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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Reactive oxygen species (ROS) activate hepatic stellate cells and enhance fibrogenesis. This study determined the role of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase deficiency in the development of hepatocellular necrosis, inflammation, and apoptosis in relation to fibrosis produced by chronic carbon tetrachloride (CCl4) administration. Wild-type (WT) mice or mice with deficiency of the gp91phox subunit of NADPH complex (gp91math image) were subjected to biweekly CCl4 injections over 8 weeks, whereas controls were given isovolumetric injections of olive oil. Serum aspartate aminotransferase (AST) was higher after CCl4 administration in gp91math image than in WT mice, correlating with increased necrosis on liver histology. By contrast, more hepatocyte apoptosis was found after CCl4 in the WT than in the gp91math image mice, which was associated with changes in components of the mitochondrial pathway of apoptosis, namely, an increase in the pro-apoptotic BAX protein in the WT, but not in the gp91math image mice and also a lower cytosolic cytochrome c in the gp91math image mice. There were fewer stellate cells and less fibrosis after CCl4 in the gp91math image as compared with the WT mice. The increase in α1(I) collagen messenger RNA (mRNA), however, was greater after CCl4 in the gp91math image mice. Matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA increased more in the gp91math image than in WT mice after CCl4. Tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2 increased after CCl4 only in the gp91math image mice. Conclusion: Decreased hepatic fibrosis after chronic CCl4 administration in mice with NADPH oxidase deficiency occurs in the setting of greater necrosis and inflammation but decreased apoptosis. (HEPATOLOGY 2009.)

Reactive oxygen species (ROS) activate stellate cells and stimulate fibrogenesis.1 Lipid peroxidation products stimulate α1(I) collagen expression and collagen synthesis by stellate cells in culture.2, 3 Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase, which generates superoxide ion (O2· −) from oxygen (O2), is a principal source of ROS. Platelet-derived growth factor–induced proliferation of hepatic stellate cells, which is mediated by ROS produced by NADPH oxidase, was abrogated by inhibition of NADPH with diphenylene iodonium pretreatment.4 Furthermore, hepatic fibrosis produced in vivo by dimethylnitrosamine was eliminated by daily diphenylene iodonium administration.4 Liver cell death attributable to necrosis or apoptosis results in inflammatory responses and in fibrogenesis.5, 6 Mice with deficiency of the p47phox subunit of NADPH oxidase complex developed less hepatic necrosis and fibrosis after bile duct ligation than wild-type (WT) mice.7 In another study, using a pan-caspase inhibitor, hepatocyte apoptosis was found to be critical for the development of liver inflammation and fibrosis after bile duct ligation.8 In prior studies, we found that mice with NADPH oxidase deficiency, because of lack of the gp91math image subunit of NADPH complex (gp91math image), developed less hepatic fibrosis after chronic carbon tetrachloride (CCl4) administration than WT mice,9 but the means of cell death were not assessed. In a more recent study, gp91math image and WT mice fed a methionine-deficient and choline-deficient diet for 8 weeks developed similar amounts of necroinflammatory change and fibrosis.10

The purpose of this study was to determine how NADPH oxidase deficiency decreases hepatic fibrosis produced by chronic CCl4 administration by examining the roles of hepatocellular necrosis, inflammation, and apoptosis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Animals and Materials.

Male WT C57BL/6J and gp91math image mice were purchased from Jackson Laboratory (Bar Harbor, ME). All animals received humane care in compliance with the guidelines from the Animal Care and Use Committee of The Johns Hopkins University. Sirius Red was obtained from Polysciences, Inc, Warrington, PA. Carbon tetrachloride (CCl4) and goat anti-mouse α-smooth muscle actin Cy3 conjugate antibody were purchased from Sigma Chemical Co., St. Louis, MO. Caspase 3 and caspase 8 fluorometric assay kits were obtained from BioVision (Mountain View, CA)

Animal Treatment.

Mice at 4 to 6 weeks of age weighing 20 to 30 g were kept in a temperature-controlled room with an alternating 12-hour dark and light cycle. Eight WT and eight gp91math image mice were given intraperitoneal injections of CCl4 biweekly as 5 μL of a 20% solution of CCl4 in olive oil per gram body weight (1.0 mL/kg CCl4). The eight control WT and eight gp91math image mice received the same isovolumetric dose of olive oil as intraperitoneal injections. The animals were sacrificed at 1 week and at 8 weeks after the start of the injections. At the time of sacrifice, blood was obtained from the aorta for measurements of aminotransferases and the samples were stored at −20°C. The liver was removed, rinsed with phosphate-buffered saline (PBS), and divided into four portions: (1) fixed in 10% buffered formaldehyde formalin and embedded in paraffin; (2) snap frozen at −70°C for sectioning and immunohistochemistry; (3) homogenized in appropriate buffer(s) and aliquots of the homogenates or the separated cytosols after centrifugation frozen at −70°C for biochemical assays; and (4) placed in RNA STAT-60 solution (from Tel-Test, Inc, Friendswood, TX) and stored in −70°C for RNA isolation.

Liver Histology and Morphometric Collagen Determination.

The liver sections embedded in paraffin were cut (5 μm) and stained with hematoxylin-eosin, Masson's trichrome, or Sirius red. The extent of necrosis and inflammation was evaluated on blinded slides by M.S.T. from our Department of Pathology. Fibrosis was determined histologically by measuring the intensity of fibrosis in four to six (×100) digital images captured from slides of each mouse stained with Sirius red. The total fibrosis density score was determined by dividing the image intensity by the image area as described previously.11

Quantitation of Stellate Cells.

Immunofluorescent staining for alpha-smooth muscle actin was done in deparaffinized liver sections. The slides were washed in deionized water for 1 minute and in PBS for 5 minutes, followed by blocking using PBS-5% fetal bovine serum. The slides were incubated with Cy3 conjugated monoclonal alpha-smooth muscle actin antibody (Sigma, 1:500 in PBS—5% fetal bovine serum) for 1 hour at room temperature and subsequently at 4°C overnight. After washing with PBS four times for 15 minutes, the slides were mounted and eight areas per slide captured by fluorescent microscopy (magnification ×100). Stellate cells were counted in eight fields per slide for each mouse.

Serum Aminotransferases.

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by the spectrophotometric method of Bergmeyer et al.12

Mitochondrial Adenosine Triphosphate.

Liver mitochondria were isolated by differential centrifugation from liver homogenates as described previously.13 Adenosine triphosphate was determined with a Bioluminescent Assay Kit obtained from Sigma Chemical Company. Mitochondrial protein was determined by the method of Lowry et al.14

Liver Malondialdehyde.

Liver slices were homogenized with cold 1.15% KCl, and malondialdehyde was determined using thiobarbituric acid by the method of Uchiyama and Mihara.15

Determination of Messenger RNA by Real Time Quantitative Polymerase Chain Reaction.

The 7900 HT (Applied Biosystems, Foster City, CA) and the SDS 2.2.1 software was used to perform real-time quantitative polymerase chain reaction at the The Johns Hopkins DNA Analysis Facility. Total cellular RNA from a portion of liver was placed in RNA STAT 60 reagent and following their protocol, RNA was purified and isolated. The concentration of the isolated RNA was determined from the optical density (OD) at 260 nm and its purity from the 260 nm/280 nm optical density ratio. The isolated RNA was stored at −80°C. Real-time quantitative polymerase chain reaction for α1(I) collagen messenger RNA (mRNA) and transforming growth factor-beta (TGF-β) were performed using sequence-specific probes from TaqMan gene expression assays of Applied Biosystems (Foster City, CA). Probes for mouse TGF-β, α1(I) collagen, β−actin (as endogenous control), mouse matrix metalloproteinase (MMP)-2, and MMP-9 were obtained from Applied Biosystems. Superscript III first-strand synthesis from Invitrogen (Carlsbad, CA) was used to synthesize first-strand complementary DNA from the purified RNA. Gene transcript levels of these probes were compared with β-actin, the housekeeping endogenous control. Variation in the amount of the transcripts was corrected by the level of expression of the β-actin gene in each individual sample.

Western Blot Analysis.

Liver sections were homogenized in 50 mM Tris-HCl buffer pH 7.6 containing 150 mM NaCl, 10 mM CaCl2, 0.25% Triton-X, 0.1 μM phenylmethanesulfonyl fluoride, 10 μM leupeptin, 10 μM pepstatin, 0.1 mM iodoacetamide, and 25 μg aprotinin, and then centrifuged at 9000g for 10 minutes at 4°C. The cytosol protein in the supernatant was initially stored at −80°C. The proteins were separated on mini-sodium dodecyl sulfate gels at 100 V for 1 hour and electrotransferred to nitrocellulose transblot membranes (BioRad, Hercules, CA). The membranes were washed in PBS, pH 7.6, containing 0.1% Tween 20 (PBS-T), blocked with 5% (wt/vol) dry nonfat milk in PBS-T for 1 hour, rinsed with PBS-T, and then incubated with either rabbit anti-mouse antibodies to Fas, Bax, BclXs/l, Bcl-2, cytochrome c, MMP-2, MMP-9, TIMP-1, TIMP-2, or β−actin, obtained from Santa Cruz Biotechnology, Inc, Santa Cruz, CA. After repeated washing, the membranes were incubated with horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G (1:10,000 dilution; Amersham Biosciences, Piscataway, NJ) at room temperature for 1 hour. The membranes were then washed again and visualized by enhanced chemiluminescence reaction (ECL Plus; Amersham Biosciences). Densitometry was determined using Image J v 1.30 obtained from the National Institutes of Health.

Terminal Deoxynucleotidyl Transferase-Mediated Nick-End Labeling Assay.

Terminal deoxynucleotidyl transferase-mediated nick-end labeling assay was performed on paraffin-embedded liver slices with the cell death detection kit from Roche Applied Science (Nutley, NJ). Fluorescence microscope using the fluorescein isothiocyanate filter revealed the apoptotic bodies, which were counted.

Apoptosis in Stellate Cells.

Apoptosis in stellate cells was determined in ultrathin liver slices by formamide-induced DNA denaturation with detection of single-stranded DNA described by Frankfurt and Krishan,16 with mouse monoclonal antibodies to single-stranded DNA (Millipore, Temecula, CA) and anti-mouse immunoglobulin G (whole molecule) fluorescein isothiocyanate as secondary antibody (Sigma). The immunostained slices were evaluated and photographed with laser confocal microscopy.

Caspase Activities.

The activities of caspase 3 and caspase 8 were determined in liver homogenates by measuring proteolytic cleavage of the specific fluorogenic substrates, DEVD (Asp-Glu-Val-Asp)-AFC (7-amino-4-trifluoromethyl coumarin) and IETD (Ile-Glu-Thr-Asp)-AFC, respectively (BioVision). The results are expressed as relative units per milligram of protein.

Statistical Analysis.

In most measurements, the mean and the standard error of the mean were calculated. The data were analyzed with the Student t test or by two-way analysis of variance when comparing means of more than two groups.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Liver Injury and Fibrosis.

The morphological changes of liver injury and fibrosis caused by CCl4 were visualized in sections stained by hematoxylin-eosin (not shown) and Sirius red. The changes include necrosis, inflammation with macrophages, lymphocytes, and fibrosis. Fatty infiltration was minimal. The grades of necroinflammatory changes were higher after CCl4 in the gp91math image mice as compared with the WT mice (P < 0.05) (Fig. 1). Liver fibrosis was more evident in the WT than in the gp91math image mice (Fig. 2A,B). The area of hepatic fibrosis detected by Sirius red staining and densitometric analysis was significantly higher in the WT mice after CCl4 as compared with the other mice groups (P < 0.01) (Fig. 2C). The number of stellate cells, identified by alpha-smooth muscle actin staining, was lower per 200× field after CCl4 in the gp91math image mice than in WT mice (Fig. 2D). The values were 33.9 ± 2.8 in gp91math image as compared with 50.0 ± 4.8 in the WT mice (P < 0.05).

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Figure 1. Necroinflammatory changes and serum aminotransferases after 8 weeks of chronic carbon tetrachloride (CCl4) administration in wild-type (WT) and gp91math image mice. Liver cell necrosis (N) was evaluated on a scale of 1-4 as follows: 1, occasional; 2, mild scattered; 3, confluent zonal; and 4, extensive. Grades for hepatic inflammation (I) were: 1, scattered; 2, mild; 3, moderate; and 4, marked. The histological changes were assessed in 20× magnification fields of slides stained with hematoxylin-eosin. The values are expressed as means ± standard error (SE) of eight determinations per group. *P < 0.05 versus WT for necroinflammatory changes. *P < 0.05 versus control for serum aminotransferases.

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Figure 2. Histology with Sirius red collagen staining of liver sections from (A) WT and (B) gp91math image mice after 8 weeks of chronic CCl4 administration (original magnification ×100). (C) Relative densitometry of fibrosis in WT and gp91math image mice after chronic CCl4 administration. The degree of fibrosis was measured by Sirius red staining and densitometry of the various groups. The density of fibrosis was determined as intensity of Sirius red staining divided by the area of the captured field. A total of 24 fields were captured from livers of each group of mice. The values are expressed as means ± SE. *P < 0.01 versus respective control. +P < 0.01 versus WT + CCl4. (D) Immunofluorescent detection of activated stellate cells in (A) WT and (B) gp91math image mice after chronic CCl4 administration. The liver slices were incubated with Cy3 monoclonal alpha-smooth muscle actin antibody (original magnification ×100).

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Serum aminotransferases were elevated after CCl4 administration (Fig. 1). The increase of AST was higher in gp91math image than in WT mice (P < 0.05). By contrast, ALT was not significantly different in the gp91math image than in the WT mice.

The increase in α1(I) collagen mRNA after CCl4 administration was greater in the gp91math image than in the WT mice (P < 0.05) (Fig. 3). The increase in TGFβ mRNA was also greater in the gp91math image than in the WT mice (P < 0.05) (Fig. 3). However, TGFβ protein on western blot analysis was not significantly changed by CCl4 in either group of mice (data not shown).

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Figure 3. Liver α1(I) collagen mRNA and transforming growth factor β (TGF-β) mRNA in WT and gp91math image mice after chronic CCl4 administration. The mRNAs were determined by real time quantitative PCR. The relative amounts of mRNA were normalized against β-actin in the same samples. The values are expressed as means ± SE of eight determinations per group. *P < 0.05 versus control. **P < 0.01 versus control. +P < 0.05 versus WT + CCl4.

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Liver malondialdehyde was markedly increased in the WT mice after CCl4 administration from a control value of 9.4 ± 1.4 to 19.1 ± 1.5 μmoles/g liver (P < 0.01). In the gp91math image animals, liver malondialdehyde increased from 5.8 ± 1.8 to 10.3 ± 0.2 (P < 0.05).

Apoptosis.

The number of apoptotic hepatocytes was highest in the WT mice after CCl4 administration (Fig. 4B). The values from 40 to 100 fields (×100) examined were 1.63 ± 0.42 and 1.67 ± 0.43 apoptotic cells per field for WT and gp91math image controls, respectively, and 69.2 ± 4.05 and 14.93 ± 2.17 for WT and gp91math image after CCl4 administration (P < 0.01).

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Figure 4. Apoptosis determined by terminal deoxynucleotidyl transferase-mediated nick-end labeling staining of liver sections from WT and gp91math image mice after chronic CCl4 administration. (A) WT-control; (B) WT + CCl4; (C) gp91math image control; (D) gp91math image + CCl4. Original magnification ×100.

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Fas (CD95/APO-1) receptor mediates apoptosis principally via the extrinsic death receptor pathway. Two Fas receptor proteins were detected by western blot in livers of the mice. CCl4 did not result in significant changes in Fas receptor proteins in the WT or gp91math image mice (Fig. 5A). Activated caspases 3 and 8 are essential in the extrinsic death receptor pathway of apoptosis. Caspase 3 was increased to a greater extent in the gp91math image mice than the WT mice after CCl4 administration (P < 0.05) (Fig. 5B), whereas the increase in caspase 8 was similar after CCl4 in the gp91math image and WT mice.

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Figure 5. Effects of chronic CCl4 on in WT and gp91math image mice on (A) FAS and (B) activities of caspase 3 and 8. FAS was determined by western blot and quantitated by densitometry. White bars represent 52 kDa and shaded bars 48 kDa Fas. All values are expressed as means ± SE of eight measurements per group. *P < 0.05 versus respective control. **P < 0.01 versus respective control; +P < 0.05 versus WT + CCl4.

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Components of the mitochondrial pathway of apoptosis that respond to intracellular stress signals were examined. Pro-apoptotic BAX protein was increased after CCl4 in the WT, but not in the gp91math image mice (P < 0.01) (Fig. 6), whereas BclX S/L was not affected by CCl4 administration in either the gp91math image or WT mice (Fig. 6). The anti-apoptotic protein Bcl-2 decreased after CCl4 in the gp91math image (P < 0.01) but not in the WT mice (Fig. 6).

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Figure 6. Effects of chronic CCl4 administration on Bax, Bcl Xs/l Bcl-2, and cytosolic cytochome c in WT and gp91math image mice. The determinations were done by western blot. The values are expressed as means ± SE of relative densitometry of eight measurements per group. *P < 0.05 versus control. **P < 0.01 versus control.

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Cytosolic cytochrome c, which is released from the mitochondria during apoptosis and is regulated by both pro-apoptotic and anti-apoptotic members of the Bcl-2 family of proteins, was decreased after CCl4 in the gp91math image CCl4 group (P < 0.05), but not in the WT mice (Fig. 6).

Stellate cell apoptosis, evaluated by the presence of single-stranded DNA under confocal microscopy, was not detected in the gp91math image or the WT mice after CCl4 (data not shown).

Matrix Metalloproteinases.

Liver MMP-2 was increased after CCl4 in the WT mice (P < 0.05) but not in the gp91math image mice (Fig. 7), whereas MMP-9 was not changed by CCl4 in either group of mice (Fig. 7). Increases in MMP-2 mRNA and in MMP-9 mRNA were greater after CCl4 in the gp91math image than in the WT mice. The increase in MMP-2 mRNA after CCl4 was 30-fold in the gp91math image mice as compared with sevenfold in the WT mice (P < 0.01), whereas the increase in MMP-9 mRNA was 111-fold in the gp91math image mice as compared with fivefold in the WT mice (P < 0.01) (data not shown).

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Figure 7. Effects of chronic CCl4 administration on matrix metalloproteinase 2 (MMP-2), 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2 in WT and gp91math image mice determined by western blot. *P < 0.05 versus respective control.

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TIMP-1 (Fig. 7) and TIMP-2 were lower in the control WT than in the control gp91math image mice (Fig. 7). After CCl4, both TIMP-1 and TIMP-2 increased only in the WT mice (P < 0.05) to levels similar to those found in the control or CCl4-treated gp91math image mice (Fig. 7).

Effects of Administration of CCl4 for One Week.

The effects of acute CCl4 on hepatic injury and fibrosis were determined in a separate experiment. After 1 week of CCl4 administration, the grade of necrosis, but not of inflammatory changes, was higher in the WT mice than in gp91math image mice (P < 0.05) (Fig. 8). Liver fibrosis detected by Sirius red staining was minimal and similar in the WT than in the gp91math image mice (not shown). Also, the number of stellate cells, identified by alpha-smooth muscle actin staining, was similar per ×200 field after CCl4 in the gp91math image mice than in WT mice. The values were 9.8 ± 1.6 and 9.3 ± 0.7 in gp91math image and the WT mice, respectively. The increase of serum ALT was higher in WT (P < 0.01) than in gp91phox mice (P < 0.05) (Fig. 8). By contrast, AST was not significantly different in the gp91math image than in the WT mice.

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Figure 8. Necroinflammatory changes and serum aminotransferases after 1 week of CCl4 administration in wild-type (WT) and gp91math image mice. Liver cell necrosis (N) was evaluated on a scale of 1-4 as follows: 1, occasional; 2, mild scattered; 3, confluent zonal; and 4, extensive. Grades for hepatic inflammation (I) were: 1, scattered; 2, mild; 3, moderate; and 4, marked. The histological changes were assessed in 20× magnification fields of slides stained with hematoxylin-eosin. The values are expressed as means ± SE of eight determinations per group. **P < 0.01 versus WT for necroinflammatory changes. *P < 0.05 and **P < 0.01 versus control for serum aminotransferases.

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The number of apoptotic hepatocytes was similar in the WT than in gp91math image mice after CCl4 administration. The values from 32 fields (×100) examined were 1.2 ± 0.5 and 1.2 ± 0.4 apoptotic cells per field for WT and gp91math image controls, respectively, and 12.4 ± 1.7 and 8.9 ± 1.5 for WT and gp91math image after CCl4 administration. Caspase 3 was increased in the gp91math image mice but not the WT mice, whereas caspase 8 was unchanged in both groups of mice (Fig. 9A) Cytosolic cytochrome c was increased, whereas mitochondrial cytochrome c was decreased, after CCl4 in the WT mice (P < 0.05), whereas no significant changes occurred in the gp91math image (Fig. 9B). There was a mean 31% decrease in mitochondrial adenosine triphosphate after CCl4 in the WT mice, and an opposite 34% increase in the gp91math image ; however, these changes were not statistically significant. The values for eight mice in each group were 16.4 ± 3.9 and 11.3 ± 1.7 nmoles/mg protein for WT mice, compared with 9.5 ± 1.8 and 12.7 ± 2.6 nmoles/mg protein for gp91math image before and after CCl4, respectively.

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Figure 9. Effects of 1 week of chronic CCl4 administration on (A) activities of caspase 3 and 8 and (B) on cytosolic and mitochondrial cytochrome c in WT and gp91math image mice. Cytochrome c was determined by western blot and quantitated by densitometry. The values are expressed as means ± SE of eight measurements per group. *P < 0.05 versus respective control.

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The increase in α1(I) collagen mRNA after CCl4 administration was greater in the gp91math image than in the WT mice (P < 0.05) (Fig. 10), and TGFβ mRNA was increased in the gp91math image but not in the WT mice (P < 0.05) (Fig. 10). MMP-2 mRNA and in MMP-9 mRNA increased after CCl4 in the gp91math image but not in the WT mice. TIMP-1 mRNA increased to a greater extent in the gp91math image than in the WT mice (Table 1). 10

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Figure 10. Liver α1(I) collagen mRNA and transforming growth factor β (TGFβ) mRNA in WT and gp91math image mice after 1 week of CCl4 administration. The mRNAs were determined by real time quantitative PCR. The relative amounts of mRNA were normalized against β-actin in the same samples. The values are expressed as means ± SE. *P < 0.05 versus respective control. †P < 0.05 versus WT + CCl4.

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Table 1. Effects of Carbon Tetrachloride (CCl4) Administration for One Week on Matrix Metalloproteinase-2 (MMP-2) mRNA, Matrix Metalloproteinase-8 (MMP-9) mRNA, and Tissue Inhibitor of Metalloproteinase 1 (TIMP 1) mRNA
mRNAWTgp91phox
ControlCCl4ControlCCl4
  • The mRNAs were determined by real time quantitative PCR. The relative amounts of mRNA were normalized against β actin in the same samples and expressed as a % of WT control for each determination. The values are expressed as means ± SE of 8 determinations per group.

  • *

    P < 0.05 versus control.

  • P<0.01 versus control.

  • P<0.05 versus WT + CCl4.

MMP-2100 ± 6.2131 ± 3.7107 ± 9.0244 ± 45.1
MMP-9100 ± 8.3148 ± 30.0111 ± 9.6198 ± 29.2*
TIMP-1100 ± 12.133,917 ± 77.6124 ± 22.946,469 ± 909

MMP-2 and MMP-9 proteins were not changed significantly after 1 week of CCl4 in either the WT or the gp91math image mice (Fig. 11). TIMP-1 protein decreased after CCl4 in the gp91math image mice but not in the WT mice, whereas TIMP2 decreased in the WT mice, remaining unchanged in the gp91math image mice (Fig. 11).

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Figure 11. Effects of 1 week of CCl4 administration on matrix metalloproteinase 2 (MMP-2), 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2 in WT and gp91math image mice determined by western blot. *P < 0.05 versus respective control.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

The development of less hepatic fibrosis after chronic CCl4 administration in the gp91phoxmath image mice than in WT mice in this study was associated with greater hepatocellular necrosis and inflammation but with decreased hepatocyte apoptosis.

The higher serum AST in the gp91math image mice, which correlated with the greater hepatocellular necrosis and inflammation, is consistent with enhanced mitochondrial injury, because approximately 80% of the AST is found in mitochondria, whereas ALT is found in the cytosol.17 Our results of greater hepatocellular necrosis and inflammation in the gp91math image mice differ from the findings of other studies using different means to produce liver injury or fibrosis. Steatosis, inflammation, and necrosis were observed in WT, but not in NADPH oxidase–deficient mice given chronic ethanol administration18; however, the effect on fibrosis was not assessed in that study because no significant fibrosis occurred. Our study also shows that the short-term administration of CCl4, which does not result in significant increase in fibrosis, results in greater hepatocellular necrosis and higher serum ALT in the WT than in the gp91math image mice.

The development of less hepatic fibrosis after bile duct ligation in mice with deficiency of the p47phox subunit of NADPH oxidase complex than in WT mice was associated with lower elevations of serum AST, and less necrosis around the biliary tracts.7 Hepatocyte apoptosis, which was not determined in the previously mentioned study, has since been demonstrated to be critical for the development of liver inflammation and fibrosis after bile duct ligation.8 In another study, gp91math image and WT mice fed a methionine-deficient and choline-deficient diet developed similar amounts of necroinflammatory change and fibrosis.10 The mechanism for the decreased apoptosis after CCl4 in the gp91phoxmath image mice is most likely attributable to a decrease in the mitochondrial pathway of apoptosis. The pro-apoptotic protein BAX protein, which was increased in the WT mice, remained unchanged in the gp91math image mice, whereas cytosolic cytochrome c, which is released from the mitochondria during apoptosis, was decreased after CCl4 in the gp91math image mice. Of note is that 1 week after CCl4 administration there was a marked increase in cytosolic cytochrome c in association with a decrease in mitochondrial cytochrome c in the WT butnot in the gp91math image mice. These changes are most likely a reflection of the more severe hepatocellular injury in the WT mice. The increase in cytosolic cytochrome probably predisposes to increased apoptosis in the WT mice, although no differences in apoptosis were observed between WT and gp91math image mice at 1 week. The Fas (CD95/APO-1) receptor, which mediates apoptosis caused by a variety of insults,19 principally via the extrinsic death receptor pathway, was not changed significantly by chronic CCl4 administration in either the WT or the gp91math image mice. Activated caspases 3 and 8 are also essential in the extrinsic death receptor pathway of apoptosis. Caspase 3 was increased to a greater extent in the gp91math image mice than in the WT mice after CCl4 administration, whereas the increases in caspase 8 were similar after CCl4 in both groups of mice. An increase in caspase 3 in the gp91math image but not the WT mice was already evident after 1 week of CCl4 administration. The significance of the greater increase in caspase 3 in the gp91math image mice than in the WT mice, in relation to the lesser increase in apoptosis after CCl4 in the gp91math image mice, is unknown.

The decrease in apoptosis in the gp91math image mice as compared with the WT mice after chronic CCl4 is most likely attributable to decreased ROS generation. Previous studies have shown that TGF-β–induced apoptosis of fetal rat hepatocytes requires ROS generation, mitochondrial permeability transition with cytochome c release, and caspase activation.20 Furthermore, blockage of the TGF-β–induced increase in ROS by diphenylene iodonium, which inhibits NADPH oxidase and other flavoproteins, abrogated the effect of TGF-β on increasing apoptosis.18 Also, relating to the role of NADPH oxidase, it has been observed that neutrophils exposed to stress stimuli from patients with chronic granulomatous disease, which are deficient in NADPH oxidase, show decreased apoptosis as compared with neutrophils from normal subjects.21 Furthermore, apoptosis induced acutely by bile salt in cultured rat hepatocytes was blunted by inhibition of NADPH oxidase.22 Increased apoptosis of hepatocytes with phagocytosis of apoptotic bodies by Kupffer and stellate cells has been linked to fibrogenesis.6 Conversely, apoptosis of stellate cells was shown to contribute to the resolution of fibrosis after discontinuation of chronic CCl4 administration in rats.23 In our study, apoptosis was not demonstrated in stellate cells from either gp91math image or WT mice after CCl4 administration.

As relates to the mechanism for the decreased hepatic fibrosis after chronic CCl4 administration in the gp91math image as compared with the WT mice, changes in both collagen formation and degradation were considered. The greater increase in the α1(I) collagen mRNA in the gp91math image mice than in the WT mice, found after 1 and 8 weeks of CCl4administration, indicates that NADPH deficiency does not decrease collagen formation. Furthermore, TGF-β mRNA was also higher in the gp91math image mice than in the WT mice. This observation agrees with prior findings indicating that TGF-β transcription is negatively regulated by NADPH oxidase–mediated oxygen radicals.24 In our study, however, the greater increase in TGF-β mRNA in the gp91math image mice than in the WT mice was not accompanied by changes in TGF-β protein. Posttranslational changes, such as feedback inhibition by procollagen pro-peptides, may be a factor for the lower accumulation of collagen in the gp91math image mice after CCl4 in the setting of increased α1(I) collagen mRNA. However, although this was shown in cultured stellate cells from normal rats, it was not found in stellate cells isolated from fibrotic livers.25

It is well established that ROS activate stellate cells and stimulate fibrogenesis.1 In our study, the lesser accumulation in malondialdehyde, a product of lipid peroxidation, after CCl4 in the gp91math image was associated with lower collagen deposition, indicating lower but not absent ROS formation from other sources, most likely the mitochondria. It is well known that chronic CCl4 administration impairs the mitochondrial electron transport chain and that this is associated with increased formation of ROS.26–28

There is evidence in this study that increased collagen degradation contributes to the lesser accumulation of hepatic fibrosis in gp91math image mice. Increased collagen formation from CCl4 administration29, 30 and from other insults31 is associated with an increase in collagen degradation. Indeed, in this study, the increases in MMP-2 mRNA and MMP-9 mRNA were greater after 1 and 8 weeks of CCl4 in the gp91math image than in the WT mice. However, this was not accompanied by higher MMP-2 and MMP-9 protein levels. More importantly, TIMP-1 and TIMP-2 proteins increased after chronic CCl4 administration in the WT mice but not in the gp91math image mice. TIMPs inhibit secretion and activation of metalloproteinases, hence inhibiting collagen degradation.32

In conclusion, this study shows decreased hepatic fibrosis after chronic CCl4 administration in NADPH oxidase–deficient mice as compared with WT mice in association with greater hepatocellular necrosis and inflammation but decreased hepatocyte apoptosis. The lower hepatic fibrosis was found in the setting of a lower number of stellate cells, a greater increase in α1(I) collagen mRNA, but a lack of an increase in TIMP in the NADPH-deficient mice as compared with the WT mice, suggesting that NADPH-deficient mice have enhanced degradation of the formed collagen.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References