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Effects of methylene blue in reducing cholestatic oxidative stress and hepatic damage after bile-duct ligation in rats
ARTICLE IN PRESS Acta histochemica 112 (2010) 259—269
www.elsevier.de/acthis
Effects of methylene blue in reducing cholestatic oxidative stress and hepatic damage after bile-duct ligation in rats Burhan Aksua,, Hasan Umitb, Mehmet Kanterc, Ahmet Guzeld, Cevat Aktasc, Sabiha Civeleke, Hafize Uzune a
Department of Pediatric Surgery, Faculty of Medicine, Trakya University, Edirne 22030, Turkey Department of Gastroenterology, Trakya University, Edirne 22030, Turkey c Department of Histology and Embryology, Trakya University, Edirne 22030, Turkey d Department of Pediatrics, Faculty of Medicine, Trakya University, Edirne 22030, Turkey e Department of Biochemistry, Cerrahpasa Faculty of Medicine, Istanbul University, Turkey b
Received 16 September 2008; received in revised form 21 November 2008; accepted 2 December 2008
KEYWORDS Methylene blue; Extrahepatic cholestasis; Oxidative stress; Hepatic fibrosis; Bile-duct ligation; a-smooth muscle actin; Rats
Summary The aim of this study was to evaluate the effects of methylene blue against cholestatic oxidative stress and liver damage after ligation of the common bile duct in male Wistar rats. Eight animals were included in each of the following five groups: untreated control, methylene blue control, sham-operated, bile-duct ligation, and bile-duct ligation plus methylene blue. Methylene blue was administered intraperitoneally for 14 days at a daily dose of 2 mg/kg per day. All rats were sacrificed 2 weeks following the experimental treatment and the livers of all groups were examined biochemically and histopathologically. The severity of cholestasis and hepatic injury were determined by changes in the plasma, including enzymatic activities: aspartate aminotransferase, alanine aminotransferase, gamma glutamine transferase, and also bilirubin levels. Malondialdehyde, nitric oxide and superoxide dismutase were measured to indicate the oxidative status in the liver tissue. Myeloperoxidase activity and levels of tissue hydroxyproline were determined as measures of neutrophil activation and collagen accumulation, respectively. Liver damage was significantly prevented in the bile-duct ligated rats treated with methylene blue compared with the control bile-duct ligated rats without methylene blue. Treatment with methylene blue markedly reduced activities of serum transaminase, gamma glutamine transferase and bilirubin levels as compared to bile-duct ligated rats without methylene blue. Positive immunolabelling for alphasmooth muscle actin (a-SMA) was increased, especially in vascular smooth muscle
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E-mail address: [email protected] (B. Aksu). 0065-1281/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2008.12.002
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B. Aksu et al. cells, fibrotic septa and also around the proliferated bile ducts, after bile-duct ligation. Only weak a-SMA immunolabelling was seen in livers of rats treated with methylene blue. These results indicate that methylene blue can attenuate hepatic damage in extrahepatic cholestasis by reducing oxidative stress and inflammatory processes. & 2008 Elsevier GmbH. All rights reserved.
Introduction Cholestasis results from structural and functional impairment of the hepatobiliary system, which occurs as a result of several environmental factors and disease processes. Evaluation of the extrahepatic biliary system for patency is a high priority when treating infants or children with cholestasis as biliary atresia occurs in a significant percentage of these young patients (Altman and Buchmiller, 2006). Obstruction of bile flow through the extrahepatic biliary system results in subsequent oxidative injury, hepatic fibrosis, biliary cirrhosis and portal hypertension (Kountouras et al., 1984). Experimental bile-duct ligation induces a form of liver fibrosis, which etiologically and pathogenically resembles biliary fibrosis in humans. Injury to hepatocytes results in the generation of lipid peroxides, which may have a direct stimulatory effect on extracellular matrix production by activated stellate cells (Serviddio et al., 2004). Complete biliary obstruction causes cholestatic injury to the liver, including hepatocellular necrosis and apoptosis, bile-duct epithelial cell proliferation, stellate cell activation and eventually liver fibrosis (Kountouras et al., 1984). Following cholestatic injury, the liver undergoes a tissue remodeling process that combines regeneration and fibrogenesis. During this repair process, the extracellular matrix contains large numbers of alphasmooth muscle actin (a-SMA) immunoreactive cells known as myofibroblasts; however their origin still remains enigmatic. Cassiman et al. (2002) and Ramm et al. (2000) demonstrated that the a-SMA immunopositive cells mainly reside in the portal ducts and fibrous septa and their location corresponds to the distribution of collagen. Although the mechanisms involved in cholestasisinduced liver fibrosis are unclear, obstruction of bile flow causes the accumulation and retention of hydrophobic bile salts in the liver, which are toxic to many cell types, including the hepatocytes and ductal biliary epithelial cells (Benedetti et al., 1997). Bile-duct ligation is characterized by increased lipid peroxidation and by a marked decline in reduced glutathione (GSH), a major cellular antioxidant (Cruz et al., 2003). It is known that
bile-duct ligation results in a shift in the oxidant/ pro-oxidant balance in favor of increased activities of free radicals (Cruz et al., 2003). Enhanced production of reactive oxygen intermediates augments lipid peroxidation by disturbing the oxidant– antioxidant balance in the hepatic mitochondrial fraction. The pattern of damage due to interference of free radicals with hepatocytes indicates peroxynitrite-mediated liver injury (Engin et al., 2003). Methylene blue is a dye that competes with molecular oxygen for the transfer of electrons from flavoenzymes, primarily xanthine oxidase (Salaris et al., 1991). The shunting of electrons to and from the colorless reduced leukomethylene blue diverts their flow from the metal sulfur center of the enzyme, where molecular oxygen is normally converted into superoxide radicals, and the generation of these cytotoxic mediators is attenuated (Salaris et al., 1991; Aksu et al., 2007). The aim of the present study was to evaluate the possible protective effects of methylene blue on oxidative damage in hepatic tissue of rats after experimental bile-duct ligation.
Materials and methods Animals All animal procedures and experimental protocols used in the study were approved by the Ethical Committee of Trakya University. A total of 40 adult male Wistar albino rats, weighing 200–250 g, were used in this study. Rats were provided by the Experimental Research Center of the Medical Faculty of Trakya University and maintained in a windowless animal facility with controlled conditions of temperature (2272 1C), illumination (lights on at 07.00 and off at 21.00) and humidity (50–55%). Rats were fed a standard rat chow and tap water ad libitum. Eight animals were included in each of the following five groups (n ¼ 8): (1) control: normal saline treatment without surgical intervention; (2) methylene blue control: methylene blue treatment without surgical intervention;
ARTICLE IN PRESS Effects of methylene blue in reducing cholestatic oxidative stress (3) sham-operated: dissection of the bile duct, treated with normal saline; (4) bile-duct ligation: double ligature and section of the extrahepatic bile duct; (5) bile-duct ligation plus methylene blue: double ligature and section of the extrahepatic bile duct, treated with methylene blue.
Experimental procedures The rats were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) intraperitoneally (i.p.) and their common bile ducts were exposed through a midline abdominal incision. The common bile duct was located and obstructive jaundice induced by a double ligation with 5/0 silk and transsection of the common bile duct in the supraduodenal region between the lowermost tributary of the bile duct and the uppermost tributary of the pancreatic duct. The shamoperated group of rats underwent opening of the abdominal cavity and dissection of the common bile duct without ligation. Methylene blue (Merck, Darmstadt, Germany) was dissolved and diluted in distilled water to a final concentration of 2%. The methylene blue stock solution was stored at 4 1C. The methylene blue was administered to the bile duct ligated plus methylene blue rat group immediately after bile-duct ligation and at the same time point to the methylene blue control rat group, and from then on for 14 days at a daily dose of 2 mg/kg i.p. The sham-operated control and bileduct ligation groups received equal amounts of normal saline intraperitoneally for 14 days. The rats were killed by decapitation after 14 days of treatment. After opening the abdominal cavity, blood was collected from all rats to determine activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamine transferase (GGT), and also the levels of total bilirubin (TB) and direct bilirubin (DB). The liver tissue samples were immediately immersion-fixed in 10% neutral buffered formalin solution for histological evaluation or stored at 80 1C for subsequent spectrophotometric determination of activities of superoxide dismutase (SOD), and levels of malondialdehyde (MDA) and nitric oxide (NO). Indirect evidence of neutrophil infiltration was determined by measuring tissue-associated myeloperoxidase activity. Collagen accumulation was assessed by measuring hydroxyproline content in liver samples.
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Nitric oxide (NO) has a half-life of only a few seconds, because it is readily oxidized to nitrite (NO 2 ) and subsequently to nitrate (NO3 ), which serve as index parameters of NO production. The method for determining plasma nitrite and nitrate levels was based on Cortas and Wakid (1990) with results expressed as mmol/g wet tissue. Myeloperoxidase activity was determined using 4-aminoantipyrine/phenol solution as the substrate for myeloperoxidase-mediated oxidation by H2O2 according to Wei and Frenkel (1993) and presented as mU/g protein. Liver malondialdehyde levels were determined by a method based on the thiobarbituric acid reactive substances reaction with thiobarbituric acid at 90–100 1C (Buege and Aust, 1978). The results are expressed as nmol/g wet tissue. The tissue samples taken for hydroxyproline determination were washed with normal saline and oven-dried at 100 1C for 72 h. Hydroxyproline levels were determined spectrophotometrically using the method of Woessner (1961). The serum activities of alanine aminotransferase, aspartate aminotransferase, gamma glutamine transferase, total bilirubin and direct bilirubin were determined using commercially available kits.
Histological evaluation Formalin-fixed liver tissues were processed for embedding in paraffin wax, sections cut at 5 mm and then stained with Masson’s trichrome technique. Lobular architecture, presence of inflammation, fibrosis and ductular proliferation were investigated. Ten areas of the liver tissues for each section were chosen within random high-power fields using a Nikon Optiphot 2 light microscope incorporating a square graticule in the eyepiece (eyepiece 10, objective 20, a total side length of 0.05 mm) and the ductular proliferation scores were grouped as: no ductular proliferation (scored as 0), mild ductular proliferation restricted to the portal area (scored as 1), marked ductular proliferation in the porta–portal bridges (scored as 2). Fibrosis was assessed in sections and scored as: no fibrosis (0), portal fibrosis (1), septal fibrosis (2), incomplete cirrhosis (3) and complete cirrhosis (4). The results for each area were calculated and recorded as the ductular proliferation and fibrosis/mm2. Histopathological examination was carried out by a pathologist who had no prior knowledge of the coding of the animal groups.
Biochemical procedures Immunohistochemical procedures Total (Cu–Zn and Mn) superoxide dismutase activity was determined according to the method of Sun et al. (1988) and expressed as U/mg protein.
Immunohistochemical procedures were performed on paraffin wax embedded sections according to the
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ABC technique described by Hsu et al. (1981). The procedures involved the following steps: (1) inhibition of endogenous peroxidase activity with 3% H2O2 in distilled water for 30 min; (2) wash in distilled water for 10 min; (3) blocking of nonspecific binding of antibodies by incubation with normal goat serum (Dako X 0907, Carpinteria, CA) diluted 1:4, for 10 min; (4) incubation with mouse monoclonal anti-actin, smooth muscle antibody (Cat. # MS-113-P, Neomarkers, USA), diluted 1:50 for 1 h, at room temperature; (5) incubation with biotinylated anti-mouse IgG (Dako LSAB 2 Kit), for 15 min; (6) incubation with avidin–biotin complex complex (Dako LSAB 2 Kit), for 15 min; (7) peroxidase was detected with aminoethylcarbazole substrate (AEC kit; Zymed Laboratories, prepared according to kit instructions), 10 min; (8) wash in tap water for 10 min; (9) counterstain of nuclei with hematoxylin; and (10) sections were washed in tap water for 5 min and mounted in Dako paramount. All dilutions and thorough washes between steps were performed using phosphate buffered saline (PBS) unless otherwise specified. All steps were carried out at room temperature unless otherwise specified. As a negative control, primary antibody was replaced with PBS.
Statistical analysis All data are presented as mean (7) standard deviations (S.D.). Normality distribution of the variables was tested using one sample Kolmogorov Smirnov test. Differences among groups were compared using the one way ANOVA test for normal, and then the Bonferroni post-hoc test was used when a significant difference was found. The Kruskal Wallis test for non-normally distributed data, and dual comparisons between groups exhibiting significant values were evaluated with Table 1.
Mann-Whitney U-test. All data for histopathological scores are expressed in a dot-scatter plot. A p-value o0.05 was considered as statistically significant. SPSS/PC+ version 11.0 statistical analysis program (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis.
Results Histopathology The sham-operated group compared to the untreated control showed mild, focal lymphocytic inflammation. After treatment with methylene blue, inflammation was absent, and similar to the control. None of the groups (control, methylene blue, and sham-operated) showed fibrosis or ductular proliferation (score 0). After bile-duct ligation, changes were observed (scores 2–3) (po0.001), including bile-duct proliferation and fibrosis in expanded portal tracts and extension of proliferated bile-ducts into lobules, with mononuclear cells and neutrophil infiltration into the widened portal areas. There was less canalicular proliferation (po0.01) and cell infiltration in the portal areas and less fibrosis (po0.01) in the bile-duct ligated group treated with methylene blue (scores 1–3). The histopathological findings are presented in Table 1 and in Figures 1–5. In the control, methylene blue and sham-operated rats, a-SMA immunoreactivity was restricted to the wall of portal veins, hepatic arterioles and terminal hepatic venules. Positive a-SMA immunoreactivity was increased, especially in vascular smooth muscle cells, fibrotic septa and also around the proliferated bile ducts. Cells with weak a-SMA immunoreactivity were observed in the livers of the bile-duct ligated rats treated with methylene blue (Figure 3a–c).
Histopathological findings: ductular proliferation and fibrosis/mm2. Control
MB+Control
Sham
BDL
BDL+MB
Ductular proliferation scores Mean7SD 0.0070.00 Median (min–max) 0 (0–0)
0.0070.00 0 (0–0)
0.0070.00 0 (0–0)
1.8870.35 2 (1–2)
1.0070.50yy, 1 (0–2)
Fibrosis Mean7SD Median (min–max)
0.0070.00 0 (0–0)
0.0070.00 0 (0–0)
2.6270.50 3(2–3)
1.8870.83yy, 2 (1–3)
0.0070.00 0 (0–0)
n ¼ 8 for each group. MB, methylene blue; BDL, bile-duct ligation. po0.001 compared with control. yy po0.001 compared with BDL. po0.01 compared with BDL.
ARTICLE IN PRESS Effects of methylene blue in reducing cholestatic oxidative stress
Figure 1. Liver sections from: (a) control, methylene blue-treated control and sham-operated rats; (b) after bile-duct ligation the rat liver showing marked ductular proliferation, cell infiltration and fibrosis; (c) bile-duct ligation-induced histopathologic changes in rat liver were reduced as a result of the methylene blue treatment. (Masson’s trichrome staining; scale bar, 50 mm).
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Figure 2. Liver sections from: (a) control, methylene blue-treated and sham-operated groups, showing regular liver parenchyma with hepatocytes and sinusoids; (b) after bile-duct ligation, rat liver developed marked fibrosis and bile-duct proliferation within the portal tract and mononuclear inflammatory infiltration in the parenchyma; (c) after bile-duct ligation and methylene blue treatment there was only moderate fibrosis, less mononuclear inflammatory infiltration and decreased bile-duct proliferation. (Masson’s trichrome staining; scale bar, 50 mm).
ARTICLE IN PRESS B. Aksu et al.
Ductular Proliferation Scores
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3
2
1
0 1
2
3
4
5
Groups
Figure 4. Ductular proliferation with histopathological scores expressed in a dot-scatter plot. The ductular proliferation scores significantly increased in the bileduct ligation group, whereas in the methylene bluetreated group, the ductular proliferation scores significantly decreased. (1) Control group; (2) methylene blue-treated control group; (3) sham-operated group; (4) bile-duct ligation group; (5) bile-duct ligation plus methylene blue-treated group (n ¼ 8 for each group).
Fibrosis Scores
3
2
1
0 1
Figure 3. a-SMA immunolocalization in rat liver tissue. (a) Control, methylene blue-treated control and shamoperated rats showed a-SMA immunolocalization only in vascular smooth muscle cells; (b) after bile-duct ligation, a-SMA immunolocalization was seen especially in vascular smooth muscle cells, fibrotic septa and also around the proliferated bile ducts; (c) treatment of bile-duct ligated rats with methylene blue resulted in decreased a-SMA immunopositivity. (immunoperoxidase, hematoxylin counterstain; scale bar, 50 mm)
2
3 4 Groups
5
Figure 5. Fibrosis with histopathological scores expressed in a dot-scatter plot. The fibrosis scores significantly increased in the bile-duct ligation group, whereas in the methylene blue-treated group, the fibrosis scores significantly decreased. (1) Control group; (2) methylene blue-treated control group; (3) shamoperated group; (4) bile-duct ligation group; (5) bile-duct ligation plus methylene blue-treated group (n ¼ 8 for each group).
Biochemical findings Serum alanine aminotransferase, aspartate aminotransferase, gamma glutamine transferase, direct bilirubin and total bilirubin were elevated significantly in the bile-duct ligated group
ARTICLE IN PRESS Effects of methylene blue in reducing cholestatic oxidative stress compared to the sham-operated group (po0.001). With methylene blue treatment following bile-duct ligation, these parameters were reduced (po0.01) (Table 2). In the bile-duct ligation group, tissue superoxide dismutase activity was significantly lower (po0.01) and malondialdehyde and nitric oxide levels were higher (po0.001, po0.05, respectively) than in the control group. Methylene blue treatment led to significant increases in mean tissue superoxide dismutase activity (po0.01). Methylene blue treatment significantly lowered the levels of malondialdehyde and nitric oxide induced by bile-duct ligation (po0.001 and po0.05, respectively) (Table 3). Myeloperoxidase levels of the liver tissue were found to be increased significantly in the bile-duct ligated group (po0.05). Treatment with methylene blue prevented neutrophilic infiltration, since myeloperoxidase levels of this group were similar to those of the control group (po0.05), (Table 3).
Table 2.
ALT AST TB DB GGT
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Bile-duct ligation produced a significant rise in hydroxyproline levels as compared to the control group after 14 days (po0.001). The increase in the liver hydroxyproline levels after bile-duct ligation was prevented by methylene blue (po0.001, Table 3).
Discussion Cholestasis, defined as a decrease in bile flow, occurs in a wide variety of human liver diseases (Trauner et al., 1998) and may result from biliary tract obstruction or hepatic parenchymal disease. Bile-duct ligation and scission induces a type of liver fibrosis that etiologically and pathogenically resembles human biliary fibrosis. Improved understanding of the cellular and molecular mechanisms of injury may enhance approaches to therapy. Injury to hepatocytes results in the generation of
Serum values of ALT (U/l), AST (U/l), TB (mg/dl), DB (mg/dl) and, GGT (U/l) for each group. Control
MB+Control
Sham
BDL
BDL+MB
50.5075.35 112.5778.12 0.3070.01 0.1070.01 3.0070.01
56.3376.09 118.1776.52 0.3770.16 0.1770.09 2.3370.41
48.7575.05 128.7677.58 0.30.70.01 0.1070.01 2.9070.08
214.00730.61 952.877166.57 8.2871.81 6.5571.71 29.7576.20
112.63713.32yy, 406.757106.34yy, 3.5270.66yy, 2.7170.72yy, 7.6372.55yy,
MB, methylene blue; BDL, bile-duct ligation; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TB, total bilirubin; DB, direct bilirubin; GGT, gamma glutamine transferase. Values are expressed as mean7SD. po0.001 compared with control. yy po0.01 compared with BDL. po0.05 compared with control.
Table 3. Liver tissue determinations for HP (mg/g wet tissue), MDA (nmol/100 mg protein), SOD (U/100 mg protein), NO (nmol/mg protein) and MPO (mU/g protein) for each group. Control SOD MDA NO HP MPO
98.78712.49 1.2770.46 399.72750.75 215.67737.66 42.8075.34
MB+Control 87.6779.04 1.0470.66 384.67748.43 235.50763.93 42.5073.87
Sham
BDL
BDL+MB
93.17711.70 1.1070.13 423.46738.98 228739.49 45.4373.87
53.1479.58 4.9171.34 584.29743.50 750.007107.20 64.0076.55
92.5077.24yy 2.6270.38yyy 413.87755.56y 315.25765.68yyy 48.3775.39y
MB, methylene blue; BDL, bile-duct ligation; SOD, superoxide dismutase; MDA, malondialdehyde; NO, nitric oxide; HP, hydroxyproline; MPO, myeloperoxidase. Values are expressed as mean7SD. po0.001 compared with control. po0.01 compared with control. po0.05 compared with control. yyy po0.001 compared with BDL. yy po0.01 compared with BDL. y po0.05 compared with BDL.
ARTICLE IN PRESS 266 lipid peroxides, which may have a direct stimulatory effect on extracellular matrix production by activated stellate cells (Kountouras et al., 1984; Liu et al., 2001). Liu et al. (2001) demonstrated excessive production of superoxide radicals and hydroxyl radicals in blood and liver in rats with obstructive jaundice induced by common bile-duct ligation. In the present study, bile-duct ligation caused significant increases in serum levels of alanine aminotransferase, aspartate aminotransferase, gamma glutamine transferase, total bilirubin and direct bilirubin, and these were attenuated following methylene blue treatment. Bile-duct ligation increased liver levels of malondialdehyde, myeloperoxidase and hydroxyproline, and these were lower following methylene blue treatment. Bile-duct ligation caused decreased superoxide dismutase activities in hepatic tissue and after methylene blue treatment this was reversed. Bile-duct ligation is associated with the development of oxidant injury. This is a dynamic process with different rates of progression or regression (Marley et al., 1999). Previous studies (Sener et al., 2005, 2007), which showed increased production of reactive oxidants in hepatic tissues monitored by chemiluminescence assay, provide additional support for the viewpoint that the generation of free radicals plays a key role in hepatic injury after bileduct ligation. Several antioxidants have been tested in experimental bile-duct obstruction models in attempts to limit the oxidative damage (Gedik et al., 2005; Sener et al., 2007). Methylene blue can act as an electron acceptor for xanthine oxidase. It might inhibit the production of superoxide in postischemic tissue by competing with molecular oxygen at the iron–sulfur centers of xanthine oxidase by enabling anaerobic oxidation of purine substrates (Kelner et al., 1988a, b), or may act as a ‘‘parasitic’’ electron acceptor, shunting electron flow from the normal pathway to the colorless, reduced form of methylene blue, i.e., leukomethylene blue, and, thus, effectively by-passing the generation of reactive oxygen species (Salaris et al., 1991). Malondialdehyde can be determined in tissues and its concentration is directly proportional to the cell damage caused by free radicals (Aksu et al., 2007). Methylene blue has been used to induce, rather than inhibit, lipid peroxidation in the presence of light by increasing singlet oxygen formation (Kamat and Devasagayam, 1996). We observed a significant increase in malondialdehyde concentrations in the liver tissue of bile-duct ligated rats, though this was prevented to a large degree after methylene blue treatment. Recent
B. Aksu et al. studies have indicated that disturbance of the oxidant–antioxidant balance may be responsible for cholestatic liver injury and that superoxide dismutase enzymatic activities are hepatoprotective and play important roles in preventing oxidative stress (Roeb et al., 2003). We found decreased superoxide dismutase activities in bile-duct ligated rats as compared to control rats and the methylene blue treatment reversed these changes. All these results may be due to the effects of methylene blue in reducing reactive oxygen species. Tissue myeloperoxidase activities have been used as biochemical markers for the tissue content of polymorphonuclear leukocytes (Gedik et al., 2005; Karaman et al., 2006). Methylene blue was shown to be beneficial in preventing the accumulation of polymorphonuclear leukocytes in the lung after intestinal ischemia-reperfusion in rats (Galili et al., 1998). The strong inhibitory effects of methylene blue on myeloperoxidase activity support the view that leukocyte-derived inflammatory mediators are integrally involved in adhesiogenesis (Heydrick et al., 2007). In our study, bile-duct ligation produced a significant increase in liver tissue myeloperoxidase activities reflecting the leukocyte accumulation in the liver tissue as compared to the control animals and this was confirmed by histopathological evaluation. We showed that methylene blue treatment produced a marked decrease in the enhancement of myeloperoxidase activity due to bile-duct ligation. The inhibitory effects of methylene blue may reflect the reduction in migration of neutrophils and other inflammatory cells in the inflamed region. Nitric oxide is a highly reactive mediator released in the liver by endothelial cells, macrophages, hepatocytes and Kupffer cells in response to various stimuli (Mayoral et al., 1999). Increased serum levels of nitric oxide are found in patients with cirrhosis (Guarner et al., 1993), as well as in bile-duct ligated cirrhotic rats (Farzaneh-Far and Moore, 2001). The main effect of methylene blue is inhibition of guanylate cyclase, the target site of nitric oxide. Methylene blue is known to have additional pharmacological actions, including the generation of oxygen radicals and direct inhibition of nitric oxide synthase (Mayer et al., 1993). The biological role of nitric oxide in the liver has been extensively studied, and the literature indicates that nitric oxide is either a primary mediator of liver cell injury or responds as a potent protective mechanism against deleterious stimuli (Clemens, 1999; Muriel., 2006). The present study indicated marked elevation in nitric oxide levels in the bileduct ligated rats, which was significantly attenuated after methylene blue treatment.
ARTICLE IN PRESS Effects of methylene blue in reducing cholestatic oxidative stress Bile-duct ligation in rats induces portal fibrosis, which begins with an early proliferation of biliary duct epithelial cells and portal periductular fibroblasts (Tuchweber et al., 1996). It is known that a consequence of bile-duct ligation is the rapid appearance of significant hepatic fibrosis (Strazzabosco et al., 2005). Hepatic fibrosis is characterized by increased production and deposition of extracellular matrix components accompanying most chronic liver disorders and is regarded as a major factor contributing to hepatic failure (Fort et al., 1998). It is known that oxidative events, especially lipid peroxidation, may play a pivotal role in the regulation of collagen accumulation. Chojkier et al. (1998) reported that products of lipid peroxidation modulate collagen gene expression and serve as a link between liver injury and fibrosis. We propose that the addition of methylene blue to antibiotic therapy may reduce fibrosis by preventing induced oxidative renal tissue damage in pyelonephritis (Aksu et al., 2007). Heydrick et al. (2007) showed that methylene blue inhibits adhesion formation via a mechanism that might involve blocking an oxidative stress-dependent decrease in peritoneal fibrinolytic activity. Methylene blue, which is an inhibitor of soluble guanylate cyclase, is known to depress the production of intracellular cyclic guanosine monophosphate, reversing the inhibition of collagen production (Houglum et al., 1991). Some studies, however, showed that methylene blue alone did not have any significant effect on collagen production (Chu and Prasad, 1999). Liver fibrosis was assessed by measuring hydroxyproline content in liver as an index of collagen accumulation. In our study, methylene blue treatment significantly reduced the increase in hydroxyproline content caused by bile-duct ligation. Its mechanism may be explained as a consequence of the antiinflammatory and antioxidant capabilities of methylene blue. In addition, this is the first study to show the protective effect of methylene blue on the development of bile-duct ligation-induced hepatic damage as determined by pathological evaluation, and measurement of inflammatory and oxidative stress parameters in the liver. This effect may be due to the inhibition of inflammatory cells accumulating in the liver and in particular may be due to the scavenging of reactive oxygen radicals by methylene blue itself. Although the mechanism of liver fibrosis is not fully understood, activated hepatic stellate cells play an important role in connective tissue synthesis and deposition during fibrogenesis (Nan et al., 2000). Fibroblasts expressing a-SMA may be derived from the transdifferentiation of quiescent hepatic stellate cells. The activation of hepatic stellate
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cells involves increased cellular proliferation, increased synthesis of extracellular matrix proteins and the expression of the activation marker a-SMA (Miyazaki et al., 1993). Extracellular matrices in liver fibrosis are known to be produced by myofibroblasts that are transformed from fatstoring cells. The development of the fibrotic process is thought to be mediated by various fibrogenic mediators (Akiyoshi and Terada, 1998). After bile-duct ligation, positive immunolabelling for a-SMA increased, especially in vascular smooth muscle cells, fibrotic septa and also around the proliferated bile ducts. A weak immunopositivity for a-SMA was observed in livers after bile-duct ligation and methylene blue treatment. In conclusion, these findings indicate that methylene blue can attenuate hepatic damage in extrahepatic cholestasis in experimental rats by reducing oxidative stress and inflammatory processes and provide an indication of the possible therapeutic use of methylene blue for cholestatic liver injury.
References Akiyoshi H, Terada T. Mast cell, myofibroblast and nerve terminal complexes in carbon tetrachloride-induced cirrhotic rat livers. J Hepatol 1998;29:112–9. Aksu B, Inan M, Kanter M, Oz Puyan F, Uzun H, DurmusAltun G, et al. The effects of methylene blue on renal scarring due to pyelonephritis in rats. Pediatr Nephrol 2007;22:992–1001. Altman P, Buchmiller TL. The jaundiced infant: biliary atresia. In: Grosfeld JL, O’Neill JA, Fonkalsrud EW, Coran AG, editors. Pediatric surgery. Mosby-Year Book; 2006. p. 1603–19. Benedetti A, Alvaro D, Bassotti C, Gigliozzi A, Ferretti G, La Rosa T, et al. Cytotoxicity of bile salts against biliary epithelium: a study in isolated bile ductule fragments and isolated perfused rat liver. Hepatology 1997;26:9–21. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302–10. Cassiman D, Libbrecht L, Desmet V, Denef C, Roskams T. Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers. J Hepatol 2002;36: 200–9. Chojkier M, Houglum K, Lee KS, Buck M. Long and shortterm D-a-tocopherol supplementation inhibits liver collagen a1(I) gene expression. Am J Physiol 1998;275: 1480–5. Chu AJ, Prasad JK. Up-regulation by human recombinant transforming growth factor beta-1 of collagen production in cultured dermal fibroblasts is mediated by the inhibition of nitric oxide signaling. J Am Coll Surg 1999;18:271–80.
ARTICLE IN PRESS 268 Clemens MG. Nitric oxide in liver injury. Hepatology 1999;30:1–5. Cortas NK, Wakid NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990;36:1440–3. Cruz A, Padillo FJ, Granados J, Tunez I, Munoz MC, Briceno J, et al. Effect of melatonin on cholestatic oxidative stress under constant light exposure. Cell Biochem Funct 2003;21:377–80. Engin A, Bozkurt B, Altan N, Memis L, Bukan N. Nitric oxide-mediated liver injury in the presence of experimental bile duct obstruction. World J Surg 2003;27: 253–5. Farzaneh-Far R, Moore K. Nitric oxide and the liver. Liver 2001;21:161–74. Fort J, Oberti F, Pilette C, Veal N, Gallois Y, Douay O, et al. Antifibrotic and hemodynamic effects of the early and chronic administration of octreotide in two models of liver fibrosis in rats. Hepatology 1998;28: 1525–31. Galili Y, Ben-Abraham R, Weinbroum A, Klausner J, Rabau M, Kluger Y, et al. Methylene blue prevents pulmonary injury following intestinal ischemia reperfusion. J Trauma 1998;45:222–5. Gedik N, Kabasakal L, Sehirli O, Ercan F, Sirvanci S, KeyerUysal M, et al. Long-term administration of aqueous garlic extract (AGE) alleviates liver fibrosis and oxidative damage induced by biliary obstruction in rats. Life Sci 2005;76:2593–606. Guarner C, Soriano G, Tomas A, Bulbena O, Novella MT, Balanzo J, et al. Increased serum nitrite and nitrate levels in patients with cirrhosis: relationship to endotoxemia. Hepatology 1993;18:1139–43. Heydrick SJ, Reed KL, Cohen PA, Aarons CB, Gower AC, Becker JM, et al. Intraperitoneal administration of methylene blue attenuates oxidative stress, increases peritoneal fibrinolysis, and inhibits intraabdominal adhesion formation. J Surg Res 2007;143:311–9 [Epub 2007 Sep 10]. Houglum K, Brenner DA, Chojkier M. d-alpha-tocopherol inhibits collagen alpha 1(I) gene expression in cultured human fibroblasts. Modulation of constitutive collagen gene expression by lipid peroxidation. J Clin Invest 1991;87:2230–5. Hsu SM, Raine L, Fanger H. Use of avidin–biotin–peroxidase complex (ABC) in immunperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29: 577–80. Kamat JP, Devasagayam TP. Methylene blue plus light induced lipid peroxidation in rat liver microsomes: inhibition by nicotinamide (vitamin B3) and other antioxidants. Chem Biol Interact 1996;99:1–16. Karaman A, Iraz M, Kirimlioglu H, Karadag N, Tas E, Fadillioglu E. Hepatic damage in biliary-obstructed rats is ameliorated by leflunomide treatment. Pediatr Surg Int 2006;22:701–8. Kelner MJ, Bagnell R, Hale B, Alexander NM. Potential of methylene blue to block oxygen radical generation in reperfusion injury. Basic Life Sci 1988a;49:895–8.
B. Aksu et al. Kelner MJ, Bagnell R, Hale B, Alexander NM. Methylene blue competes with paraquat for reduction by flavoenzymes resulting in decreased superoxide production in the presence of heme proteins. Arch Biochem Biophys 1988b;262:422–6. Kountouras J, Billing BH, Scheuer PJ. Prolonged bileduct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 1984;65:305–11. Liu TZ, Lee KT, Chern CL, Cheng JT, Stern A, Tsai LY. Free radical-triggered hepatic injury of experimental obstructive jaundice of rats involves overproduction of proinflammatory cytokines and enhanced activation of nuclear factor kappaB. Ann Clin Lab Sci 2001;31:383–90. Marley R, Holt S, Fernando B, Harry D, Anand R, Goodier D, et al. Lipoic acid prevents development of the hyperdynamic circulation in anesthetized rats with biliary cirrhosis. Hepatology 1999;29:1358–63. Mayer B, Brunner F, Schmidt K. Inhibition of nitric oxide synthesis by methylene blue. Biochem Pharmacol 1993;45:367–74. Mayoral P, Criado M, Hidalgo F, Flores O, Are´valo MA, Eleno N, et al. Effects of chronic nitric oxide activation or inhibition on early hepatic fibrosis in rats with bile duct ligation. Clin Sci 1999;96:297–305. Miyazaki H, Van Eyken P, Roskams T, De Vos R, Desmet VJ. Transient expression of tenascin in experimentally induced cholestatic fibrosis in rat liver: an immunohistochemical study. J Hepatol 1993;19:353–66. Muriel P. Role of nitric oxide in liver disorders. In: Shakir A, Friedman SL, Derek AM, editors. Liver diseases: biochemical mechanisms and new therapeutic insights. New Delhi: Oxford & IBH Publishing; 2006. p. 127–43. Nan JX, Park EJ, Kim HJ, Ko G, Sohn DH. Antifibrotic effects of the methanol extract of Polygonum aviculare in fibritic rats induced by bile duct ligation and scission. Biol Pharm Bull 2000;23:240–3. Ramm GA, Carr SC, Bridle KR, Li L, Britton RS, Crawford DH, et al. Morphology of liver repair following cholestatic liver injury: resolution of ductal hyperplasia, matrix deposition and regression of myofibroblasts. Liver 2000;20:387–96. Roeb E, Purucker E, Gartung C, Geier A, Jansen B, Winograd R, et al. Effect of glutathione depletion and hydrophilic bile acids on hepatic acute phase reaction in rats with extrahepatic cholestasis. Scand J Gastroenterol 2003;38:878–85. Salaris SC, Babbs CF, Voorhees WD III. Methylene blue as an inhibitor of superoxide generation by xanthine oxidase. A potential new drug for the attenuation of ischemia/reperfusion injury. Biochem Pharmacol 1991;42:499–506. Sener G, Kabasakal L, Yuksel M, Gedik N, Alican Y. Hepatic fibrosis in biliary-obstructed rats is prevented by Ginkgo biloba treatment. World J Gastroenterol 2005;11:5444–9. Sener G, Sehirli AO, Toklu HZ, Yuksel M, Ercan F, Gedik N. Erdosteine treatment attenuates oxidative stress and fibrosis in experimental biliary obstruction. Pediatr Surg Int 2007;23:233–41.
ARTICLE IN PRESS Effects of methylene blue in reducing cholestatic oxidative stress Serviddio G, Pereda J, Pallardo FV, Carretero J, Borras C, Cutrin J, et al. Ursodeoxycholic acid protects against secondary biliary cirrhosis in rats by preventing mitochondrial oxidative stress. Hepatology 2004;39: 711–20. Strazzabosco M, Fabris L, Spirli C. Pathophysiology of cholangiopathies. J Clin Gastroenterol 2005;39: 90–102. Sun Y, Oberley LW, Li YA. Simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497–500. Trauner M, Meier PJ, Boyer JL. Molecular pathogenesis of cholestasis. N Engl J Med 1998;339:1217–27.
269
Tuchweber B, Desmoulie `re A, Bochaton-Piallat ML, Rubbia-Brandt L, Gabbiani G. Proliferation and phenotypicmodulation of portal fibroblasts in the early stages of cholestatic fibrosis in the rat. Lab Invest 1996;74:265–78. Wei H, Frenkel K. Relationship of oxidative events and DNA oxidation in SENCAR mice to in vivo promoting activity of phorbol ester-type tumor promoters. Carcinogenesis 1993;14:1195–201. Woessner JB. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys 1961;93:440–7.
www.elsevier.de/acthis
Effects of methylene blue in reducing cholestatic oxidative stress and hepatic damage after bile-duct ligation in rats Burhan Aksua,, Hasan Umitb, Mehmet Kanterc, Ahmet Guzeld, Cevat Aktasc, Sabiha Civeleke, Hafize Uzune a
Department of Pediatric Surgery, Faculty of Medicine, Trakya University, Edirne 22030, Turkey Department of Gastroenterology, Trakya University, Edirne 22030, Turkey c Department of Histology and Embryology, Trakya University, Edirne 22030, Turkey d Department of Pediatrics, Faculty of Medicine, Trakya University, Edirne 22030, Turkey e Department of Biochemistry, Cerrahpasa Faculty of Medicine, Istanbul University, Turkey b
Received 16 September 2008; received in revised form 21 November 2008; accepted 2 December 2008
KEYWORDS Methylene blue; Extrahepatic cholestasis; Oxidative stress; Hepatic fibrosis; Bile-duct ligation; a-smooth muscle actin; Rats
Summary The aim of this study was to evaluate the effects of methylene blue against cholestatic oxidative stress and liver damage after ligation of the common bile duct in male Wistar rats. Eight animals were included in each of the following five groups: untreated control, methylene blue control, sham-operated, bile-duct ligation, and bile-duct ligation plus methylene blue. Methylene blue was administered intraperitoneally for 14 days at a daily dose of 2 mg/kg per day. All rats were sacrificed 2 weeks following the experimental treatment and the livers of all groups were examined biochemically and histopathologically. The severity of cholestasis and hepatic injury were determined by changes in the plasma, including enzymatic activities: aspartate aminotransferase, alanine aminotransferase, gamma glutamine transferase, and also bilirubin levels. Malondialdehyde, nitric oxide and superoxide dismutase were measured to indicate the oxidative status in the liver tissue. Myeloperoxidase activity and levels of tissue hydroxyproline were determined as measures of neutrophil activation and collagen accumulation, respectively. Liver damage was significantly prevented in the bile-duct ligated rats treated with methylene blue compared with the control bile-duct ligated rats without methylene blue. Treatment with methylene blue markedly reduced activities of serum transaminase, gamma glutamine transferase and bilirubin levels as compared to bile-duct ligated rats without methylene blue. Positive immunolabelling for alphasmooth muscle actin (a-SMA) was increased, especially in vascular smooth muscle
Corresponding author. Tel.: +902842357641; fax: +902842352730.
E-mail address: [email protected] (B. Aksu). 0065-1281/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2008.12.002
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B. Aksu et al. cells, fibrotic septa and also around the proliferated bile ducts, after bile-duct ligation. Only weak a-SMA immunolabelling was seen in livers of rats treated with methylene blue. These results indicate that methylene blue can attenuate hepatic damage in extrahepatic cholestasis by reducing oxidative stress and inflammatory processes. & 2008 Elsevier GmbH. All rights reserved.
Introduction Cholestasis results from structural and functional impairment of the hepatobiliary system, which occurs as a result of several environmental factors and disease processes. Evaluation of the extrahepatic biliary system for patency is a high priority when treating infants or children with cholestasis as biliary atresia occurs in a significant percentage of these young patients (Altman and Buchmiller, 2006). Obstruction of bile flow through the extrahepatic biliary system results in subsequent oxidative injury, hepatic fibrosis, biliary cirrhosis and portal hypertension (Kountouras et al., 1984). Experimental bile-duct ligation induces a form of liver fibrosis, which etiologically and pathogenically resembles biliary fibrosis in humans. Injury to hepatocytes results in the generation of lipid peroxides, which may have a direct stimulatory effect on extracellular matrix production by activated stellate cells (Serviddio et al., 2004). Complete biliary obstruction causes cholestatic injury to the liver, including hepatocellular necrosis and apoptosis, bile-duct epithelial cell proliferation, stellate cell activation and eventually liver fibrosis (Kountouras et al., 1984). Following cholestatic injury, the liver undergoes a tissue remodeling process that combines regeneration and fibrogenesis. During this repair process, the extracellular matrix contains large numbers of alphasmooth muscle actin (a-SMA) immunoreactive cells known as myofibroblasts; however their origin still remains enigmatic. Cassiman et al. (2002) and Ramm et al. (2000) demonstrated that the a-SMA immunopositive cells mainly reside in the portal ducts and fibrous septa and their location corresponds to the distribution of collagen. Although the mechanisms involved in cholestasisinduced liver fibrosis are unclear, obstruction of bile flow causes the accumulation and retention of hydrophobic bile salts in the liver, which are toxic to many cell types, including the hepatocytes and ductal biliary epithelial cells (Benedetti et al., 1997). Bile-duct ligation is characterized by increased lipid peroxidation and by a marked decline in reduced glutathione (GSH), a major cellular antioxidant (Cruz et al., 2003). It is known that
bile-duct ligation results in a shift in the oxidant/ pro-oxidant balance in favor of increased activities of free radicals (Cruz et al., 2003). Enhanced production of reactive oxygen intermediates augments lipid peroxidation by disturbing the oxidant– antioxidant balance in the hepatic mitochondrial fraction. The pattern of damage due to interference of free radicals with hepatocytes indicates peroxynitrite-mediated liver injury (Engin et al., 2003). Methylene blue is a dye that competes with molecular oxygen for the transfer of electrons from flavoenzymes, primarily xanthine oxidase (Salaris et al., 1991). The shunting of electrons to and from the colorless reduced leukomethylene blue diverts their flow from the metal sulfur center of the enzyme, where molecular oxygen is normally converted into superoxide radicals, and the generation of these cytotoxic mediators is attenuated (Salaris et al., 1991; Aksu et al., 2007). The aim of the present study was to evaluate the possible protective effects of methylene blue on oxidative damage in hepatic tissue of rats after experimental bile-duct ligation.
Materials and methods Animals All animal procedures and experimental protocols used in the study were approved by the Ethical Committee of Trakya University. A total of 40 adult male Wistar albino rats, weighing 200–250 g, were used in this study. Rats were provided by the Experimental Research Center of the Medical Faculty of Trakya University and maintained in a windowless animal facility with controlled conditions of temperature (2272 1C), illumination (lights on at 07.00 and off at 21.00) and humidity (50–55%). Rats were fed a standard rat chow and tap water ad libitum. Eight animals were included in each of the following five groups (n ¼ 8): (1) control: normal saline treatment without surgical intervention; (2) methylene blue control: methylene blue treatment without surgical intervention;
ARTICLE IN PRESS Effects of methylene blue in reducing cholestatic oxidative stress (3) sham-operated: dissection of the bile duct, treated with normal saline; (4) bile-duct ligation: double ligature and section of the extrahepatic bile duct; (5) bile-duct ligation plus methylene blue: double ligature and section of the extrahepatic bile duct, treated with methylene blue.
Experimental procedures The rats were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) intraperitoneally (i.p.) and their common bile ducts were exposed through a midline abdominal incision. The common bile duct was located and obstructive jaundice induced by a double ligation with 5/0 silk and transsection of the common bile duct in the supraduodenal region between the lowermost tributary of the bile duct and the uppermost tributary of the pancreatic duct. The shamoperated group of rats underwent opening of the abdominal cavity and dissection of the common bile duct without ligation. Methylene blue (Merck, Darmstadt, Germany) was dissolved and diluted in distilled water to a final concentration of 2%. The methylene blue stock solution was stored at 4 1C. The methylene blue was administered to the bile duct ligated plus methylene blue rat group immediately after bile-duct ligation and at the same time point to the methylene blue control rat group, and from then on for 14 days at a daily dose of 2 mg/kg i.p. The sham-operated control and bileduct ligation groups received equal amounts of normal saline intraperitoneally for 14 days. The rats were killed by decapitation after 14 days of treatment. After opening the abdominal cavity, blood was collected from all rats to determine activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamine transferase (GGT), and also the levels of total bilirubin (TB) and direct bilirubin (DB). The liver tissue samples were immediately immersion-fixed in 10% neutral buffered formalin solution for histological evaluation or stored at 80 1C for subsequent spectrophotometric determination of activities of superoxide dismutase (SOD), and levels of malondialdehyde (MDA) and nitric oxide (NO). Indirect evidence of neutrophil infiltration was determined by measuring tissue-associated myeloperoxidase activity. Collagen accumulation was assessed by measuring hydroxyproline content in liver samples.
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Nitric oxide (NO) has a half-life of only a few seconds, because it is readily oxidized to nitrite (NO 2 ) and subsequently to nitrate (NO3 ), which serve as index parameters of NO production. The method for determining plasma nitrite and nitrate levels was based on Cortas and Wakid (1990) with results expressed as mmol/g wet tissue. Myeloperoxidase activity was determined using 4-aminoantipyrine/phenol solution as the substrate for myeloperoxidase-mediated oxidation by H2O2 according to Wei and Frenkel (1993) and presented as mU/g protein. Liver malondialdehyde levels were determined by a method based on the thiobarbituric acid reactive substances reaction with thiobarbituric acid at 90–100 1C (Buege and Aust, 1978). The results are expressed as nmol/g wet tissue. The tissue samples taken for hydroxyproline determination were washed with normal saline and oven-dried at 100 1C for 72 h. Hydroxyproline levels were determined spectrophotometrically using the method of Woessner (1961). The serum activities of alanine aminotransferase, aspartate aminotransferase, gamma glutamine transferase, total bilirubin and direct bilirubin were determined using commercially available kits.
Histological evaluation Formalin-fixed liver tissues were processed for embedding in paraffin wax, sections cut at 5 mm and then stained with Masson’s trichrome technique. Lobular architecture, presence of inflammation, fibrosis and ductular proliferation were investigated. Ten areas of the liver tissues for each section were chosen within random high-power fields using a Nikon Optiphot 2 light microscope incorporating a square graticule in the eyepiece (eyepiece 10, objective 20, a total side length of 0.05 mm) and the ductular proliferation scores were grouped as: no ductular proliferation (scored as 0), mild ductular proliferation restricted to the portal area (scored as 1), marked ductular proliferation in the porta–portal bridges (scored as 2). Fibrosis was assessed in sections and scored as: no fibrosis (0), portal fibrosis (1), septal fibrosis (2), incomplete cirrhosis (3) and complete cirrhosis (4). The results for each area were calculated and recorded as the ductular proliferation and fibrosis/mm2. Histopathological examination was carried out by a pathologist who had no prior knowledge of the coding of the animal groups.
Biochemical procedures Immunohistochemical procedures Total (Cu–Zn and Mn) superoxide dismutase activity was determined according to the method of Sun et al. (1988) and expressed as U/mg protein.
Immunohistochemical procedures were performed on paraffin wax embedded sections according to the
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ABC technique described by Hsu et al. (1981). The procedures involved the following steps: (1) inhibition of endogenous peroxidase activity with 3% H2O2 in distilled water for 30 min; (2) wash in distilled water for 10 min; (3) blocking of nonspecific binding of antibodies by incubation with normal goat serum (Dako X 0907, Carpinteria, CA) diluted 1:4, for 10 min; (4) incubation with mouse monoclonal anti-actin, smooth muscle antibody (Cat. # MS-113-P, Neomarkers, USA), diluted 1:50 for 1 h, at room temperature; (5) incubation with biotinylated anti-mouse IgG (Dako LSAB 2 Kit), for 15 min; (6) incubation with avidin–biotin complex complex (Dako LSAB 2 Kit), for 15 min; (7) peroxidase was detected with aminoethylcarbazole substrate (AEC kit; Zymed Laboratories, prepared according to kit instructions), 10 min; (8) wash in tap water for 10 min; (9) counterstain of nuclei with hematoxylin; and (10) sections were washed in tap water for 5 min and mounted in Dako paramount. All dilutions and thorough washes between steps were performed using phosphate buffered saline (PBS) unless otherwise specified. All steps were carried out at room temperature unless otherwise specified. As a negative control, primary antibody was replaced with PBS.
Statistical analysis All data are presented as mean (7) standard deviations (S.D.). Normality distribution of the variables was tested using one sample Kolmogorov Smirnov test. Differences among groups were compared using the one way ANOVA test for normal, and then the Bonferroni post-hoc test was used when a significant difference was found. The Kruskal Wallis test for non-normally distributed data, and dual comparisons between groups exhibiting significant values were evaluated with Table 1.
Mann-Whitney U-test. All data for histopathological scores are expressed in a dot-scatter plot. A p-value o0.05 was considered as statistically significant. SPSS/PC+ version 11.0 statistical analysis program (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis.
Results Histopathology The sham-operated group compared to the untreated control showed mild, focal lymphocytic inflammation. After treatment with methylene blue, inflammation was absent, and similar to the control. None of the groups (control, methylene blue, and sham-operated) showed fibrosis or ductular proliferation (score 0). After bile-duct ligation, changes were observed (scores 2–3) (po0.001), including bile-duct proliferation and fibrosis in expanded portal tracts and extension of proliferated bile-ducts into lobules, with mononuclear cells and neutrophil infiltration into the widened portal areas. There was less canalicular proliferation (po0.01) and cell infiltration in the portal areas and less fibrosis (po0.01) in the bile-duct ligated group treated with methylene blue (scores 1–3). The histopathological findings are presented in Table 1 and in Figures 1–5. In the control, methylene blue and sham-operated rats, a-SMA immunoreactivity was restricted to the wall of portal veins, hepatic arterioles and terminal hepatic venules. Positive a-SMA immunoreactivity was increased, especially in vascular smooth muscle cells, fibrotic septa and also around the proliferated bile ducts. Cells with weak a-SMA immunoreactivity were observed in the livers of the bile-duct ligated rats treated with methylene blue (Figure 3a–c).
Histopathological findings: ductular proliferation and fibrosis/mm2. Control
MB+Control
Sham
BDL
BDL+MB
Ductular proliferation scores Mean7SD 0.0070.00 Median (min–max) 0 (0–0)
0.0070.00 0 (0–0)
0.0070.00 0 (0–0)
1.8870.35 2 (1–2)
1.0070.50yy, 1 (0–2)
Fibrosis Mean7SD Median (min–max)
0.0070.00 0 (0–0)
0.0070.00 0 (0–0)
2.6270.50 3(2–3)
1.8870.83yy, 2 (1–3)
0.0070.00 0 (0–0)
n ¼ 8 for each group. MB, methylene blue; BDL, bile-duct ligation. po0.001 compared with control. yy po0.001 compared with BDL. po0.01 compared with BDL.
ARTICLE IN PRESS Effects of methylene blue in reducing cholestatic oxidative stress
Figure 1. Liver sections from: (a) control, methylene blue-treated control and sham-operated rats; (b) after bile-duct ligation the rat liver showing marked ductular proliferation, cell infiltration and fibrosis; (c) bile-duct ligation-induced histopathologic changes in rat liver were reduced as a result of the methylene blue treatment. (Masson’s trichrome staining; scale bar, 50 mm).
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Figure 2. Liver sections from: (a) control, methylene blue-treated and sham-operated groups, showing regular liver parenchyma with hepatocytes and sinusoids; (b) after bile-duct ligation, rat liver developed marked fibrosis and bile-duct proliferation within the portal tract and mononuclear inflammatory infiltration in the parenchyma; (c) after bile-duct ligation and methylene blue treatment there was only moderate fibrosis, less mononuclear inflammatory infiltration and decreased bile-duct proliferation. (Masson’s trichrome staining; scale bar, 50 mm).
ARTICLE IN PRESS B. Aksu et al.
Ductular Proliferation Scores
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3
2
1
0 1
2
3
4
5
Groups
Figure 4. Ductular proliferation with histopathological scores expressed in a dot-scatter plot. The ductular proliferation scores significantly increased in the bileduct ligation group, whereas in the methylene bluetreated group, the ductular proliferation scores significantly decreased. (1) Control group; (2) methylene blue-treated control group; (3) sham-operated group; (4) bile-duct ligation group; (5) bile-duct ligation plus methylene blue-treated group (n ¼ 8 for each group).
Fibrosis Scores
3
2
1
0 1
Figure 3. a-SMA immunolocalization in rat liver tissue. (a) Control, methylene blue-treated control and shamoperated rats showed a-SMA immunolocalization only in vascular smooth muscle cells; (b) after bile-duct ligation, a-SMA immunolocalization was seen especially in vascular smooth muscle cells, fibrotic septa and also around the proliferated bile ducts; (c) treatment of bile-duct ligated rats with methylene blue resulted in decreased a-SMA immunopositivity. (immunoperoxidase, hematoxylin counterstain; scale bar, 50 mm)
2
3 4 Groups
5
Figure 5. Fibrosis with histopathological scores expressed in a dot-scatter plot. The fibrosis scores significantly increased in the bile-duct ligation group, whereas in the methylene blue-treated group, the fibrosis scores significantly decreased. (1) Control group; (2) methylene blue-treated control group; (3) shamoperated group; (4) bile-duct ligation group; (5) bile-duct ligation plus methylene blue-treated group (n ¼ 8 for each group).
Biochemical findings Serum alanine aminotransferase, aspartate aminotransferase, gamma glutamine transferase, direct bilirubin and total bilirubin were elevated significantly in the bile-duct ligated group
ARTICLE IN PRESS Effects of methylene blue in reducing cholestatic oxidative stress compared to the sham-operated group (po0.001). With methylene blue treatment following bile-duct ligation, these parameters were reduced (po0.01) (Table 2). In the bile-duct ligation group, tissue superoxide dismutase activity was significantly lower (po0.01) and malondialdehyde and nitric oxide levels were higher (po0.001, po0.05, respectively) than in the control group. Methylene blue treatment led to significant increases in mean tissue superoxide dismutase activity (po0.01). Methylene blue treatment significantly lowered the levels of malondialdehyde and nitric oxide induced by bile-duct ligation (po0.001 and po0.05, respectively) (Table 3). Myeloperoxidase levels of the liver tissue were found to be increased significantly in the bile-duct ligated group (po0.05). Treatment with methylene blue prevented neutrophilic infiltration, since myeloperoxidase levels of this group were similar to those of the control group (po0.05), (Table 3).
Table 2.
ALT AST TB DB GGT
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Bile-duct ligation produced a significant rise in hydroxyproline levels as compared to the control group after 14 days (po0.001). The increase in the liver hydroxyproline levels after bile-duct ligation was prevented by methylene blue (po0.001, Table 3).
Discussion Cholestasis, defined as a decrease in bile flow, occurs in a wide variety of human liver diseases (Trauner et al., 1998) and may result from biliary tract obstruction or hepatic parenchymal disease. Bile-duct ligation and scission induces a type of liver fibrosis that etiologically and pathogenically resembles human biliary fibrosis. Improved understanding of the cellular and molecular mechanisms of injury may enhance approaches to therapy. Injury to hepatocytes results in the generation of
Serum values of ALT (U/l), AST (U/l), TB (mg/dl), DB (mg/dl) and, GGT (U/l) for each group. Control
MB+Control
Sham
BDL
BDL+MB
50.5075.35 112.5778.12 0.3070.01 0.1070.01 3.0070.01
56.3376.09 118.1776.52 0.3770.16 0.1770.09 2.3370.41
48.7575.05 128.7677.58 0.30.70.01 0.1070.01 2.9070.08
214.00730.61 952.877166.57 8.2871.81 6.5571.71 29.7576.20
112.63713.32yy, 406.757106.34yy, 3.5270.66yy, 2.7170.72yy, 7.6372.55yy,
MB, methylene blue; BDL, bile-duct ligation; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TB, total bilirubin; DB, direct bilirubin; GGT, gamma glutamine transferase. Values are expressed as mean7SD. po0.001 compared with control. yy po0.01 compared with BDL. po0.05 compared with control.
Table 3. Liver tissue determinations for HP (mg/g wet tissue), MDA (nmol/100 mg protein), SOD (U/100 mg protein), NO (nmol/mg protein) and MPO (mU/g protein) for each group. Control SOD MDA NO HP MPO
98.78712.49 1.2770.46 399.72750.75 215.67737.66 42.8075.34
MB+Control 87.6779.04 1.0470.66 384.67748.43 235.50763.93 42.5073.87
Sham
BDL
BDL+MB
93.17711.70 1.1070.13 423.46738.98 228739.49 45.4373.87
53.1479.58 4.9171.34 584.29743.50 750.007107.20 64.0076.55
92.5077.24yy 2.6270.38yyy 413.87755.56y 315.25765.68yyy 48.3775.39y
MB, methylene blue; BDL, bile-duct ligation; SOD, superoxide dismutase; MDA, malondialdehyde; NO, nitric oxide; HP, hydroxyproline; MPO, myeloperoxidase. Values are expressed as mean7SD. po0.001 compared with control. po0.01 compared with control. po0.05 compared with control. yyy po0.001 compared with BDL. yy po0.01 compared with BDL. y po0.05 compared with BDL.
ARTICLE IN PRESS 266 lipid peroxides, which may have a direct stimulatory effect on extracellular matrix production by activated stellate cells (Kountouras et al., 1984; Liu et al., 2001). Liu et al. (2001) demonstrated excessive production of superoxide radicals and hydroxyl radicals in blood and liver in rats with obstructive jaundice induced by common bile-duct ligation. In the present study, bile-duct ligation caused significant increases in serum levels of alanine aminotransferase, aspartate aminotransferase, gamma glutamine transferase, total bilirubin and direct bilirubin, and these were attenuated following methylene blue treatment. Bile-duct ligation increased liver levels of malondialdehyde, myeloperoxidase and hydroxyproline, and these were lower following methylene blue treatment. Bile-duct ligation caused decreased superoxide dismutase activities in hepatic tissue and after methylene blue treatment this was reversed. Bile-duct ligation is associated with the development of oxidant injury. This is a dynamic process with different rates of progression or regression (Marley et al., 1999). Previous studies (Sener et al., 2005, 2007), which showed increased production of reactive oxidants in hepatic tissues monitored by chemiluminescence assay, provide additional support for the viewpoint that the generation of free radicals plays a key role in hepatic injury after bileduct ligation. Several antioxidants have been tested in experimental bile-duct obstruction models in attempts to limit the oxidative damage (Gedik et al., 2005; Sener et al., 2007). Methylene blue can act as an electron acceptor for xanthine oxidase. It might inhibit the production of superoxide in postischemic tissue by competing with molecular oxygen at the iron–sulfur centers of xanthine oxidase by enabling anaerobic oxidation of purine substrates (Kelner et al., 1988a, b), or may act as a ‘‘parasitic’’ electron acceptor, shunting electron flow from the normal pathway to the colorless, reduced form of methylene blue, i.e., leukomethylene blue, and, thus, effectively by-passing the generation of reactive oxygen species (Salaris et al., 1991). Malondialdehyde can be determined in tissues and its concentration is directly proportional to the cell damage caused by free radicals (Aksu et al., 2007). Methylene blue has been used to induce, rather than inhibit, lipid peroxidation in the presence of light by increasing singlet oxygen formation (Kamat and Devasagayam, 1996). We observed a significant increase in malondialdehyde concentrations in the liver tissue of bile-duct ligated rats, though this was prevented to a large degree after methylene blue treatment. Recent
B. Aksu et al. studies have indicated that disturbance of the oxidant–antioxidant balance may be responsible for cholestatic liver injury and that superoxide dismutase enzymatic activities are hepatoprotective and play important roles in preventing oxidative stress (Roeb et al., 2003). We found decreased superoxide dismutase activities in bile-duct ligated rats as compared to control rats and the methylene blue treatment reversed these changes. All these results may be due to the effects of methylene blue in reducing reactive oxygen species. Tissue myeloperoxidase activities have been used as biochemical markers for the tissue content of polymorphonuclear leukocytes (Gedik et al., 2005; Karaman et al., 2006). Methylene blue was shown to be beneficial in preventing the accumulation of polymorphonuclear leukocytes in the lung after intestinal ischemia-reperfusion in rats (Galili et al., 1998). The strong inhibitory effects of methylene blue on myeloperoxidase activity support the view that leukocyte-derived inflammatory mediators are integrally involved in adhesiogenesis (Heydrick et al., 2007). In our study, bile-duct ligation produced a significant increase in liver tissue myeloperoxidase activities reflecting the leukocyte accumulation in the liver tissue as compared to the control animals and this was confirmed by histopathological evaluation. We showed that methylene blue treatment produced a marked decrease in the enhancement of myeloperoxidase activity due to bile-duct ligation. The inhibitory effects of methylene blue may reflect the reduction in migration of neutrophils and other inflammatory cells in the inflamed region. Nitric oxide is a highly reactive mediator released in the liver by endothelial cells, macrophages, hepatocytes and Kupffer cells in response to various stimuli (Mayoral et al., 1999). Increased serum levels of nitric oxide are found in patients with cirrhosis (Guarner et al., 1993), as well as in bile-duct ligated cirrhotic rats (Farzaneh-Far and Moore, 2001). The main effect of methylene blue is inhibition of guanylate cyclase, the target site of nitric oxide. Methylene blue is known to have additional pharmacological actions, including the generation of oxygen radicals and direct inhibition of nitric oxide synthase (Mayer et al., 1993). The biological role of nitric oxide in the liver has been extensively studied, and the literature indicates that nitric oxide is either a primary mediator of liver cell injury or responds as a potent protective mechanism against deleterious stimuli (Clemens, 1999; Muriel., 2006). The present study indicated marked elevation in nitric oxide levels in the bileduct ligated rats, which was significantly attenuated after methylene blue treatment.
ARTICLE IN PRESS Effects of methylene blue in reducing cholestatic oxidative stress Bile-duct ligation in rats induces portal fibrosis, which begins with an early proliferation of biliary duct epithelial cells and portal periductular fibroblasts (Tuchweber et al., 1996). It is known that a consequence of bile-duct ligation is the rapid appearance of significant hepatic fibrosis (Strazzabosco et al., 2005). Hepatic fibrosis is characterized by increased production and deposition of extracellular matrix components accompanying most chronic liver disorders and is regarded as a major factor contributing to hepatic failure (Fort et al., 1998). It is known that oxidative events, especially lipid peroxidation, may play a pivotal role in the regulation of collagen accumulation. Chojkier et al. (1998) reported that products of lipid peroxidation modulate collagen gene expression and serve as a link between liver injury and fibrosis. We propose that the addition of methylene blue to antibiotic therapy may reduce fibrosis by preventing induced oxidative renal tissue damage in pyelonephritis (Aksu et al., 2007). Heydrick et al. (2007) showed that methylene blue inhibits adhesion formation via a mechanism that might involve blocking an oxidative stress-dependent decrease in peritoneal fibrinolytic activity. Methylene blue, which is an inhibitor of soluble guanylate cyclase, is known to depress the production of intracellular cyclic guanosine monophosphate, reversing the inhibition of collagen production (Houglum et al., 1991). Some studies, however, showed that methylene blue alone did not have any significant effect on collagen production (Chu and Prasad, 1999). Liver fibrosis was assessed by measuring hydroxyproline content in liver as an index of collagen accumulation. In our study, methylene blue treatment significantly reduced the increase in hydroxyproline content caused by bile-duct ligation. Its mechanism may be explained as a consequence of the antiinflammatory and antioxidant capabilities of methylene blue. In addition, this is the first study to show the protective effect of methylene blue on the development of bile-duct ligation-induced hepatic damage as determined by pathological evaluation, and measurement of inflammatory and oxidative stress parameters in the liver. This effect may be due to the inhibition of inflammatory cells accumulating in the liver and in particular may be due to the scavenging of reactive oxygen radicals by methylene blue itself. Although the mechanism of liver fibrosis is not fully understood, activated hepatic stellate cells play an important role in connective tissue synthesis and deposition during fibrogenesis (Nan et al., 2000). Fibroblasts expressing a-SMA may be derived from the transdifferentiation of quiescent hepatic stellate cells. The activation of hepatic stellate
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cells involves increased cellular proliferation, increased synthesis of extracellular matrix proteins and the expression of the activation marker a-SMA (Miyazaki et al., 1993). Extracellular matrices in liver fibrosis are known to be produced by myofibroblasts that are transformed from fatstoring cells. The development of the fibrotic process is thought to be mediated by various fibrogenic mediators (Akiyoshi and Terada, 1998). After bile-duct ligation, positive immunolabelling for a-SMA increased, especially in vascular smooth muscle cells, fibrotic septa and also around the proliferated bile ducts. A weak immunopositivity for a-SMA was observed in livers after bile-duct ligation and methylene blue treatment. In conclusion, these findings indicate that methylene blue can attenuate hepatic damage in extrahepatic cholestasis in experimental rats by reducing oxidative stress and inflammatory processes and provide an indication of the possible therapeutic use of methylene blue for cholestatic liver injury.
References Akiyoshi H, Terada T. Mast cell, myofibroblast and nerve terminal complexes in carbon tetrachloride-induced cirrhotic rat livers. J Hepatol 1998;29:112–9. Aksu B, Inan M, Kanter M, Oz Puyan F, Uzun H, DurmusAltun G, et al. The effects of methylene blue on renal scarring due to pyelonephritis in rats. Pediatr Nephrol 2007;22:992–1001. Altman P, Buchmiller TL. The jaundiced infant: biliary atresia. In: Grosfeld JL, O’Neill JA, Fonkalsrud EW, Coran AG, editors. Pediatric surgery. Mosby-Year Book; 2006. p. 1603–19. Benedetti A, Alvaro D, Bassotti C, Gigliozzi A, Ferretti G, La Rosa T, et al. Cytotoxicity of bile salts against biliary epithelium: a study in isolated bile ductule fragments and isolated perfused rat liver. Hepatology 1997;26:9–21. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302–10. Cassiman D, Libbrecht L, Desmet V, Denef C, Roskams T. Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers. J Hepatol 2002;36: 200–9. Chojkier M, Houglum K, Lee KS, Buck M. Long and shortterm D-a-tocopherol supplementation inhibits liver collagen a1(I) gene expression. Am J Physiol 1998;275: 1480–5. Chu AJ, Prasad JK. Up-regulation by human recombinant transforming growth factor beta-1 of collagen production in cultured dermal fibroblasts is mediated by the inhibition of nitric oxide signaling. J Am Coll Surg 1999;18:271–80.
ARTICLE IN PRESS 268 Clemens MG. Nitric oxide in liver injury. Hepatology 1999;30:1–5. Cortas NK, Wakid NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990;36:1440–3. Cruz A, Padillo FJ, Granados J, Tunez I, Munoz MC, Briceno J, et al. Effect of melatonin on cholestatic oxidative stress under constant light exposure. Cell Biochem Funct 2003;21:377–80. Engin A, Bozkurt B, Altan N, Memis L, Bukan N. Nitric oxide-mediated liver injury in the presence of experimental bile duct obstruction. World J Surg 2003;27: 253–5. Farzaneh-Far R, Moore K. Nitric oxide and the liver. Liver 2001;21:161–74. Fort J, Oberti F, Pilette C, Veal N, Gallois Y, Douay O, et al. Antifibrotic and hemodynamic effects of the early and chronic administration of octreotide in two models of liver fibrosis in rats. Hepatology 1998;28: 1525–31. Galili Y, Ben-Abraham R, Weinbroum A, Klausner J, Rabau M, Kluger Y, et al. Methylene blue prevents pulmonary injury following intestinal ischemia reperfusion. J Trauma 1998;45:222–5. Gedik N, Kabasakal L, Sehirli O, Ercan F, Sirvanci S, KeyerUysal M, et al. Long-term administration of aqueous garlic extract (AGE) alleviates liver fibrosis and oxidative damage induced by biliary obstruction in rats. Life Sci 2005;76:2593–606. Guarner C, Soriano G, Tomas A, Bulbena O, Novella MT, Balanzo J, et al. Increased serum nitrite and nitrate levels in patients with cirrhosis: relationship to endotoxemia. Hepatology 1993;18:1139–43. Heydrick SJ, Reed KL, Cohen PA, Aarons CB, Gower AC, Becker JM, et al. Intraperitoneal administration of methylene blue attenuates oxidative stress, increases peritoneal fibrinolysis, and inhibits intraabdominal adhesion formation. J Surg Res 2007;143:311–9 [Epub 2007 Sep 10]. Houglum K, Brenner DA, Chojkier M. d-alpha-tocopherol inhibits collagen alpha 1(I) gene expression in cultured human fibroblasts. Modulation of constitutive collagen gene expression by lipid peroxidation. J Clin Invest 1991;87:2230–5. Hsu SM, Raine L, Fanger H. Use of avidin–biotin–peroxidase complex (ABC) in immunperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29: 577–80. Kamat JP, Devasagayam TP. Methylene blue plus light induced lipid peroxidation in rat liver microsomes: inhibition by nicotinamide (vitamin B3) and other antioxidants. Chem Biol Interact 1996;99:1–16. Karaman A, Iraz M, Kirimlioglu H, Karadag N, Tas E, Fadillioglu E. Hepatic damage in biliary-obstructed rats is ameliorated by leflunomide treatment. Pediatr Surg Int 2006;22:701–8. Kelner MJ, Bagnell R, Hale B, Alexander NM. Potential of methylene blue to block oxygen radical generation in reperfusion injury. Basic Life Sci 1988a;49:895–8.
B. Aksu et al. Kelner MJ, Bagnell R, Hale B, Alexander NM. Methylene blue competes with paraquat for reduction by flavoenzymes resulting in decreased superoxide production in the presence of heme proteins. Arch Biochem Biophys 1988b;262:422–6. Kountouras J, Billing BH, Scheuer PJ. Prolonged bileduct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 1984;65:305–11. Liu TZ, Lee KT, Chern CL, Cheng JT, Stern A, Tsai LY. Free radical-triggered hepatic injury of experimental obstructive jaundice of rats involves overproduction of proinflammatory cytokines and enhanced activation of nuclear factor kappaB. Ann Clin Lab Sci 2001;31:383–90. Marley R, Holt S, Fernando B, Harry D, Anand R, Goodier D, et al. Lipoic acid prevents development of the hyperdynamic circulation in anesthetized rats with biliary cirrhosis. Hepatology 1999;29:1358–63. Mayer B, Brunner F, Schmidt K. Inhibition of nitric oxide synthesis by methylene blue. Biochem Pharmacol 1993;45:367–74. Mayoral P, Criado M, Hidalgo F, Flores O, Are´valo MA, Eleno N, et al. Effects of chronic nitric oxide activation or inhibition on early hepatic fibrosis in rats with bile duct ligation. Clin Sci 1999;96:297–305. Miyazaki H, Van Eyken P, Roskams T, De Vos R, Desmet VJ. Transient expression of tenascin in experimentally induced cholestatic fibrosis in rat liver: an immunohistochemical study. J Hepatol 1993;19:353–66. Muriel P. Role of nitric oxide in liver disorders. In: Shakir A, Friedman SL, Derek AM, editors. Liver diseases: biochemical mechanisms and new therapeutic insights. New Delhi: Oxford & IBH Publishing; 2006. p. 127–43. Nan JX, Park EJ, Kim HJ, Ko G, Sohn DH. Antifibrotic effects of the methanol extract of Polygonum aviculare in fibritic rats induced by bile duct ligation and scission. Biol Pharm Bull 2000;23:240–3. Ramm GA, Carr SC, Bridle KR, Li L, Britton RS, Crawford DH, et al. Morphology of liver repair following cholestatic liver injury: resolution of ductal hyperplasia, matrix deposition and regression of myofibroblasts. Liver 2000;20:387–96. Roeb E, Purucker E, Gartung C, Geier A, Jansen B, Winograd R, et al. Effect of glutathione depletion and hydrophilic bile acids on hepatic acute phase reaction in rats with extrahepatic cholestasis. Scand J Gastroenterol 2003;38:878–85. Salaris SC, Babbs CF, Voorhees WD III. Methylene blue as an inhibitor of superoxide generation by xanthine oxidase. A potential new drug for the attenuation of ischemia/reperfusion injury. Biochem Pharmacol 1991;42:499–506. Sener G, Kabasakal L, Yuksel M, Gedik N, Alican Y. Hepatic fibrosis in biliary-obstructed rats is prevented by Ginkgo biloba treatment. World J Gastroenterol 2005;11:5444–9. Sener G, Sehirli AO, Toklu HZ, Yuksel M, Ercan F, Gedik N. Erdosteine treatment attenuates oxidative stress and fibrosis in experimental biliary obstruction. Pediatr Surg Int 2007;23:233–41.
ARTICLE IN PRESS Effects of methylene blue in reducing cholestatic oxidative stress Serviddio G, Pereda J, Pallardo FV, Carretero J, Borras C, Cutrin J, et al. Ursodeoxycholic acid protects against secondary biliary cirrhosis in rats by preventing mitochondrial oxidative stress. Hepatology 2004;39: 711–20. Strazzabosco M, Fabris L, Spirli C. Pathophysiology of cholangiopathies. J Clin Gastroenterol 2005;39: 90–102. Sun Y, Oberley LW, Li YA. Simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497–500. Trauner M, Meier PJ, Boyer JL. Molecular pathogenesis of cholestasis. N Engl J Med 1998;339:1217–27.
269
Tuchweber B, Desmoulie `re A, Bochaton-Piallat ML, Rubbia-Brandt L, Gabbiani G. Proliferation and phenotypicmodulation of portal fibroblasts in the early stages of cholestatic fibrosis in the rat. Lab Invest 1996;74:265–78. Wei H, Frenkel K. Relationship of oxidative events and DNA oxidation in SENCAR mice to in vivo promoting activity of phorbol ester-type tumor promoters. Carcinogenesis 1993;14:1195–201. Woessner JB. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys 1961;93:440–7.