1 Department of Pathology, The University of Hong Kong and Queen Mary Hospital and 2 Centre for the Study of Liver Diseases and 7 Department of Anatomy, The University of Hong Kong, Hong Kong; 3 Department of Pathology, Harvard Medical School, Boston 02115; and 6 Department of Surgery, Boston University School of Medicine, Boston, Massachusetts 02118; 4 Research Unit of Alcohol Diseases, Helsinki University Central Hospital, Helsinki, Finland; 5 Department of Nutrition, McGill University, Montreal, Quebec, Canada H9X 3V9; 8 Department of Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109; and 9 Department of Medicine, Weill Medical College, Cornell University and Anne Fisher Nutrition Center, Strang Cancer Prevention Center, New York, New York 10021
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ABSTRACT |
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Alcoholic liver
injury is more severe and rapidly developing in women than men.
To evaluate the reason(s) for these gender-related differences, we
determined whether pathogenic mechanisms important in alcoholic liver
injury in male rats were further upregulated in female rats. Male and
age-matched female rats (7/group) were fed ethanol and a diet
containing fish oil for 4 wk by intragastric infusion. Dextrose
isocalorically replaced ethanol in control rats. We analyzed liver
histopathology, lipid peroxidation, cytochrome P-450
(CYP)2E1 activity, nonheme iron, endotoxin, nuclear factor-B (NF-
B) activation, and mRNA levels of cyclooxygenase-1 (COX-1) and
COX-2, tumor necrosis factor-
(TNF-
), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2). Alcohol-induced liver injury was more severe in female vs. male rats.
Female rats had higher endotoxin, lipid peroxidation, and nonheme iron
levels and increased NF-
B activation and upregulation of the
chemokines MCP-1 and MIP-2. CYP2E1 activity and TNF-
and COX-2
levels were similar in male and female rats. Remarkably, female rats
fed fish oil and dextrose also showed necrosis and inflammation. Our
findings in ethanol-fed rats suggest that increased endotoxemia and
lipid peroxidation in females stimulate NF-
B activation and
chemokine production, enhancing liver injury. TNF-
and COX-2
upregulation are probably important in causing liver injury but do not
explain gender-related differences.
cytokines; cyclooxygenase-2; tumor necrosis factor-
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INTRODUCTION |
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WOMEN DEVELOP ALCOHOL-INDUCED liver injury more rapidly than men (5, 33, 55). Additionally, the progression of liver injury is greater in women with alcoholic hepatitis who stop or reduce drinking (40, 50). The basis for this increased sensitivity to alcohol-induced liver injury in women is uncertain, but gender-dependent differences in absorption, disposition, and metabolism of alcohol have been postulated to be important (11, 39, 56). Gender-related differences in pharmacokinetics of alcohol lead to higher levels of ethanol in women after drinking (55). Differences in alcohol elimination rates also increase the plasma levels of acetaldehyde in women (55). Higher levels of endotoxin and proinflammatory cytokines are detected in livers of female ethanol-fed rats compared with male rats (28). Estrogen has been implicated as one factor that helps to explain the enhanced severity of alcoholic liver injury in females (37). For example, ovariectomized female rats fed ethanol develop less severe liver injury than normal female rats; estrogen replacement increases the severity of pathology in ovariectomized rats to levels observed in normal female rats.
The establishment of the continuous intragastric in vivo feeding model represents a major advance in terms of relating biochemical and physiological alterations to pathological changes in alcohol-induced liver injury (17, 62, 64). Several potential pathogenic mechanisms have been identified that are important in alcoholic liver injury in male rats. The goal of the present study was to determine whether these same mechanisms contribute to the enhanced severity of alcoholic liver injury in female rats.
Several lines of investigation (3, 27, 45, 61) indicate that endotoxemia and oxidative stress are important pathogenic mechanisms in alcohol-induced liver injury. It is well known that endotoxin is hepatotoxic and that concentrations of endotoxin are increased in experimental and human alcoholic liver disease (1, 19, 42). Lipid peroxides are also potentially hepatotoxic, and increased levels are detected after alcohol administration (13, 30, 52). Endotoxin and lipid peroxidation are believed to act in a concerted manner to promote alcoholic liver injury. It was important, therefore, to compare levels of endotoxin and oxidative stress in male and female rats.
One factor that might link endotoxemia, oxidative stress, and liver
injury is the transcription factor nuclear factor-B (NF-
B) (6, 38). NF-
B can stimulate the expression of a variety of genes, including those believed to be important in alcoholic liver
injury (7, 8, 41). For example, activation of NF-
B and
increased expression of tumor necrosis factor-
(TNF-
) and cyclooxygenase-2 (COX-2) were detected in Kupffer cells in alcohol-fed male rats exhibiting necroinflammatory changes (43, 47).
Recently, Kono et al. (28) detected elevated levels of
endotoxin and NF-
B binding activity and increased CD14 expression in
female rats fed ethanol. Elevated levels of cytochrome P-450
(CYP)2E1 and nonheme iron may also contribute to ethanol-induced
oxidative stress (2, 64). Ethanol induces CYP2E1, and
CYP2E1 levels correlate with the degree of lipid peroxidation
(2). Furthermore, chronic ethanol administration leads to
increased concentrations of nonheme iron in the liver, and the highest
levels are seen in rats with pathological liver injury
(54).
On the basis of prior studies in male rats (18, 42), we postulated that the enhanced susceptibility of females to alcohol-induced liver injury could be a consequence of higher levels of previously investigated inciting factors such as oxidative stress and endotoxemia. In the present work, the intragastric feeding model was used to investigate this possibility. The severity of necrosis, inflammation, and fibrosis was greater in ethanol-fed female than male rats. Gender-dependent differences in pathological injury were accompanied by higher levels of endotoxin, lipid peroxidation, and the chemokines monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2). Interestingly, female dextrose-fed rats developed changes in the liver resembling those seen in nonalcoholic steatohepatitis (NASH).
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MATERIALS AND METHODS |
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Animals.
Male (275-300 g) and age-matched female Wistar rats (200-215
g) were fed a liquid diet by continuous infusion through permanently implanted gastric tubes, as described previously (17, 63). The rats were given their total nutrient intake by intragastric infusion. The diet provided 250 kcal · kg1 · day
1 and was
modified to include a salt and vitamin mix (Dyets, Bethlehem, PA)
(18). The source of protein was lactalbumin. The
percentage of total calories derived from fat (fish oil) was 35%. The
diet was supplemented with choline and methionine. The amount of
ethanol was initially 10 g · kg
1 · day
1 and was
increased to 16 g · kg
1 · day
1 as tolerance
developed. Each ethanol-fed rat had at least two measurements of blood
alcohol; all samples for blood alcohol were obtained at 10 AM.
Histopathological analysis, including Sirius red staining for
collagen.
A small sample of liver was obtained and formalin fixed when the rats
were killed. Hematoxylin and eosin stain was used for light microscopy.
The severity of liver pathology was assessed as follows: steatosis (the
% of liver cells containing fat), 1+, 25% of cells; 2+,
26-50% of cells; 3+, 51-75% of cells; and 4+, >75% of
cells. Necrosis was quantified as the number of necrotic foci per
square millimeter, and inflammation was scored as the number of
inflammatory cells per square millimeter. At least three different
sections were examined per sample of liver. The pathologist evaluating
these sections was unaware of the treatment the rats had received.
Measurement of blood alcohol levels. Blood was collected from the tail vein. Ethanol concentration was then measured using the alcohol dehydrogenase kit from Sigma Chemical (St. Louis, MO).
Measurement of plasma endotoxin levels. Blood samples were collected in endotoxin-free vials (Sigma Chemical) and centrifuged at 400 g for 15 min at 4°C. Samples were then diluted 1:10 in pyrogen-free water and heated to 75°C for 30 min to remove inhibitors of endotoxin from plasma. The limulus amoebocyte lysate test (Kinetic-QLC; Whittaker Bioproducts, Walkersville, MD) was used for measurements of endotoxin. Samples were incubated at 37°C for 10 min with limulus amebocyte lysate. The substrate solution was added, and the incubation was continued for 20 min. The reaction was stopped with 25% acetic acid. Samples were read spectrophotometrically at 410 nm.
Determination of thiobarbituric acid-reactive substances. The levels of liver thiobarbituric acid-reactive substances (TBARS) were measured according to the method of Ohkawa et al. (49). Briefly, 0.2 ml SDS (8.1%), 1.5 ml 20% acetic acid, and 1.5 ml 0.8% thiobarbituric acid were added to 200 µl liver homogenate. After addition of distilled water, the tubes were vortexed and placed in boiling water for 1 h. The reaction was stopped by immersion of tubes in a cold water bath. After addition of 15:1 (vol/vol) butanol-pyridine and centrifugation, the upper phase was removed, and absorbance at 532 nm was determined. Butylated hydroxytoluene (90 µmol/l) was added to prevent the formation of TBARS in vitro.
Measurement of conjugated dienes. Conjugated dienes in the total lipid extracted from liver homogenates were quantified by measurements of optical density between 220 and 300 nm, as described previously (52).
Determination of nonheme iron.
Nonheme iron was determined in liver homogenate, with Ferene S, as an
indicator with the molar absorptivity of 35,500 M1 · cm
1 at 594 nm4.
The liver was homogenized in NaC1 solution (7 mM NaC1/100 mg tissue)
and centrifuged at 1,000 g for 10 min. The clear supernatant (150 µl) was mixed with distilled H2O (150 µl) and 150 µl of thiourea-ascorbate solution (4.4% and 2.68%, respectively, in
distilled H2O). TCA (150 µl of 40% solution )
was added to the mixture, vortexed, and centrifuged for 30-60 s.
The supernatant (500 µl) was then mixed with 125 µl of fresh Ferene
S solution (35 mg Ferene S in 10 ml of 50% ammonium acetate solution).
The mixture was incubated at room temperature for 5-10 min, and
the absorbance was read at 594 nm. Control experiments were carried out
to ensure that the measured nonheme iron was not from nonspecific iron
released from ferruginous compounds during the procedures.
Determination of aniline hydroxylase activity. Aniline hydroxylase assays were performed as described previously (44), according to the method of Imai et al. (23).
Determination of NF-B.
Electrophoretic mobility shift assays were used to determine the
binding activity of NF-
B and were performed essentially as described
previously (26, 31, 32). Equal amounts of protein were
incubated with a 5' 32P-labeled oligonucleotide containing
an NF-
B consensus site. The incubation mixtures were separated on a
7% nondenaturing polyacrylamide gel, and bands were detected by
autoradiography. The specificity of binding was determined by prior
addition of 100-fold excess of unlabeled competitor consensus
oligonucleotide. Supershift experiments were performed using antiserum
directed against the p50 subunit of NF-
B (Santa Cruz Biotechnology,
Santa Cruz, CA).
Analysis of mRNAs for COX-1, COX-2, TNF-, MCP-1, MIP-2,
cytokine-induced neutrophil chemoattractant, and
-actin by RT-PCR.
To examine the expression of COX-1, COX-2, TNF-
, MCP-1, MIP-2,
cytokine-induced neutrophil chemoattractant (CINC), and
-actin in
liver tissue, we isolated total RNA according to the guanidinium isothiocyanate method (14). The integrity of RNA was
assessed by agarose gel electrophoresis and ethidium bromide staining. RT-PCR was performed essentially as previously described
(43). The sequences of primer pairs, 5' and 3', and
predicted sizes of the amplified PCR fragments of COX-1, COX-2,
TNF-
, MCP-1, MIP-2, CINC, and
-actin have been reported
previously (41, 43, 44, 46). PCR products and molecular
weight markers were subjected to electrophoresis on 1% agarose gels
and visualized by means of ethidium bromide staining. The gels were
analyzed by laser scanning densitometry using a densitometer and
ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Each
experiment included a negative control (sample RNA that had not been
subjected to RT). This sample did not yield a PCR product, confirming
the absence of extraneous genomic DNA or PCR products contaminating the
samples. Varying the number of PCR cycles did not change the relative
differences between the samples, indicating that the PCR conditions
were not within the plateau phase of amplication. All amplification
reactions of one experiment were performed in parallel in the same
heating block to ensure compatible conditions.
Measurement of cytokines in plasma.
TNF- and MIP-2 were assayed by the solid phase enzyme-linked
immunosorbent technique according to the manufacturer's suggested protocol (Biosource, Camarillo, CA). Samples were compared with the
standard curve. The cross-reactivity with other cytokines was <10% (Biosource).
Statistical analysis. All data are expressed as means ± SD unless otherwise indicated. Differences between groups were analyzed using ANOVA with post hoc analysis using the Bonferroni test.
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RESULTS |
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The weight gain over the 4-wk period in male and female rats was 34 ± 5, 29 ± 4, 20 ± 3, and 18 ± 2 g for MFD, MFE, FFD, and FFE, respectively. Although the weight gain was numerically smaller in female compared with male rats, the weight gain as a percentage of the original body weight was similar among the groups. The blood alcohol levels (in means ± SE) were similar in the ethanol-fed groups: 231 ± 26 and 220 ± 31 mg/dl for MFE and FFE, respectively. The amount of alcohol required to achieve these levels was similar in the groups. In the first week of ethanol administration, the daily intake of ethanol in the two groups was 11.5 ± 0.9 and 11.3 ± 0.7 g/kg body wt for MFE and FFE, respectively. During the subsequent 3 wk, the daily ethanol intake was 15.2 ± 0.7 and 14.7 ± 1.1 g/kg body wt for MFE and FFE, respectively.
The most severe pathology was seen in the FFE rats (Table
1). In particular, the degree of necrosis
and inflammation was much more severe in FFE than in MFE rats (Fig.
1, A and B). Also, fatty liver, necrosis, and inflammation were observed in FFD but not in
MFD rats (Fig. 1, C and D). The inflammatory
cells were mainly mononuclear cells with occasional neutrophils. The
degree of fibrosis (collagen and pericellular fibrosis) was also more severe in female rats compared with male rats. In fact, bridging fibrosis was seen in four of the seven FFE rats, but only in one of the
seven MFE rats (Fig. 2).
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The highest levels of lipid peroxidation and endotoxin were seen in the
FFE group (Table 2). Levels of endotoxin
were also higher in FFD compared with MFD rats. Ethanol induced CYP2E1
activity (aniline hydroxylase) in both male and female rats but
gender-dependent differences were not observed. Levels of nonheme iron
were significantly higher in FFE rats compared with MFE rats.
Furthermore, the levels of nonheme iron in FFD rats were similar to
those seen in MFE rats and higher than those in MFD rats
(P < 0. 05) (Table 2).
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Activation of NF-B correlates with pathological liver injury.
To evaluate activation of NF-
B, electrophoretic mobility shift
assays of nuclear extracts from whole liver were carried out. NF-
B
binding activity was increased in the FFD group and both ethanol-fed
groups. Furthermore, the degree of NF-
B activation was greater in
female ethanol-fed rats compared with male ethanol-fed rats. (Fig.
3A). The protein-DNA complex
was further characterized by using competition and supershift
assays. Addition of a 100-fold excess of unlabeled oligonucleotide
containing an NF-
B consensus site abrogated complex formation, while
addition of an oligonucleotide containing signal transducer and
activator of transcription 5 had no effect (Fig. 3B).
Supershift analysis identified p50 protein in the binding complex. In
association with increased NF-
B binding activity, reduced levels of
IkB
were detected (Fig. 3A).
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Chemokine expression is greater in female vs. male ethanol-fed
rats.
Based on a previous study (41) showing increased chemokine
expression in experimental alcoholic liver disease, we hypothesized that alterations in chemokine expression in the liver might contribute to the differences in the severity of pathological changes between male
and female ethanol-fed groups. MCP-1 mRNA was significantly increased
(P < 0.01) in MFE compared with MFD rats; the level in
female ethanol-fed rats was higher than in male ethanol-fed rats
(P < 0.01) (Fig. 4).
Levels of MIP-2 mRNA were similarly increased in male ethanol-fed rats
compared with dextrose-fed controls (P < 0.01), with
much higher levels found in female ethanol-fed rats (P < 0.01). The levels of MIP-2 protein in plasma mirrored the levels of
MIP-2 mRNA in the livers of the different experimental groups (Fig. 4).
Also of interest is that levels of MIP-2 mRNA and protein were higher
in female dextrose-fed rats compared with their male counterparts.
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Expression of TNF- and COX-2 correlates with inflammatory
changes, endotoxemia, and lipid peroxidation but does not explain
gender-dependent differences.
We (43) previously proposed that endotoxin and lipid
peroxidation induce TNF-
and COX-2 in alcohol-induced liver injury. An effort was made to measure mRNAs for TNF-
and COX-2 by Northern blot and RNase protection assays, but levels of both mRNAs were too low
to be detected by these analyses (46). Therefore, RT-PCR was used for these measurements. Data for TNF-
and COX-2 are shown
in Table 3. Elevated levels of both
TNF-
and COX-2 mRNAs were detected only in rats that exhibited
necrosis and inflammation (MFE, FFD, and FFE). The increase in TNF-
mRNA was accompanied by increased levels of TNF-
protein in plasma;
the highest levels were seen in rats fed ethanol (Table 3). Remarkably,
FFD rats also had increased TNF-
and COX-2 expression, which
correlated with the presence of necroinflammatory changes. Although
both TNF-
and COX-2 were upregulated in the livers of rats
exhibiting necroinflammatory injury, the expression of COX-2 and
TNF-
was not different in male vs. female ethanol-fed rats.
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DISCUSSION |
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The pathological severity of alcohol-induced liver injury in female rats was much greater than in male rats and correlated with increased levels of endotoxin, lipid peroxidation, and chemokines. It is also of considerable interest that FFD rats exhibited necrosis and inflammation. Although the exact cellular mechanism(s) responsible for enhanced liver injury in female rats is unknown, our results provide potentially significant insights.
Important factors in gender-related differences in alcohol-induced
pathological changes.
Several measured parameters were significantly different between male
and female rats and potentially account for the enhanced severity of
liver injury in female rats. These factors included endotoxin, lipid
peroxidation, and chemokines. TNF- and COX-2 were similar in both
male and female ethanol-fed rats. The potential contribution of each of
these factors to liver injury is discussed below.
Endotoxin and lipid peroxidation. Levels of endotoxin in plasma were higher in female than in male rats. This finding is consistent with the results of Iimuro et al. (21) in rats fed ethanol with corn oil. The mechanisms responsible for endotoxemia after alcohol administration include increased gut permeability and decreased hepatic clearance of endotoxin (45). Because estrogen receptors exist in the intestinal epithelium, estrogen could affect the permeability of the gut and in turn lead to higher endotoxin levels in female rats (60). Lipid peroxidation in alcohol-fed animals is believed to be a consequence of enhanced oxidative stress due to increased CYP2E1 activity and free iron as well as decreased amounts of antioxidants and antioxidant enzymes (27, 45). Lipid peroxidation was increased in females compared with males and was associated with higher levels of free iron but not CYP2E1 activity. The reason for higher levels of free iron in female rats is unknown, but increased iron absorption has been proposed as one factor (51). The concentration of polyunsaturated fatty acids is higher in female than male rat liver (12), which potentially provide the substrate for enhanced lipid peroxidation. Other factors that could contribute to enhanced levels of lipid peroxidation in females, such as decreased levels of antioxidants, were not evaluated.
Activation of NF-B is associated with inflammatory changes in
liver.
One factor that might link endotoxemia, oxidative stress, and liver
injury is NF-
B (6-8). In many cells, including
those in the liver, NF-
B is found in an inactive form in the
cytoplasm bound to an inhibitory protein, I
B (25). In
response to activating signals, the inhibitory protein is degraded by
the proteosome and NF-
B translocates to the nucleus. Activation of
NF-
B was observed in MFE, FFD, and FFE rats, all of which exhibited
necroinflammatory changes (Fig. 3). NF-
B activation was greater in
female ethanol-fed rats compared with male ethanol-fed rats. These
observations are in agreement with those of Kono et al.
(28).
Increased chemokine expression is associated with enhanced liver injury in female ethanol-fed rats. Chemokines are generally divided into C, CC, CXC, and CX3C subfamilies based on their NH2-terminal cysteine motifs (20, 24). A more recent classification (53) uses physiological features to distinguish between inflammatory and homeostatic cytokines. Most inflammatory chemokines are upregulated in nonlymphoid tissue under inflammatory conditions and recruit effector T cells (20). Several types of liver cells can produce chemokines; these cells include hepatocytes, Kupffer cells, biliary cells, and hepatic stellate cells. Of note is that exposure of Kupffer cells to conditioned media from hepatocytes results in chemokine production (10, 36). We (41) and others (9, 10, 36) have previously shown that the chemokines MCP-1 and MIP-2 are upregulated in the presence of alcohol-induced necroinflammatory changes in the liver. It was therefore of interest to determine whether these inflammatory chemokines could explain, at least in part, gender-related differences in the severity of alcoholic liver injury. In this study, we show that levels of MCP-1 and MIP-2 are higher in female ethanol-fed rats compared with males. In addition to provoking an inflammatory response, MIP-2 is cytotoxic to hepatocytes previously exposed to ethanol in vivo (9). Chemokines can also stimulate the release of reactive oxygen species from Kupffer cells and neutrophils (10, 62). CINC, another chemokine of importance in alcoholic liver injury, induces neutrophil accumulation and hepatocyte damage in liver (36). Mononuclear cells rather than neutrophils are the predominant inflammatory cells in the livers of rats with alcoholic liver injury. There was no difference in CINC expression in the liver between males and females, suggesting that CINC expression probably does not explain the enhanced severity of inflammatory changes in female rat liver. It is also not known whether cells such as hepatocytes isolated from females or those exposed to estrogen are more sensitive to the effects of chemokines; the role of chemokines in enhancing liver injury in females needs further analysis.
Expression of TNF- and COX-2 is upregulated in association with
necroinflammatory changes.
A growing body of evidence places the activity of TNF-
and COX-2 at
the center of multiple mechanisms of tissue injury (16, 43). Importantly, COX-2-deficient mice are protected against the
toxic effects of endotoxin (16). Although the causal
relationship between the interaction of TNF-
and COX-2 and liver
injury remains speculative, this hypothesis is strongly supported by
the finding that activation of NF-
B and increased expression of
TNF-
and COX-2 were associated with necrosis and inflammation. The
results of the present investigation show that TNF-
and COX-2 levels are similar in male and female rats and therefore do not explain the
gender-related differences in liver pathology. This interpretation has
to be viewed with some caution because mRNA levels in whole liver do
not reflect the contribution of the different cell types. Kupffer
cells, for example, are a major source of proinflammatory cytokines and
metabolites. Also, the level of mRNA may not reflect the quantity or
activity of TNF-
or COX-2 protein.
FFD rats as a model for NASH.
Also of interest is the unexpected finding that FFD rats developed
fatty liver, necrosis, and inflammation. Increased NF-B binding
activity and elevated levels of COX-2 and TNF-
were detected in
these livers. These changes were accompanied by higher levels of
endotoxin and lipid peroxidation compared with male rats fed a similar
diet. The pathological and biochemical findings in FFD rats are similar
to those found in humans with NASH (57). It is generally
accepted that the histological criteria for NASH include steatosis,
lobular inflammation, and perivenular fibrosis (57).
Factors thought to be relevant to the pathogenesis of NASH include
female gender, oxidant stress, endotoxemia, obesity, and insulin
resistance (65). As described above, some of these factors
were associated with pathological liver injury in FFD rats. It is
tempting to speculate that a longer period of feeding could lead to
significant fibrosis. Although the molecular basis for NASH remains
uncertain, the present work suggests a possible basis for the
inflammatory reaction. Moreover, the results of this study indicate
that the intragastric model could be used to evaluate the pathogenesis
or treatment of NASH. In a previously used model (29, 65)
of NASH, the leptin-deficient obese mouse, increased expression of
COX-2, oxidants, and proinflammatory cytokines was observed in macrophages.
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ACKNOWLEDGEMENTS |
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We thank D. Peters, T. Cloutier, and L. Miao for technical assistance.
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FOOTNOTES |
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This study was supported by grants from the Alcohol Beverages Medical Research Foundation, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-2075, and the University of Hong Kong. K. Jokelainen was supported by grants from the Academy of Finland, Finnish Cultural Foundation, and Yrjö Jahnsson Foundation.
Address for reprint requests and other correspondence: A. A. Nanji, Clinical Biochemistry Unit, LG 136, Block K, Queen Mary Hospital, 102 Pokfulam Rd., Hong Kong (E-mail: ananji{at}pathology.hku.hk).
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 27 April 2001; accepted in final form 17 August 2001.
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