our results are consistent with these observations, since we found that resuscitation with a solution containing the ROS scavenger ethyl pyruvate (51, 53)
downregulated NF-
B activation in liver and colonic mucosa in mice
subjected to HS/R.
Previous studies (13, 46, 47, 50) have shown that HS/R
leads to activation of a number of stress-related and proinflammatory genes. Although it is likely that more than one mechanism is
responsible for activation of these genes after HS/R, one important
factor appears to be increased formation of ROS and activation of
various redox-sensitive signaling cascades. Treatment of experimental animals with ROS scavengers has been shown to inhibit HS/R-mediated upregulation of TNF (1, 17, 42, 50), IL-6
(50), and macrophage inflammatory protein-2
(47). In the present study, we confirmed and extended
these findings by showing that treatment with REPS downregulated
HS/R-induced expression of TNF, iNOS, COX-2, and IL-6 to varying
degrees, depending on the tissue examined. It is noteworthy that
resuscitation with REPS significantly inhibited HS/R-induced
upregulation of IL-6 in ileal mucosa, since NF-B activation in this
tissue was largely unchanged by HS/R (irrespective of whether RLS or
REPS was used for resuscitation). These data suggest that ethyl
pyruvate is capable of modulating one or more signal transduction
pathways not involving NF-
B that are important in control of the
inflammatory response to HS/R in at least some tissues.
Although Tawadrous et al. (51) and others
(53) previously showed that ethyl pyruvate is capable of
functioning as an ROS scavenger in vivo or in vitro, the possibility
exists that this molecule might be beneficial in hemorrhagic shock for
other reasons. For example, it is possible that when used as a
component of resuscitation fluid for hemorrhagic shock, ethyl pyruvate
functions as a metabolic substrate to decrease the cytosolic
[NADH]/[NAD+] ratio and maintain the cellular
phosphorylation potential, [ATP]/[ADP][Pi]; this
mechanism has been proposed to explain some of the beneficial effects
of the parent compound pyruvate in a porcine model of hemorrhagic shock
(30). However, it also possible that ethyl pyruvate
functions in ways that are quite distinct from those of pyruvate. This
notion is supported by studies showing that a related pyruvate ester,
methyl pyruvate, stimulates insulin secretion by isolated pancreatic
islets (29, 56), whereas pyruvate is not insulinogenic
(44). To explain the differential effects of these two
closely related compounds, it was speculated that the more lipophilic
compound, methyl pyruvate, might penetrate the mitochondrial matrix
better than pyruvate and thereby support supranormal rates of ATP
production. However, recently reported data refute this hypothesis and
suggest that pyruvate and methyl pyruvate have distinct biochemical
effects in pancreatic -cells that are unrelated to ATP biosynthesis
(21). It is unknown at present whether ethyl pyruvate also
has biochemical actions that are clearly distinct from those of
pyruvate. However, in an earlier study (48), we observed
that ethyl pyruvate provided better protection than pyruvate against
gut mucosal damage caused by mesenteric I/R in rats.
In summary, we showed herein that HS/R in mice is associated with intestinal barrier dysfunction, hepatocellular injury, and increased expression of a number of stress-related and/or proinflammatory genes. These effects of HS/R were all attenuated, if not blocked completely, when the experimental animals were resuscitated with REPS, instead of a conventional crystalloid resuscitation fluid. These data support the view that REPS warrants further evaluation as a therapeutic agent for the prevention of organ injury and systemic inflammation after resuscitation from hemorrhagic shock.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of General Medical Sciences Grants GM-53789, GM-37631, and GM-58484; and Defense Advanced Research Projects Agency Grant N65236-00-1-5434.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. P. Fink, Dept. of Critical Care Medicine, Univ. of Pittsburgh Medical School, 616 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (E-mail: finkmp{at}ccm.upmc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 27, 2002;10.1152/ajpgi.00022.2002
Received 16 January 2002; accepted in final form 1 February 2002.
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