1 Department of Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh 15213; and 2 The Center for Biologic Imaging and 3 Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Sustained upregulation of inducible nitric
oxide (NO) synthase in the liver after endotoxin [lipopolysaccharide
(LPS)] challenge may result in hepatocellular injury. We hypothesized
that administration of a NO scavenger, NOX, may attenuate LPS-induced
hepatocellular injury. Sprague-Dawley rats received NOX or saline via
subcutaneous osmotic pumps, followed 18 h later by LPS challenge.
Hepatocellular injury was assessed using biochemical assays, light, and
transmission electron microscopy (TEM). Interleukin (IL)-6 mRNA was
measured by RT-PCR. Tumor necrosis factor (TNF)- protein expression
was determined by immunohistochemistry. NOX significantly reduced serum
levels of ornithine carbamoyltransferase and aspartate
aminotransferase. TNF-
and IL-6 expression were increased in the
livers of saline-treated but not NOX-treated rats. Although there was
no difference between groups by light microscopy, TEM revealed
obliteration of the space of Disse in saline-treated but not in
NOX-treated animals. Electron paramagnetic resonance showed the
characteristic mononitrosyl complex in NOX-treated rats. We conclude
that NOX reduces hepatocellular injury after endotoxemia. NOX may be
useful in the management of hepatic dysfunction secondary to sepsis or
other diseases associated with excessive NO production.
inducible nitric oxide synthase; endotoxemia; dithiocarbamate; interleukin-6; ornithine carbamoyltransferase
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INTRODUCTION |
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THE LIVER PERFORMS a
variety of important host defense and metabolic functions that include
synthesis of acute phase proteins, gluconeogenesis, detoxification, and
clearance of endogenous mediators, as well as secretion of
proinflammatory cytokines (41). Hepatic dysfunction after
sepsis is a frequent event that is characterized by loss of synthetic
function, hepatocellular necrosis, and release of inflammatory
mediators such as tumor necrosis factor- (TNF-
), interleukin
(IL)-1, IL-6, prostaglandins, and nitric oxide (NO) (3, 7, 13,
25). The specific role of these various cytokines in the
pathogenesis of hepatocellular dysfunction or necrosis after
endotoxemia is still undefined.
Whereas sustained production of NO in the gut has been shown to induce derangement in intestinal barrier function (9, 51, 55), the role of NO as a putative mediator of hepatic injury after endotoxic shock remains controversial. Several authors have shown that nonspecific inhibition of all three isoforms of NO synthase (NOS) during endotoxemia may augment hepatocellular injury (12, 18, 39). NO donors have been shown to preserve hepatic perfusion during endotoxemia and to prevent inflammatory changes in the microcirculation (37, 42). However, a growing body of evidence suggests that sustained production of NO resulting from upregulation of inducible NOS (iNOS) after lipopolysaccharide (LPS) challenge may cause hepatocellular injury, either directly (57), or indirectly, by forming reactive nitrogen intermediates (35). Menezes et al. (33) recently demonstrated that a NO scavenger, NOX, decreased hepatocellular injury and improved survival after hemorrhagic shock. We hypothesized that NOX may prevent hepatocellular injury after endotoxic shock.
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MATERIALS AND METHODS |
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Experimental Design
The experimental protocol was approved by the Animal Research and Care Committee of the Children's Hospital of Pittsburgh, in accordance with the National Institutes of Health guidelines for animal care. Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing between 250 and 300 g were acclimatized for a minimum of 1 wk before experimentation. After pentobarbital sodium (50 mg/kg) was administered intraperitoneally to the animals, osmotic pumps (Alzet model 2ML1, 10 ml/h, 7-day pump, Alza, Palo Alto, CA) were placed subcutaneously in the back of each rat, and the delivery catheter was tunneled subcutaneously in the neck. The animals were randomized to receive either 2 ml of normal saline (NS) or NOX (450 mg) in a final volume of 2 ml via the osmotic pumps. NOX was a kind gift of Dr. Ching-San Lai (Medinox, San Diego, CA). Additional doses of NOX (112.5 mg) or NS (0.5 ml) were given concomitant with LPS administration and at 4-h intervals thereafter for a total of three doses. A dose-response curve was generated for NOX before the final dose used in these experiments was selected (9). Eighteen hours later, the animals were challenged with 10 mg/kg ip of LPS (Escherichia coli 0111:B4, Difco, Detroit, MI). In our posttreatment groups, the same protocol was used except that the delivery catheter was not tunneled until 4 and 8 h after LPS administration. All animals were killed 24 h after LPS challenge.Electron Paramagnetic Resonance Spectrometry
Sprague-Dawley rats were randomized to receive either NOX or NS as described for 24 h before LPS challenge (10 mg/kg ip). Additional doses of NOX or NS were given as described. At 6, 8, and 24 h after LPS challenge, the rats were killed, and their livers were harvested and perfused with NS via the portal vein until the perfusate in the right atrium was free of any blood. Sections of the perfused liver weighing 1-2 g were then placed in capillary tubing and stored atThe EPR spectra were recorded with an E4 spectrometer (Varian Associates, Palo Alto, CA) at 77°K to allow detection of nitrosyl-hemoglobin signals. The scans were run at full microwave power (~100 mW), a modulation amplitude of 10 G, a microwave frequency of ~9.10 GHz, a scan time of 4 min, and a time constant of 0.3 s. Identification of the carrier of each spectrum was made by measuring its g value. This measurement was performed by using an electronic counter to determine the microwave frequency to one part in 106 and comparing the field position of the sample with that of 1,1-diphenyl-2-picrylhydrazyl, which has a known g value (2.0036). A standard solution of 5 mM S-nitroso-N-acetyl-D,L-penicillamine (SNAP), 1 mM FeSO4, and 10 mM NOX dissolved in water was used to verify the spectrum of a mononitrosyl-iron-NOX complex in our system. The spectrum for each rat was then determined by EPR and assessed by a single investigator (D. W. Pratt) who was blinded to the treatment groups. The spectra were evaluated for the presence or absence of a nitrosyl-hemoglobin signal or a mononitrosyl-iron complex signal.
Biochemical Assays
Plasma was harvested by cardiac puncture. Ornithine carbamoyltransferase (OCT) was measured directly from the serum according to the method published by Ohshita et al. (38). Serum glucose and aspartate aminotransferase (AST) levels were measured by routine clinical chemistry. Fibrinogen levels were determined by the modified heat precipitation reaction (30). Briefly, capillary tubes were filled with blood specimens and centrifuged for 3 min in an international microhematocrit (model MB, Inter Equipment, Needham, MA) and then placed in a water bath at 56°C for 3 min. The tubes were centrifuged for 5 min, and the height of the plasma column and the layer of fibrinogen sedimented on the packed red blood cells were measured to the nearest 0.1 mm and expressed as volumes per milliliter.RNA Preparation and RT-PCR
Individual livers were homogenized in guanidinium isothiocyanate using a Polytron homogenizer (Kinematica, Switzerland), and total RNA was extracted according to the method of Chomczynski and Sacchi (8). The amount of RNA was determined spectrophotometrically. Two micrograms of RNA from each sample were subjected to first-strand cDNA synthesis using oligo(dT) primer and 100 µl murine Maloney leukemia virus (MMLV). Samples were incubated at 37°C for 60 min. To test the efficacy of reverse transcriptase, RT-PCR was performed forLight Microscopy
The tissue was fixed in 10% neutral buffered Formalin and stained with hematoxylin and eosin (H + E). Ten specimens from each group were reviewed by a Children's Hospital of Pittsburgh pathologist (M. Parizhkaya) who was blinded to the treatment group.Transmission Electron Microscopy
The portal vein was cannulated, and retrograde fixation of the liver was performed with 2.5% gluteraldehyde. The liver was fixed in 2.5% gluteraldehyde overnight and then washed with 0.1 mol/l PBS. The tissue was sectioned in 1-mm blocks and postfixed for 6 h in 1% osmium tetroxide, dehydrated through graded alcohols, and embedded in epoxy resin (Epon) (Energy Beam Sciences, Agawam, MA). After embedment, thin sections (60 nm) were cut by a microtome (Reichert Ultracut S, Lieca, Deerfield, IL), mounted on copper grids, counterstained with 2% uranyl acetate for 10 min and 1% lead citrate for 7 min, dried, and analyzed using a transmission electron microscope (TEM; JEOL 100CX). Thick sections were cut (300 nm) and stained with 1% toluidine blue. The TEM pictures were reviewed by two investigators (D. Beer-Stolz and S. C. Watkins) who were blinded to the experimental groups.TUNEL Assay
The TdT-mediated dUTP nick end labeling (TUNEL) protocol was conducted as per standard published procedure and is briefly described below (10). After the liver specimens were sectioned, three washes in PBS were performed, followed by fixation in cold methanol for 30 min. The specimens were washed twice more in PBS. Ten microliters of terminal transferase (TdT) reaction mixture containing cobalt chloride and biotinylated dUTP were added to the slides and incubated at 37°C for 90 min. Slides were then washed with PBS three more times and labeled with Streptavidin-conjugated Alexa 488 (Molecular Probes, Eugene, Oregon) for detection of DNA strand break. Gelvatol (1% gelatin) (Monsata, St. Louis, MO) was then added, and the slides were coverslipped.Immunocytochemical Labeling for Kupffer cells,
TNF-, and iNOS
Determination of Neutrophil Infiltration in the Liver
Sections of liver were fixed in 2% paraformaldehyde and 30% sucrose, and 5-µm sections were cut with the cryostat. The tissue was fixed on slides with 2% paraffin. The slides were incubated in 1% hydrogen peroxide for 2 min and then washed three times with PBS. Hanker-Yates (Sigma, St. Louis, MO) solution was added to the sections for 30 min, and they were then washed with PBS. Gelvatol was added and the slides were coverslipped. Neutrophils were identified by light microscopy at ×40 magnification and quantitated.Statistical Analysis
Data are presented as means ± SE. Analyses were discrete comparisons between groups, and significance was determined using the unpaired Student's t-test for normally distributed data. A P value of <0.05 was considered statistically significant. ![]() |
RESULTS |
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EPR Spectrometry
To confirm that NOX scavenged excess NO produced in the liver, we evaluated liver samples from rats challenged with LPS for the presence of nitrosylated hemoglobin or mononitrosyl-iron-NOX complexes by EPR. Mixture of the NO donor SNAP, FeSO4, and NOX in vitro resulted in a characteristic mononitrosyl-iron spectrum by EPR (Fig. 1A). Specimens from rats challenged with LPS and treated with NS consistently demonstrated the presence of nitrosylated hemoglobin, but without the mononitrosyl-iron complex. The strongest nitrosylated hemoglobin signals were detected in rats killed at 6 or 8 h after LPS administration (Fig. 1B). In contrast, specimens from NOX-treated rats showed marked suppression or total absence of the nitrosylated hemoglobin signal and an EPR spectrum similar to that generated by the mixture of SNAP, FeSO4, and NOX (Fig. 1C). The standard solution had a calculated g value of 2.037 ± 0.010, whereas the signal in the NOX-treated rats had a g value of 2.030 ± 0.010. These two values are equal within experimental error.
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Effect of NOX on LPS-Mediated Hepatocellular Injury
Hepatocellular injury was evaluated in vivo by measuring the serum levels of AST and OCT. Endotoxin administration resulted in increased AST levels in animals receiving NS compared with controls (P < 0.001, Student's t-test); however, this effect was substantially diminished by NOX pretreatment (Table 1). OCT is an enzyme that is located in the mitochondria of hepatocytes and is highly specific for hepatocellular injury. Endotoxemia also resulted in a notable increase in OCT levels over control values (P = 0.002, Student's t-test); NOX administration reduced serum OCT to near baseline (Table 1).
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In a separate set of experiments, we attempted to determine whether NOX
could prevent hepatocellular injury when administered after LPS
challenge (Table 2). There was a trend
toward decreased AST levels (P = 0.10) when NOX was
administered 4 h after LPS challenge. By 8 h, NOX was
ineffective in preventing AST elevation. Although OCT levels after LPS
challenge were slightly higher in this set of experiments, NOX was
effective in reducing serum OCT levels when administered 4 h, but
not 8 h, after LPS challenge (Table 2).
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Hepatocellular injury after endotoxemia may be associated with decreased serum glucose and impaired gluconeogenesis (21). LPS-challenged animals had serum glucose levels that were significantly decreased compared with those found in control animals (P = 0.002). NOX pretreatment reduced the fall in serum glucose (Table 1). Because endotoxemia has been shown to increase levels of fibrinogen and the acute-phase reactants (47, 50), we measured serum fibrinogen levels 24 h after LPS challenge. Fibrinogen levels were significantly elevated in saline-treated animals challenged with LPS compared with controls (P = 0.001). NOX pretreatment significantly reduced serum fibrinogen levels after endotoxemia (Table 1).
Effect of NOX on Expression of Proinflammatory Mediators
Inflammatory mediators are released in response to LPS challenge and may impair hepatic function (6). We examined iNOS, IL-6, and TNF-
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Immunohistochemical analysis of freshly harvested sections of
liver from animals in both groups revealed that iNOS protein was found
in Kupffer cells and in hepatocytes in all LPS-challenged animals
regardless of treatment. There was no difference in expression of iNOS
protein between the NOX and NS groups 24 h after LPS challenge (data not shown). Immunohistochemical analysis revealed that control animals that did not receive LPS demonstrated low baseline levels of
TNF- expression (Fig. 2A).
At 24 h after LPS challenge, significant TNF-
expression can be
detected in the NS-treated group (Fig. 2B). However,
NOX-treated animals show relatively little expression of TNF-
(Fig.
2C).
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Morphological Analysis
Random sections of liver obtained from NS-treated and NOX-treated rats 24 h after LPS challenge were stained with H + E and examined by light microscopy. Patchy areas of hepatocellular necrosis, accompanied by hemorrhage and neutrophil infiltration, were detected in both NS-treated and NOX-treated animals without any apparent difference between the two groups (data not shown).When examined by TEM, the necrotic areas from NS-treated rats after LPS
challenge revealed swelling of the mitochondria and endoplasmic
reticulum, obliteration of the hepatic sinusoids, and necrosis of
endothelial cells and hepatocytes (Fig.
3D). We also examined
normal-appearing areas by H + E staining in the same liver using
TEM. The space of Disse, an area that is normally filled with
extracellular matrix, is important for communication between
hepatocytes and sinusoids. In the NS group, the space of Disse was
significantly reduced because of an increased number of microvilli on
the basolateral surfaces of the hepatocytes (Fig. 3C). These
changes were not evident in the NOX-treated (Fig. 3B) or
control (Fig. 3A) groups.
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Neutrophil and Kupffer Cell Response to LPS Challenge
Neutrophils and Kupffer cells have been implicated in hepatocellular injury after LPS challenge. Kupffer cells were identified by immunohistochemistry. At 24 h there were few Kupffer cells in the control animals (Fig. 2D) and the NOX-treated group (Fig. 2F). The NS-treated rats had a significant increase in the number of Kupffer cells (Fig. 2E). Histochemical staining for myeloperoxidase showed relatively few neutrophils in the liver under normal physiological conditions (Fig. 4A). Endotoxemia resulted in a significant increase in neutrophils in the hepatic sinusoids of the NS-treated group (Fig. 4B), but not in the NOX-treated group (Fig. 4C). Quantitative analysis showed 2.7 ± 0.6 neutrophils per field (40× magnification) in the control animals and 3.2 ± 1.8 in the NOX-treated group, compared with 8.9 ± 2.4 neutrophils per field (40× magnification) in the NS-treated group (P < 0.001).
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To determine if apoptosis plays a role in the mechanism of hepatocellular injury after LPS challenge, we qualitatively measured apoptosis in the liver by the TUNEL assay. There was no increased rate of apoptosis in the liver in animals treated with NS after LPS challenge (Fig. 2, G and H), suggesting that necrosis was the predominant mechanism of injury. NOX administration had no effect on the low rate of apoptosis (Fig. 2I).
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DISCUSSION |
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Multiorgan dysfunction represents a major cause of morbidity and
mortality in sepsis (11). Numerous studies suggest that proinflammatory mediators such as TNF-, IL-6, or NO may play an
important role in the systemic response to infections in general, and
in hepatic dysfunction during sepsis in particular (15, 28,
56). The present study demonstrates that NO mediates
hepatocellular injury after endotoxic shock. Administration of the NO
scavenger, NOX, reduced hepatocellular injury after LPS challenge as
evidenced by a reduction in serum levels of the liver enzymes OCT and
AST. These findings were associated with diminished Kupffer cell
proliferation, decreased neutrophil infiltration, and attenuation of
morphological changes seen on TEM, although not by light microscopy.
There was a concomitant decrease in the mRNA levels of IL-6 and in
immunoreactivity to TNF-
. The protective effect of NOX on serum
liver enzymes was seen even when administered up to 4 h after the
onset of sepsis.
NOX is a 300-Da water-soluble dithiocarbamate derivative that chelates reduced iron and binds NO in vitro and in vivo (9, 23, 27). Several classes of dithiocarbamate derivatives that differ in their NO binding properties exist, including diethyldithiocarbamate, which is hydrophobic, as well as N-methyl-D-glucamine dithiocarbamate (MGD) and proline dithiocarbamate, which are hydrophilic (26, 34, 40). Dithiocarbamate-iron complexes effectively bind NO, resulting in the formation of a paramagnetic mononitrosyl-iron complex that can be detected by EPR (26, 27). NOX, which most closely resembles MGD in its physical and biochemical properties, is less likely to bind peroxynitrite or superoxide in vitro (44). We used EPR to verify that NOX scavenged excess NO produced in the liver. After LPS challenge, liver sections from rats treated with NS demonstrated the characteristic nitrosylated hemoglobin signals by EPR. However, specimens from NOX-treated rats revealed diminution or absence of the nitrosylated hemoglobin signal and formation of a characteristic mononitrosyl-iron dithiocarbamate signal. The morphology of the EPR spectra in NOX-treated rats is similar to that of other mononitrosyl-iron dithiocarbamate signals published by other investigators (34), which suggests that NOX indeed acted as a NO scavenger in our system. When dissolved in water, NOX produces a yellow solution that interferes with the Greiss reagent, which detects nitrite and nitrate by a colorimetric reaction; thus we did not assess NO production in the serum (9).
NO production is normally regulated by three isoforms of NO synthase
(NOS). NOS-1 (nNOS) and NOS-3 (eNOS) are calcium dependent and are
produced constitutively in tissues at low levels. Inhibition of eNOS
leads to decreased hepatic perfusion and increased hepatocellular injury in a model of hemorrhagic shock (14). NOS-2 (iNOS)
is calcium independent and may be induced in large quantities by inflammatory stimuli, including LPS (2, 53). Small
quantities of NO derived from eNOS may exert a protective role in the
liver by 1) preserving hepatic arterial and portal blood
flow (42, 43), 2) preventing inflammation in
the hepatic microcirculation (37), or 3)
inhibiting reactive oxygen intermediate release and limiting
TNF--mediated liver injury (4, 20, 24). However, excess
NO produced in inflammation may be deleterious, as we have previously
shown in the gut. Although there have been conflicting reports
regarding the role of NO in hepatocellular damage, our findings
corroborate those of several authors who have shown that NO, or its
reactive nitrogen intermediates, may promote liver injury after
endotoxemia, ischemia/reperfusion, or hemorrhagic shock
(32, 33, 36). Mustafa et al. (36) used
platelet-activating factor receptor antagonists to inhibit NO formation
and prevent hepatic injury in LPS-challenged livers and in Kupffer cell
culture. Menezes et al. (33) showed that NOX prevented
hepatic dysfunction in a model of hemorrhagic shock. Although these
authors did not show that the NOX effect was due to local scavenging of
NO in the liver, NOX reduced serum OCT elevation when infused
continuously during the hypotensive insult. The hepatocellular injury
attributed to NO may be due to its direct cytotoxicity or its
diffusion-limited reaction with superoxide to produce the toxic
nitrogen metabolite peroxynitrite (45). Ma et al.
(32) pretreated mice with endotoxin to induce hepatic NO
production before ischemia/reperfusion, which resulted in
increased hepatocellular injury, implicating peroxynitrite as a
causative agent (32). However, we could not demonstrate evidence of peroxynitrite-mediated injury in our study because of
marked background autofluorescence in the liver tissue. Nonetheless, given this limitation, we cannot exclude the possibility that NOX
prevented hepatocellular injury by decreasing peroxynitrite generation,
consistent with our observations in the rat intestine after LPS
challenge (9, 51).
The mechanism of NO-mediated hepatocellular injury also remains somewhat controversial. Early reports suggested that LPS-induced hepatic dysfunction was primarily due to necrosis rather than apoptosis (56). However, Redmond et al. (46) used LPS in conjunction with antioxidants to induce hepatocellular apoptosis. Inhibition of NO production reduced both hepatocyte necrosis and apoptosis in this model. Wang et al. (57) confirmed these results by illustrating that the NO donor, SNAP, could induce hepatocellular apoptosis. However, in the presence of reactive oxygen intermediates, NO led to hepatic necrosis. In our study, LPS challenge induced hepatic injury via necrosis rather than apoptosis. We were unable to detect apoptosis in the liver in any of our treatment groups. By light microscopy, LPS challenge resulted in variable areas of necrosis, predominantly in the midzonal region of the hepatic lobule, in both NS-treated and NOX-treated rats at 24 h. It is possible that morphological changes may have been detected between the groups by light microscopy had we been able to study later time points (48 h), because in NS-treated rats, even areas that appeared to be normal by light microscopy (at 24 h) revealed significant abnormalities when examined by TEM. The space of Disse, the region between hepatocytes and sinusoidal endothelial cells, is an area that is normally filled with extracellular matrix. The spaces are vital to hepatocyte and endothelial cell function because they modulate cell growth, differentiation, migration, and apoptosis (19). These spaces are obscured in liver sections from rats challenged with LPS and treated with NS by an increased number of microvilli on the basolateral surfaces of the hepatocytes. Similar changes have been noted after massive hepatectomy in regenerating livers (52). Compared with control rats, NOX-treated rats did not show obliteration of the space of Disse after LPS challenge, although there were no demonstrable differences by routine H + E staining.
Both TNF- and IL-6 have been implicated in hepatocellular
dysfunction and necrosis associated with sepsis (48, 49,
58). Our data show that NOX downregulates IL-6 mRNA expression
and TNF-
protein production in the liver (although TNF-
mRNA was only mildly suppressed). These findings are consistent with those of
Hierholzer et al. (16), who demonstrated that upregulation of these cytokines in hemorrhagic shock was dependent on iNOS (16). The decreased levels of TNF-
and IL-6 in animals
receiving NOX may reflect in part a decrease in Kupffer cell
proliferation or a decrease in neutrophil infiltration because both
types of cells are capable of secreting these inflammatory mediators
(29). Furthermore, neutrophils have been shown to promote
hepatocellular injury in vitro and in vivo (17). In our
study, we observed a threefold increase in neutrophils in the hepatic
sinusoids of NS-treated rats after LPS challenge. Again, NOX abrogated
this effect. We cannot exclude the possibility that posttranscriptional modification of TNF-
mRNA in the NOX-treated animals resulted in
decreased TNF-
protein production, although we did not test this
hypothesis. Alternatively, because NOX is a dithiocarbamate that binds
nuclear factor-
B, it may block signaling via this transcription
factor, which has been shown to upregulate both TNF-
and IL-6
production (1, 22). In contrast to TNF-
and IL-6,
neither iNOS mRNA nor protein expression was affected by NOX. However,
these findings are not surprising because a NO scavenger would not be
expected to affect iNOS mRNA or protein expression. The EPR data are
consistent with NOX acting as a NO scavenger in the liver.
In addition to decreased serum liver enzyme levels and morphological damage, further evidence that NOX maintained hepatocyte viability during endotoxemia includes decreased serum fibrinogen levels and preservation of normal serum glucose levels. Serum fibrinogen levels were increased during endotoxemia, similar to observations in humans (5, 47). NOX administration reduced fibrinogen levels, although not to baseline. Glucose levels are normally decreased during sepsis (21). Titheradge et al. (54) reported that the inhibition of gluconeogenesis after LPS challenge is the result of inhibition of phosphoenolpyruvate carboxykinase due to the sustained production of NO (54). Thus NOX therapy may reduce synthesis of the acute-phase proteins and improve gluconeogenesis.
In conclusion, our data corroborate previous reports suggesting that
excess NO production after LPS challenge results in increased hepatocellular injury. Hepatic injury was associated with sustained upregulation of iNOS, TNF-, and IL-6 mRNA, as well as increased serum fibrinogen levels and decreased serum glucose. The novel NO
scavenger NOX attenuated LPS-induced hepatocellular dysfunction. Thus
NOX may have important implications for the treatment of septic shock
and other inflammatory conditions associated with sustained production
of NO.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grant RO1-AI-14032 and the Benjamin R. Fisher Endowed Chair in Pediatric Surgery.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. R. Ford, Children's Hospital of Pittsburgh, 3705 Fifth Ave., Pittsburgh, PA 15213 (E-mail: FordH{at}chplink.chp.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.
Received 8 May 2000; accepted in final form 30 January 2001.
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