Potential conflict of interest: Nothing to report.
The role of nitric oxide (NO) in liver injury and fibrosis is unclear. The purpose of this study was to determine whether inducible NO synthase deficiency (iNOS−/−) affects liver injury and fibrosis produced in mice by chronic carbon tetrachloride (CCl4) administration. Wild-type (WT) or iNOS−/− mice were subjected to biweekly CCl4 injections over 8 weeks, whereas controls were given isovolumetric injections of olive oil. Serum aminotransferases were lower after CCl4 in the iNOS−/− than in the WT mice, which correlated with decreased necrosis on liver histology. There was increased apoptosis, a lower number of stellate cells, and a lesser degree of fibrosis after CCl4 in the iNOS−/− as compared with the WT mice. α1(I) collagen messenger RNA (mRNA) was markedly increased after CCl4 in the WT and to a significantly lesser extent in the iNOS−/− mice. Liver matrix metalloproteinase-9 (MMP-9) mRNA and MMP-2 mRNA were increased more in the WT than in the iNOS−/− mice after CCl4. Also tissue inhibitor metalloproteinase 1 (TIMP-1) mRNA was increased to a much greater extent in the WT than in the iNOS−/− mice after CCl4 (P < 0.05). However, MMP-9 and TIMP-1 protein, determined by western blot, were similarly increased after CCl4 in both groups of mice. Conclusion: NO protects against CCl4-induced apoptosis. In the absence of iNOS, there is decreased necrosis, increased apoptosis, and reduced liver fibrosis. (HEPATOLOGY 2008;47:2051–2058.)
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
Nitric oxide (NO) is a free radical that has a broad range of functions. It is synthesized from L-arginine by three known NO synthase isoforms: neuronal NO synthase, inducible NO synthase (iNOS-2), and endothelial NO synthase. NO has many roles ranging from neurotransmission, prevention of blood clotting, and regulation of blood pressure (mainly through the constitutive expression of neuronal NO synthase or endothelial NO synthase). In the liver, NO production by endothelial NO synthase has a protective role by maintaining perfusion and preventing platelet aggregation, whereas the exact role of iNOS remains unclear. There are differing results on whether NO production by iNOS is hepatoprotective or detrimental, depending on the level, intensity, and duration of an insult. In response to inflammation, iNOS is up-regulated in hepatocytes and macrophages.1 In the presence of iNOS inhibitors, apoptotic cell death is increased, suggesting that NO has antiapoptotic properties.2
Interactions between NO and reactive oxygen species (ROS) are important in their ultimate effects. NO can inhibit the generation of oxygen radicals and decrease lipid peroxidation. However, cytotoxic actions of NO can occur at high levels of ROS because of the interaction of either O2 or O2− to generate dinitrogen trioxide (N2O3) or peroxinitrite. With regards to collagen regulation, a few studies show that NO inhibits ROS-stimulated collagen formation. Stimulation of iNOS by lipopolysaccharide inhibited collagen synthesis in cultured mesangial cells,3 whereas NO inhibition with NG-nitro-L-arginine methyl ester in transgenic mice harboring the α1(I) collagen promoter increased activation of this promoter in glomeruli and afferent arterioles.4In vitro exposure of human stellate cells to ROS increased rat α1(I) procollagen messenger RNA (mRNA), and this effect was enhanced by inhibition of NO formation by NG-nitro-L-arginine methyl ester.5 In in vivo studies, after chronic CCl4 administration, the development of hepatic fibrosis was similar in iNOS−/− and in control mice in one study,6 whereas in another study, from our laboratory, the amount of hepatic fibrosis deposited was less in the iNOS−/− than in control mice.7
The purpose of this study was to determine further the role of iNOS in the pathogenesis of liver injury and fibrosis produced by chronic CCl4 administration in mice.
Male iNOS−/− (B6129P2-Nos2tm1/lau/J), and wild-type (WT) (B6129PF2J) 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. Dulbecco's modified Eagle's medium and fetal bovine serum were purchased from Life Technologies Inc. (Gaithersburg, MD). Caspase 3 and Caspase 8 fluorometric assay kits were obtained from BioVision (Mountain View, CA).
Mice 4 to 6 weeks of age and 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 iNOS−/− mice were given intraperitoneal injections of CCl4biweekly as 5 μL of a 20% solution of CCl4in olive oil per gram body weight (1.0 mL/kg CCl4). The eight control WT and eight iNOS−/− mice received the same isovolumetric dose of olive oil as intraperitoneal injections. The animals were sacrificed 8 weeks after the start of these injections.
At the time of sacrifice, blood was obtained from the aorta for measurement 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: (a) fixed in 10% buffered formaldehyde formalin and embedded in paraffin; (b) snap frozen at −70°C for sectioning and immunohistochemistry; (c) homogenized in appropriate buffer(s) and aliquots frozen at −70°C for biochemical assays; and (d) placed in RNA STAT-60 (from Tel-Test, INC, Friendswood, TX) solution and stored in −70°C for RNA isolation.
Liver Histology and Morphometric Collagen Determination.
The liver sections imbedded 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.8
Quantitation of Stellate Cells.
Immunofluorescent staining for alpha-smooth muscle actin (α-SMA) 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 α-SMA 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 4 times for 15 minutes, the slides were mounted and eight areas per slide captured by fluorescent microscopy (magnification ×100). Stellate cells were counted in the eight fields per slide for each mouse.
Alanine aminotransferase and aspartate aminotransferase were determined by the spectrophotometric method of Bergmeyer et al.9
Liver slices were homogenized with cold 1.15% KCl, and malondialdehyde was determined using thiobarbituric acid by the method of Uchiyama and Mihara.10
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 in the DNA analysis facility at 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 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 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-β1, α1(I) collagen, β-actin (as endogenous control), mouse matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) 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 aprotonin and then centrifuged at 3000g 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 (Bio-Rad, 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 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 that 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 (ssDNA) described by Frankfurt and Krishan,11 with mouse monoclonal antibodies to ssDNA (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.
The activities of caspase 3 and caspase 8 were determined in liver homogenates by measuring proteolytic cleavage of the specific fluorogenic substrates DEVD-AFC (Asp-Glu-Val-Asp) and IETD (Ile-Glu-Thr-Asp)-AFC (AFC: 7-amino-4-trifluoromethyl coumarin, respectively; BioVision). The results are expressed as relative units per milligram of protein.
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.
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 grade of necrosis after CCl4 was less in the iNOS−/− mice as compared with the WT mice (P < 0.05) (Fig. 1). The grade of inflammation was not significantly different in the iNOS−/− and in the WT mice. Liver fibrosis was less evident in the iNOS−/− than in the WT mice (Fig. 2). The amount of hepatic fibrosis after CCl4 administration, detected by Sirius red staining and densitometry analysis, was 50% less in iNOS−/− mice than in WT mice (P < 0.01) (Fig. 3). The number of stellate cells, identified by α-smooth muscle actin staining was also lower, per 200× field after CCl4 in the iNOS−/− mice than in WT mice (Fig. 4). The values were 34.5 ± 2.8 in the iNOS−/− as compared with 69.5 ± 4.0 in the WT mice (P < 0.01).
Serum aminotransferases were elevated after CCl4 administration. The increases of aspartate aminotransferase and of alanine aminotransferase were less in the iNOS than in the WT mice (Fig. 5).
α1(I) Collagen mRNA was markedly increased after CCl4 administration in the WT mice (P < 0.01) and to a much lesser extent in the iNOS−/− mice (P < 0.05), whereas TGF-β mRNA was increased after CCl4 administration in the WT (P < 0.05) but not in the iNOS−/− mice (Fig. 6).
Liver malondialdehyde was markedly increased in the WT mice after CCl4 administration from a control value of 8.5 ± 0.5 to 29.3 ± 1.9 μmol/g liver (P < 0.001). By contrast, in the iNOS−/− mice, liver malondialdehyde did not increase significantly after CCl4 administration. The values were 7.4 ± 0.5 and 11.3 ± 2.0 μmol/g liver for the iNOS control and for iNOS−/− after CCl4 administration, respectively.
The number of apoptotic hepatocytes was highest in the iNOS−/− after CCl4 administration (Fig. 7). The values from 40 to 100 fields (×100) examined were 2.7 ± 1.0 and 0.9 ± 0.3 apoptotic cells per field for WT and iNOS−/− control, respectively, and 49.5 ± 4.2 and 29.3 ± 1.9 for iNOS−/− and WT after CCl4 administration (P < 0.01).
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, and both were increased by CCl4administration in both iNOS−/− and WT mice (Fig. 8). Activated caspases 3 and 8, which are essential in the extrinsic death receptor pathway of apoptosis, were increased to a lesser extent after CCl4 administration in the iNOS−/− than in the WT mice (Fig. 9).
Components of the mitochondrial pathway of apoptosis that responds to intracellular stress signals were also examined. Proapoptotic BAX protein increased to a greater extent in the iNOS−/− than in WT mice (Fig. 10), whereas BclXS/L was not affected by CCl4administration in either the iNOS−/− or WT mice (Fig. 11). The antiapoptotic protein Bcl-2 increased after CCl4 administration in the iNOS−/− (P < 0.05) but not in the WT mice (Fig. 10). Cytosolic cytochrome C, which is released from mitochondria during apoptosis and is regulated by both proapoptotic and antiapoptotic members of the Bcl-2 family of proteins, was not increased by CCl4 administration in either the iNOS−/− or WT mice (data not shown).
Stellate cell apoptosis evaluated by the presence of ssDNA under confocal microscopy was detected in a few stellate cells in the iNOS−/− mice after CCl4 (Fig. 12), but not in the WT mice.
Liver MMP-2 was not changed significantly by CCl4 in either the iNOS−/− or WT mice (Fig. 13), whereas MMP-9 was increased to a similar extent after CCl4 in the iNOS−/− or WT mice (P < 0.05) (Fig. 13).
TIMP-1 was increased to a similar extent after CCl4 in the iNOS−/− or WT mice (P < 0.05) (Fig. 14), whereas TIMP-2 was not changed significantly by CCl4 in either the iNOS−/− or WT mice (data not shown).
Increases in MMP-2 mRNA and MMP-9 mRNA were greater after CCl4 in the WT than in the iNOS−/− mice (P < 0.01) (data not shown). TIMP-1 mRNA was also increased in both WT and iNOS−/− mice, and the increase after CCl4 was much greater in the WT (1000-fold increase) as compared with the iNOS−/− mice (350-fold increase); P < 0.05 (data not shown).
This study shows that iNOS-deficient mice develop less hepatic fibrosis produced by chronic CCl4administration than WT mice. These results confirm our previous observations7 but differ from the study of Moreno and Murriel6 showing similar accumulation of fibrosis in iNOS−/− and WT mice. The most likely explanation for this difference in results is that we used the appropriate B6129PF2J WT controls for the iNOS−/− mice, and Moreno and Muriel6 used C57BL/6J C as their WT controls. We found in our previous study that the B6129PF2 WT mice develop a significantly higher amount of hepatic fibrosis by Sirius red staining and hydroxyproline content after chronic CCl4administration than the WT C57BL/6J C mice.7
The lesser accumulation of fibrosis in the iNOS−/− mice after CCl4administration in our study was associated with a lower number of stellate cells. A combination of decreased formation and increased degradation of collagen was considered as a cause of the lesser accumulation of collagen in the liver. The increase in α1(I) collagen mRNA in the WT but not in the iNOS−/− mice indicates decreased type I collagen synthesis in the iNOS−/− mice. The effect of iNOS on collagen accumulation did not seem to be mediated by TGF-β because TGF-β mRNA was increased to a similar extent after CCl4 in the iNOS−/− and WT mice.
There is no evidence in this study that the lesser accumulation of hepatic fibrosis in iNOS−/− mice is caused by increased collagen degradation. It is well known that increased collagen formation from CCl4 administration12, 13 and from other insults14 is associated with an increase in collagen degradation. Indeed, in this study, CCl4 administration resulted in increases in MMP-2 mRNA and MMP-9 mRNA in the WT, but not the iNOS−/− mice, whereas MMP-2 protein was increased to a similar extent in the WT and iNOS−/− mice. Although TIMP-1 mRNA was increased to a greater extent in the WT mice, the increases in TIMP-1 protein were similar in the WT and iNOS−/− mice.
The lesser accumulation of collagen after CCl4 administration in iNOS−/− mice was associated with less hepatocellular necrosis in association with lower elevation of the serum aminotransferases but with increased hepatocyte apoptosis.
Apoptosis of stellate cells was demonstrated only in iNOS−/− but not in WT mice after CCl4. Iredale et al.15 showed that apoptosis of stellate cells contributes to resolution of fibrosis after discontinuation of chronic CCl4 administration in rats. The mechanism for the increased apoptosis in the iNOS−/− mice is unclear. Both the proapoptotic protein BAX and the antiapoptotic protein Bcl-2 were increased to a greater extent in the iNOS−/− than in the WT mice. The Fas (CD95/APO-1) receptor, which mediates apoptosis caused by a variety of insults,16 principally via the extrinsic death receptor pathway, was increased after chronic CCl4 in both the iNOS−/− and WT mice. Two Fas receptor proteins were detected by western blot in livers of the mice, and both were increased. Previous studies have demonstrated alternate Fas transcripts arising from alternatively spliced mRNA, in human mononuclear cells17 and in human hepatocytes,18 one the Fas membrane protein and another a soluble form of Fas. The soluble form of Fas has the ability to block Fas-mediated apoptosis.17 The molecular weights of the two FAS forms found in our study correspond to purified human recombinant baculovirus-expressed Fas membrane proteins of 48 and 52 kDa demonstrated previously.19
Previous studies showed that NO can inhibit caspase activities and apoptosis through S-nitrosylation of caspases.20 In this study, after CCl4, however, the activities of caspase 3 and caspase 8 were increased to a lesser extent in the iNOS−/− than in the WT mice.
This study shows that, in the iNOS−/− mice treated with CCl4, there is a lack of significant increase in malondialdehyde, a product of lipid peroxidation. This lack of lipid peroxidation is most likely the result of a lack of formation of the strong oxidant peroxynitrite radical peroxinitrite−, which results from the interaction between NO (absent in the iNOS−/− mice) and superoxide ion after CCl4 administration. In a prior study, we showed a lack of immunostaining for 3-nitrotyrosine protein adducts (formed when peroxinitrite interacts with tyrosine residues) in iNOS−/− as compared with WT mice after CCl4 treatment.7 Reactive oxygen species (ROS) enhance stellate cell activation and stimulate fibrogenesis.21 Lipid peroxidation products, such as malondialdehyde, stimulate α1(I) collagen expression and collagen synthesis by stellate cells in culture.22, 23 ROS is generated by increased nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase, which produces superoxide anion (O2•−) from oxygen (O2), and the amount of hepatic fibrosis after chronic CCl4 administration is less in NADPH-deficient mice than in WT mice. Hence, most likely the decrease in collagen formation in the iNOS−/− mice is attributable to a decrease in oxygen radicals principally because of the absence of peroxynitrite radical. The finding by Koruk et al.24 of elevated serum NO in patients with cirrhosis suggests that NO contributes to the progression of cirrhosis. In conclusion, this study shows that iNOS deficiency has a protective effect on hepatic fibrosis induced by CCl4 in mice. The principal mechanism for this effect is a decrease in the synthesis of collagen, which is not accompanied by increased collagen degradation. Lack of formation of the peroxynitrite radical and increased apoptosis are mechanisms for the lesser hepatic fibrosis after CCl4 administration in iNOS deficiency.