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Toxicology and Applied Pharmacology 241 (2009) 311321

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Toxicology and Applied Pharmacology


j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p

Liver brosis in mice induced by carbon tetrachloride and its reversion by luteolin
Robert Domitrovi a,, Hrvoje Jakovac b, Jelena Tomac c, Ivana ain d
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Department of Chemistry and Biochemistry, School of Medicine, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia Department of Physiology and Immunology, School of Medicine, University of Rijeka, Rijeka, Croatia c Department of Histology and Embriology, School of Medicine, University of Rijeka, Rijeka, Croatia d School of Medicine, University of Rijeka, Rijeka, Croatia
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a r t i c l e

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a b s t r a c t
Hepatic brosis is effusive wound healing process in which excessive connective tissue builds up in the liver. Because specic treatments to stop progressive brosis of the liver are not available, we have investigated the effects of luteolin on carbon tetrachloride (CCl4)-induced hepatic brosis. Male Balb/C mice were treated with CCl4 (0.4 ml/kg) intraperitoneally (i.p.), twice a week for 6 weeks. Luteolin was administered i.p. once daily for next 2 weeks, in doses of 10, 25, and 50 mg/kg of body weight. The CCl4 control group has been observed for spontaneous reversion of brosis. CCl4-intoxication increased serum aminotransferase and alkaline phosphatase levels and disturbed hepatic antioxidative status. Most of these parameters were spontaneously normalized in the CCl4 control group, although the progression of liver brosis was observed histologically. Luteolin treatment has increased hepatic matrix metalloproteinase-9 levels and metallothionein (MT) I/II expression, eliminated brinous deposits and restored architecture of the liver in a dosedependent manner. Concomitantly, the expression of glial brillary acidic protein and -smooth muscle actin indicated deactivation of hepatic stellate cells. Our results suggest the therapeutic effects of luteolin on CCl4-induced liver brosis by promoting extracellular matrix degradation in the brotic liver tissue and the strong enhancement of hepatic regenerative capability, with MTs as a critical mediator of liver regeneration. 2009 Elsevier Inc. All rights reserved.

Article history: Received 22 June 2009 Revised 26 August 2009 Accepted 2 September 2009 Available online 8 September 2009 Keywords: Carbon tetrachloride Liver brosis Luteolin -Smooth muscle actin Glial brillary acidic protein Matrix metalloproteinase Metallothionein

Introduction Liver brosis is a frequent event which follows a repeated or chronic insult of sufcient intensity to trigger a wound healing-like reaction, characterized by excessive connective tissue deposition in extracellular matrix (ECM). Chronic carbon tetrachloride (CCl4) intoxication is a well-known model for producing oxidative stress and chemical hepatic injury. Its biotransformation produces hepatotoxic metabolites, the highly reactive trichloromethyl free radical, which are further converted to the peroxytrichloromethyl radical (Williams and Burk, 1990). Reactive oxidant species likely contribute to both onset and progression of brosis (Poli, 2000). Antioxidant treatment in vivo seems to be effective in preventing or reducing chronic liver damage and brosis (Parola and Robino, 2001). Polyphenols, naturally occurring antioxidants in fruits, vegetables, and plant-derived beverages such as tea and wine, have been associated with a variety of benecial properties (Havsteen, 2002). The avone luteolin (3,4,5,7-tetrahydroxyavone) is an important member of the avonoid family, present in glycosylated forms and as aglycone in various plants (Shimoi et al., 1998). Luteolin is reported to have antiinammatory (Ziyan et al., 2007; Veda et al., 2002), antioxidant (Perez Corresponding author. Fax: +385 51 651 135. E-mail address: robertd@medri.hr (R. Domitrovi). 0041-008X/$ see front matter 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2009.09.001

Garcia et al., 2000), antiallergic (Veda et al., 2002), antitumorigenic (Ju et al., 2007), anxiolytic-like (Coleta et al., 2008), and vasorelaxative properties (Woodman and Chan, 2004). Previously, we have shown a hepatoprotective activity of luteolin in acute liver damage in mice (Domitrovi et al., 2008a, Domitrovi et al., 2009). Hepatic stellate cells (HSCs) are a minor cell type most commonly found in the space of Disse, intercalated between hepatocytes and cells lining the sinusoid, projecting their dendritic processes to nearby hepatocytes and endothelial cells (Blouin et al., 1977, Mermelstein et al., 2001). Upon liver injury, HSCs become activated, converting into myobroblast-like cells. Activated HSCs proliferate and produce extracellular matrix (ECM), playing a major role in hepatic brosis and regeneration (Friedman, 2000). ECM, which consists of collagens and other matrix components such as proteoglycans, bronectins, and hyaluronic acid (Arthur, 1994), is regulated by a balance of synthesis and enzymatic degradation of ECM. The key enzymes responsible for degradation of all the protein components of ECM and basement membrane are matrix metalloproteinases (MMPs), a zinc-dependent family of endopeptidases. Previous studies have demonstrated that the activity of these enzymes is altered during the processes of brogenesis and brinolysis (Knittel et al., 2000). The metallothioneins (MTs), small cysteine-rich heavy metalbinding proteins, participate in an array of protective stress responses. In mice, among the four known MT genes, the MT I and MT II genes are

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most widely expressed. Transcription of these genes is rapidly and dramatically up-regulated in response to agents which cause oxidative stress and/or inammation (Andrews, 2000). The induction of MT synthesis can protect animals from hepatotoxicity induced by various toxins including CCl4, but also play a role in repair and regeneration of injured liver (Cherian and Kang, 2006). Because the specic treatments to stop progressive brosis of the liver are not available, the objective of the present study was to investigate the therapeutic effect and mechanisms of action of luteolin in chemically induced liver brosis in mice. Materials and methods Materials. Luteolin, carbon tetrachloride (CCl4), olive oil, dimethyl sulfoxide (DMSO), nitric acid (HNO3), hydrogen peroxide (H2O2), and gelatin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and solvents were of the highest grade commercially available. Animals. Male Balb/c mice from our breeding colony, 23 months old, were divided into 6 groups with 5 animals per group. Mice were fed a standard rodent diet (pellet, type 4RF21 GLP, Mucedola, Italy) containing 19.4% protein, 5.5% ber, 11.1% water, 54.6% carbohydrates, 6.7% ash, and 2.6% by weight of lipids (native soya oil) to prevent essential fatty acid deciency. Total energy of the diet was 16.4 MJ/kg. The animals were maintained at 12 h light/dark cycle, at constant temperature (20 1 C) and humidity (50 5%). All experimental procedures were approved by the Ethical Committee of the Medical Faculty, University of Rijeka. Experimental design. Mice were given CCl4 intraperitoneally (i.p.) at a dose of 0.4 ml/kg, dissolved in olive oil, twice a week for 6 weeks (the CCl4 group), except the control group which received vehicle only. Seventy-two hours after the last CCl4 injection, the CCl4 group was killed. The CCl4 control group was observed for spontaneous resolution of hepatic brosis for next 2 weeks. In the luteolin-treated groups, the polyphenolic compound was administered i.p. at a dose of 10, 25, or 50 mg/kg daily for 2 weeks, respectively. These doses were selected on the basis of preliminary studies (Domitrovi et al., 2008a, Domitrovi et al., 2009). Luteolin was dissolved in DMSO and diluted in saline to the nal concentrations. The nal concentration of DMSO was less than 1%. Mice from the control and CCl4 control groups received diluted DMSO solution daily for 2 weeks. Animals were terminated 24 h after the last dose of luteolin or diluted solvent by cervical dislocation. The blood was taken from orbital sinus of ether anesthetized mice. The abdomen of terminated animal was cut open quickly and the liver perfused with isotonic saline, excised, blotted dry, weighed, and divided into samples. The samples were used to assess biochemical parameters, and another was preserved in a 4% phosphate-buffered formalin solution to obtain histological sections. Hepatotoxicity study. Serum levels of ALT, AST, and ALP as markers of hepatic function, were measured by using a Bio-Tek EL808 Ultra Microplate Reader (BioTek Instruments, Winooski, VT, USA) according to the manufacturer's instructions. Determination of Cu/Zn SOD activity and GSH concentration. Cu/Zn SOD activity and total GSH, indicators of oxidative stress, were measured spectrophotometrically, using Superoxide Dismutase Assay Kit and Glutathione Assay Kit (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer's instructions. Protein content in supernatants was estimated by Bradford's method, with bovine serum albumin used as a standard (Bradford, 1976). Determination of hepatic hydroxyproline. The tissue samples (50 mg) were hydrolyzed in 4 ml 6 M HCl at 110 C for 24 h. After

being ltered through a 0.45-m lter, 2 ml of samples was extracted and analyzed according to the procedure of Bergman and Loxley (1963). Briey, sample neutralization was obtained with 10 M NaOH and 3 M HCl. After neutralization, subsequent steps were made in duplicate for each sample. To a 200 l of the above solution, 400 l of isopropanol in citrate-acetate-buffered Chloramine T was added. After 4 min, 2.5 ml of Ehrlich reagent was added. Tubes were wrapped in aluminum foil and incubated for 25 min in a water-bath at 60 C, cooling each sample in tap water, and measuring the absorbance of each sample spectrophotometrically at 550 nm (Cary 100, Varian, Mulgrave, Australia). Determination of trace elements. Hepatic zinc (Zn) and copper (Cu) content were determined by ion coupled plasma spectrometry (ICPS) using Prodigy ICP Spectrometer (Leeman Labs, Hudson, NH, USA), according to the method previously described (Domitrovi et al., 2008a). Determination of retinol. The hepatic levels of retinol were analyzed by high-performance liquid chromatography (HPLC) according to Hosotani and Kitagawa (2003), as described previously (Domitrovi et al., 2008b). Histopathology. Liver specimens were xed in 4% phosphatebuffered formalin, embedded in parafn, and cut into 4 m thick sections. Sections for histopathological examination were stained with hematoxylin and eosin (H&E) and Mallory trichrome stain using standard procedure. Immunohistochemical determination of GFAP, -SMA, and MT I/II. Immunohistochemical studies were performed on parafn embedded liver tissues using mouse monoclonal anti-MT I+II antibody diluted 1:50 (clone E9; DakoCytomation, Carpinteria, CA, USA), mouse monoclonal anti-GFAP antibody diluted 1:100 (clone 1B4; BD Pharmingen, San Diego, CA, USA), and mouse monoclonal antibody to -SMA diluted 1:100 (SPM332; Abcam, Cambridge, UK), employing DAKO EnVision+ System, Peroxidase/DAB kit according to the manufacturer's instructions (DAKO Corporation, Carpinteria, CA, USA). Briey, slides were incubated with peroxidase block to eliminate endogenous peroxidase activity. After washing, monoclonal antibodies diluted in phosphate-buffered saline supplemented with bovine serum albumin were added to tissue samples and incubated overnight at 4 C in a humid environment, followed by incubation with peroxidase labeled polymer conjugated to secondary antibodies containing carrier protein linked to Fc fragments to prevent nonspecic binding. The immunoreaction product was visualized by adding substrate-chromogen diaminobenzidine (DAB) solution, resulting with brownish coloration at antigen sites. Tissues were counterstained with hematoxylin, dehydrated in gradient of alcohol, and mounted with mounting medium. The intensity of staining was graded as weak, moderate, and intense. The specicity of the reaction was conrmed by substitution of primary antibodies with irrelevant immunoglobulins of matched isotype, used in the same conditions and dilutions as primary antibodies. Stained slides were analyzed by light microscopy (Olympus BX51, Tokyo, Japan). MMP zymography. MMP-2 and MMP-9 activities were analyzed by gelatin zymography assays as described (Kuo et al., 2003), with modications. After tissue homogenization in radioimmunoprecipitation assay buffer (4 ml of buffer per gram of tissue) containing 50 mM TrisHCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM PMSF, 1 mM sodium orthovanadate, and 2 g/ml of each aprotinin, leupeptin and pepstatin. 10 g of liver tissue protein lysates were separated by a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) gels containing 0.1% gelatin, at 4 C and 150 V for 4 h. Gels were washed for 30 min in 2.5% Triton X-100 to remove the SDS, and briey

R. Domitrovi et al. / Toxicology and Applied Pharmacology 241 (2009) 311321 Table 1 Relative liver weight, hydroxyproline content, and serum markers of liver damage. Control Relative liver weight (g/100 g body weight) Hydroxyproline (g/g liver) AST (U/l) ALT (U/l) ALP (U/l) 5.31 0.40a 189 37a 22.7 2.5a 16.0 3.7a 77.4 1.1a CCl4 6.94 0.23b 846 159 b 276.5 4.1b 226.1 20.2b 114.0 4.0b CCl4 control 6.17 0.19b 1188 205c 20.4 1.6a 13.1 2.1a 87.9 7.9b Luteolin 10 mg/kg 5.83 0.65a 443 82d 18.6 3.3a 15.1 2.9a 79.8 4.1a Luteolin 25 mg/kg 5.34 0.20a 294 34e 23.3 4.9a 14.0 2.3a 66.7 3.4c

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Luteolin 50 mg/kg 4.95 0.72a 231 31a 27.0 10.3a 12.0 1.2a 60.3 5.0c

Each value represents the mean SD for 5 mice. Values not sharing a common letter superscript (a, b, c) are signicantly different (p b 0.05). Statistical difference was determined using nonparametric ManWhitney and KruskalWallis tests.

washed in the reaction buffer containing 50 mM TrisHCl, pH 7.5, 5 mM CaCl2, 1 M ZnCl2, and 0.02% NaN3. The reaction buffer was changed to a fresh one, and the gels were incubated at 37 C for 48 h. Gelatinolytic activity was visualized by staining the gels with 0.1% Coomassie blue R-350, destained with methanol-acetic acid water (30:10:60 v/v) two times for 20 min. The clear zones in the background of blue staining indicate the presence of gelatinase activities. The intensity of the bands was assayed by scanning video densitometry (Bio-Rad GS-710 Calibrated Imaging Densitometer, BioRad Laboratories, UK). Statistical analysis. Data were analyzed using StatSoft STATISTICA version 7.1 software. Differences between the groups were assessed by a nonparametric ManWhitney and KruskalWallis tests. Values in the text are means standard deviation (SD). Differences with p b 0.05 were considered to be statistically signicant. Results Hepatotoxicity Serum AST, ALT, and ALP activities were changed signicantly in mice receiving CCl4 twice a week for 6 weeks and terminated 72 h later (Table 1). The relative liver weight signicantly increased in the CCl4 group when compared to controls. In the CCl4 control group, observed for the spontaneous regression of brosis for 2 weeks, the relative liver weight and ALP activity were still increased; however, AST and ALT activities were normalized. Luteolin administration attenuated the elevation of ALP activity in the mice treated with CCl4 and decreased relative liver weight to control values. Higher doses of luteolin, 25 and 50 mg/kg, decreased ALP activity below control values. However, AST activity in mice treated with 50 mg/kg of luteolin was signicantly increased compared to the control group and groups treated with luteolin. Hepatic hydroxyproline The liver hydroxyproline content was vefold higher in the CCl4 group than in controls and progressively increased in the CCl4 control group (Table 1). Luteolin therapy signicantly decreased the hepatic hydroxyproline level, which was returned to normal values in the group receiving 50 mg/kg of luteolin.

Cu/Zn SOD activity and GSH concentration There was no evidence of oxidative stress in the CCl4 control group when compared to the CCl4 group. The hepatic total GSH level remained at normal values throughout the treatment period, except in the group treated with 50 mg/kg of luteolin, where it was signicantly increased (Table 2). Cu/Zn SOD activity was increased in the CCl4 group; however, its level was normalized in the CCl4 control group and groups treated with luteolin. The liver total retinol concentration was markedly decreased in the CCl4 group, then increased in the CCl4 control group, returning to normal values in the group treated with the highest dose of luteolin. Cu and Zn content The Cu content was decreased in all experimental groups, compared to controls (Table 2). The hepatic Zn content has not been signicantly changed by CCl4 intoxication or during luteolin therapy. The liver trace element content was expressed as g/g dry liver weight (ppm). Histopathology Liver sections from control mice stained with hematoxylin and eosin (H&E) showed normal hepatic architecture. Liver specimens from the CCl4 group showed remnants of degenerated and balooned/ necrotic hepatocytes containing acidophilic hyaline inclusions (Fig. 1B). Mild mononuclear cell inltration in these areas was also present. Hepatocytes in the vicinity of hepatic lesions, particularly those situated immediately alongside the lesion border, showed features of pronounced macrovesicular or microvesicular steatosis. Distended sinusoidal spaces lled with erythrocytes as a morphological hallmark of congestion (disturbed blood ow) were also observed. Hepatic lesions, fatty degeneration, and hyaline deposits as well as morphological signatures of congestion were more pronounced in the CCl4 control group (Fig. 1C). Hepatic lesions and hyaline deposits were also present in the livers of mice treated with 10 mg/kg of luteolin (Fig. 1D), but they were signicantly reduced in extent and were less frequent compared to the CCl4 control group, whereas macrovesicular steatosis was still present in the surrounding liver parenchyma, with noticeable signs of congestion. The livers of mice receiving 25 mg/kg of luteolin showed only sporadic, markedly small hepatic lesions, hyaline

Table 2 Hepatic indicators of oxidative stress, liver retinol, and metal content. Control Cu/Zn SOD (U/mg protein) GSH (M/g liver) Retinol (M/g liver) Zn (ppm) Cu (ppm) 0.95 0.04a 9.71 0.51a 1.51 0.13a 75.3 2.6 22.5 1.7a CCl4 1.50 0.10b 9.64 0.21a 0.74 0.09b 85.3 9.2 14.0 1.3b CCl4 control 0.91 0.23a 9.51 0.63a 1.22 0.11c 84.8 10.4 16.7 2.1b,c Luteolin 10 mg/kg 0.86 0.08a 9.56 0.79a 1.02 0.19c 79.3 6.1 16.3 0.7c Luteolin 25 mg/kg 0.88 0.08a 10.57 0.60a 1.19 0.12c 81.3 9.1 16.1 0.8c Luteolin 50 mg/kg 0.89 0.15a 11.37 0.65b 1.32 0.23a,c 85.3 11.6 15.8 0.4c

Each value represents the mean SD for 5 mice. Values not sharing a common letter superscript (a, b, c) are signicantly different (p b 0.05). Statistical difference was determined using nonparametric ManWhitney and KruskalWallis tests.

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Fig. 1. The effect of CCl4 and luteolin therapy on liver histology. (A) The livers from control mice showed normal hepatic architecture. (B) The livers of mice receiving CCl4 twice a week for 6 weeks, terminated 72 h after the last CCl4 injection, showed severe hepatic lesions, degenerated and balooned/necrotic hepatocytes with cellular hyaline inclusions, and mild mononuclear cell inltration (arrowheads). (C) In the CCl4 control group, observed for spontaneous regression of brosis for additional 2 weeks, hepatic lesions, balooned hepatocytes, and hyaline bodies were more frequent. (D) Hepatic lesions and hyaline deposits in mice receiving 10 mg/kg luteolin for 2 weeks were signicantly reduced in extent and were less frequent. (E) The livers of mice receiving 25 mg/kg of luteolin showed sporadic, small hepatic lesions, hyaline changes, and microvesicular steatosis. (F) Mice treated with 50 mg/kg of luteolin showed maintained hepatic architecture, with only weak microvesicular steatosis. Arrows show balooned hepatocytes with hyaline inclusions. Original magnication 100, insets 1000. H&E stain.

changes, and microvesicular steatosis in the periportal areas, but without morphological signs of hemodynamic disturbance (Fig. 1E). In animals treated with 50 mg/kg of luteolin, the livers showed maintained histoarchitecture, almost similar to controls, with only weak microvesicular steatosis of periportal and perilobular hepatocytes (Fig. 1F). There were no changes which could be attributed to hepatic hemodynamic disturbances in these animals. The livers from control mice stained with Mallory trichrome stain showed traces of collagen only in the vascular walls. Liver section from

the CCl4 group showed multiple brotic nodules and extensive brosis predominantly in the periportal areas (Fig. 2B). The pericellular collagen deposition and the extent of brotic changes were even more pronounced in the CCl4 control group (Fig. 2C). Fibrotic lesions were still present in the livers of mice treated with 10 mg/kg of luteolin (Fig. 2D), but they were signicantly reduced in extent and were less frequent compared to the CCl4 control group, whereas mild pericellular brosis was observed in the surrounding liver parenchyma. The livers of mice receiving 25 mg/kg of luteolin showed only sporadic, markedly

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small brotic deposits in the periportal areas, with a discrete pericellular brosis (Fig. 2E). Animals treated with 50 mg/kg of luteolin showed only minor and sporadic pericellular brotic changes (Fig. 2F). Immunohistochemistry of GFAP, -SMA, and MT I/II Activated HSCs could be distinguished from the other myobroblasts of the liver (such as portal myobroblasts) by their specic position in the liver parenchyma (Cassiman et al., 2002). With regard to the distribution of -SMA-positive brogenic cells, in the livers of

control animals, -SMA immunopositivity was restricted to the smooth musculature belonging to the arterial tunica media, as well as to the wall of majority of portal and central veins, while other liver cells remain negative (Fig. 3A). CCl4 strongly induced perisinusoidal -SMA expression, which was recognized as activated HSCs, through affected lobuli, connected between themselves with thin, bridging immunopositivity (Fig. 3B). The HSCs varied in shape and size. In the CCl4 control group, perisinusoidal immunopositivity was withdrawn, but emerged in hepatocytes located at the scar-parenchyma interface (Fig. 3C). The livers of mice receiving 10 and 25 mg/kg of luteolin showed only

Fig. 2. The histopathologic detection of collagen in the livers. Collagen bers of the connective tissues are identied by their blue color. (A) Liver section from a control mice. (B) Mice receiving CCl4 twice a week for 6 weeks, terminated 72 h after the last CCl4 injection, had developed extensive brosis in the periportal areas. (C) The CCl4 control group, observed for spontaneous regression of brosis for additional 2 weeks, had more intense collagen staining. (D) The collagen deposits in mice receiving 10 mg/kg luteolin for 2 weeks were greatly reduced in size. (E) The livers of mice receiving 25 mg/kg of luteolin showed sporadic, small brotic lesions in the periportal zone. (F) In mice treated with 50 mg/kg of luteolin there were observed only minor pericellular brotic changes (arrow). Original magnication 100, insets 1000. Mallory trichrome stain.

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sporadic -SMA immunopositivity in the hepatocytes in the vicinity of brotic lesions (Figs. 3D and E). The livers of mice receiving 50 mg/kg of luteolin showed staining pattern similar to control animals. Immunohistochemical staining using monoclonal anti GFAP antibody showed thin, irregular immunopositive bands lining the hepatic sinusoids of control animals (staining pattern considered as a characteristic for HSC) (Fig. 4A). In the CCl4 and CCl4 control groups, moderate and strong GFAP expressions, respectively, were limited to the scar areas (Figs. 4B and C). Treatment with 10 and 25 mg/kg of luteolin decreased GFAP expression in the brotic areas, but induced

sporadic assembling of GFAP positive cells in the surrounding tissue (Figs. 4D and E). The livers of mice receiving 50 mg/kg of luteolin showed staining pattern similar to control animals, characterized by moderate GFAP expression alongside the sinusoidal border (Fig. 4F). Control animals had moderate cytoplasmic MT I/II expression in the perilobular areas (Fig. 5A). Liver tissues from the CCl4 group showed weak cytoplasmic immunostaining of fatty changed hepatocytes surrounding the brotic areas, but strong immunopositivity within balooned hepatocytes (Fig. 5B). In the CCl4 control group, MT I/II immunopositivity was found to be only sporadic and restricted to

Fig. 3. The expression and specic tissue distribution of -SMA. (A) In the livers of control animals, -SMA immunopositivity was restricted to smooth musculature (arrow), while other liver cells remain negative. (B) In mice receiving CCl4 twice a week for 6 weeks, terminated 72 h after the last CCl4 injection, many -SMA-containing myobroblasts were strongly and diffusely stained in affected lobuli, connected between themselves with thin, bridging immunopositivity (arrows). (C) In the CCl4 control group, observed for spontaneous regression of brosis for additional 2 weeks, clusters of moderately -SMA-immunopositive hepatocytes located at the scar-parenchyma interface were found (arrows). The livers of mice receiving 10 mg/kg (D) and 25 mg/kg (E) of luteolin showed only sporadic -SMA immunopositivity in the hepatocytes in vicinity to brotic lesions (arrows). (F) The livers of mice receiving 50 mg/kg of luteolin showed staining pattern similar to control animals. Original magnication 400, inset 1000. Immunohistochemical stain for -SMA.

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Fig. 4. The expression and specic tissue distribution of GFAP. (A) The liver sections from control mice showed thin GFAP immunopositive bands lining the hepatic sinusoids. (B) In the livers of mice receiving CCl4 twice a week for 6 weeks, terminated 72 h after the last CCl4 injection, moderate GFAP expression, and (C) in mice from the CCl4 control group, observed for spontaneous regression of brosis for additional 2 weeks, strong GFAP expression were limited to the scar areas. Mice receiving 10 mg/kg (D) and (E) 25 mg/kg luteolin for 2 weeks had decreased GFAP expression in the brotic areas, with sporadic expression in the surrounding tissue. (F) The livers of mice receiving 50 mg/kg of luteolin showed staining pattern similar to the control animals. Original magnication 1000. Immunohistochemical stain for GFAP.

hyaline bodies in the context of brous lesions, while in the remnant liver tissue was not detected (Fig. 5C). The livers of animals receiving 10 mg/kg of luteolin showed weak cytoplasmic MT I/II expression in the remaining hepatic parenchyma and more frequent appearance of MT I/II immunopositive hyaline deposits (Fig. 5D). Concomitantly with the histoarchitecture improvement, moderate perilobular MT I/II immunopositivity was observed in the livers of animals treated with 25 mg/kg of luteolin (Fig. 5E). Liver sections from mice treated with 50 mg/kg of luteolin have shown strong cytoplasmic and nuclear immunopositivity, remarkably in the periportal and perilobular areas (Fig. 5F). Fig. 5G shows the extent of MT I/II expression.

MMP zymography MMPs were identied by their molecular weights. Liver extracts contained mainly the latent form of MMP-9 at about 92 kDa (Fig. 6). CCl4-intoxication decreased MMP-9 expression, which was even more pronounced in the CCl4 control group. Treatment with lutolin resulted in an increase in MMP-9 expression in a dose-dependent manner, being the most prominent in mice treated with 25 and 50 mg/kg of luteolin. Active form of MMP-9 was barely detectable in groups treated with 25 and 50 mg/kg of luteolin. In contrast to MMP-9, both latent and active forms of MMP-2 expression were below detection limit in all groups.

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Fig. 6. A representative SDS-PAGE gelatin zymogram of liver specimens from the control and treated animals. (A) Expression of latent form of 92 kDa MMP-9 in equal protein quantities of hepatic homogenates from control mice (lane 1), mice treated with CCl4 (lane 2), mice left for spontaneous recovery (lane 3), and from mice treated with luteolin 10 mg/kg (lane 4), 25 mg/kg (lane 5), and 50 mg/kg (lane 6). Weak expression of the active 86 kDa form of MMP-9 was noticeable only in lanes 5 and 6. No signicant effect of CCl4-intoxication or the treatment with luteolin on the MMP-2 expression or proMMP-2 activation has been detected. (B) Densitometric analysis of bands detected in gelatin zymograms. The bar graph shows averages of band intensity of proMMP-9 expression (mean SD, n = 5). Values not sharing a common letter superscript (a, b, c, d, e) are signicantly different (p b 0.05). MMPs were identied by their molecular weights.

Discussion Liver brosis represents chronic wound repair following liver injury (Friedman, 1999). Independently from the nature of the injury, the usual initiating event of brogenesis is represented by tissue necrosis of variable entity. Activation of either resident or bloodderived phagocytes increases the steady-state concentration of brogenic cytokines, which recruit a large amount of broblasts and broblast-like cells for excess production of ECM. The terminal outcome of liver brosis is the formation of nodules encapsulated by brillar scar matrix (Tsukamoto, 1999). Irreversible brosis develops when the appropriate cellular mediators as the source of MMPs are absent (Issa et al., 2004). The progression of brosis observed in the CCl4 control group suggests development of an irreversible brosis. Additionally, numerous eosinophilic hyaline globules of varying sizes have been found in brotic lesions. The amount of hyaline bodies has correlated with abundance of collagen deposits and the degree of liver brosis. Hyaline deposits, such as Mallory bodies, could be occasionally found in the hepatocytes in alcoholic hepatitis but also in different nonalcoholic liver disorders (Denk et al., 2000). These inclusions consist of aggregates of abnormally phosphorylated, ubiquitinated, and cross-linked keratins and nonkeratin components (Stumptner et al., 2002). Protein aggregates could be reverted to normal state by molecular chaperones

or degradated by proteasomes (Kopito, 2000). However, if reparation or degradation fails, abnormal proteins became segregated in the cytoplasm as inclusion bodies. Hepatic indicators of oxidative stress and hepatic damage in this study were markedly changed in CCl4-intoxicated group, 72 h after discontinuation of the toxin. Two weeks later, in the CCl4 control group and the groups treated with luteolin, most of the biochemical parameters, Cu/Zn SOD activity, GSH, and aminotransferase levels did not indicate signicant oxidative stress or hepatocellular damage. The possible reason for this is that mammalian cells respond quickly to the initial oxidative damage by means of a characteristic adaptative phenomenon (Radice et al., 1998). Acquisition of adaptation to tissue damage is a common response to insults in order to reduce the vulnerability of the cell or tissue, which depends on the activity of various stress sensors, signal transduction and effectors of protection (Hagberg et al., 2004). Therefore, biochemical parameters may not reect the degree of tissue damage (Murayama et al., 2007). These results are of the signicant clinical importance, suggesting that the classical biochemical parameters such as serum aminotransferase activities are not a reliable indicators of chronic liver damage. The accumulation of ECM observed in brosis and cirrhosis is considered to be the result of activation of broblasts, which acquire a myobroblastic phenotype. Myobroblasts are produced by the activation of precursor cells, such as HSCs and portal broblasts. Signicant differences between these two brogenic cell populations have been reported, in the mechanisms leading to myobroblast differentiation, their activation and deactivation (Guyot et al., 2006). However, the pathophysiological mechanisms of hepatic brogenesis are not yet fully understood. In particular, the role of HSCs remains unclear. HSCs have been also found to migrate in vitro (Marra et al., 1999), suggesting that they may migrate to the brotic lesions and take part in the repair process (Kinnman et al., 2000). Stimulation of HSCs with various cytokines or type I collagen, results in an increase in their migratory capacity, which could be accompanied by or independent of HCS proliferation (Yang et al., 2003). In the quiescent state, HSCs express desmin and GFAP (Yokoi et al., 1984, Neubauer et al., 1996). Upon HSCs activation, GFAP expression decreases, whereas the levels of desmin and -SMA increase, due to their transformation into myobroblast-like cells (Campbell et al., 2005). In this study, deposition of collagen in damaged hepatic areas, associated with increased -SMA and decreased GFAP perisinusoidal immunopositivity, indicates that activated HSCs are responsible for the brosis seen in the CCl4-intoxicated mice. Additionally, increased GFAP immunopositivity in brotic scars suggests the accumulation of quiescent HSCs at the places of active brogenesis. Changes in the activation marker expression in the CCl4 control group, however, indicate deactivation of HSCs, which is critical for resolution of brosis (Iredale, 2001). Nevertheless, -SMA immunopositive hepatocytes located at the scar-parenchyma interface in the CCl4 control group suggest that hepatocytes have acquired myobroblast-like phenotype. Indeed, Zeisberg et al. (2007) have recently shown that adult hepatocytes could be actively engaged in the epithelial to mesenchymal transition mediated by TGF-1, by expressing broblast-specic protein-1 (FSP1), suggesting that hepatocyte-derived broblasts could be an additional lineage of mesenchymal cells that contribute to the progression of liver brosis. Furthermore, Dooley et al. (2008) have observed hepatocyte transdifferentiation immediately adjacent to the brotic lesions, which is in agreement to our ndings. However, to our knowledge, this is for the rst time that the -SMA expression,

Fig. 5. The expression and specic tissue distribution of MT I/II. (A) The livers from control mice showed weak perilobular MT I/II immunopositivity. (B) The livers of mice receiving CCl4 twice a week for 6 weeks, terminated 72 h after the last CCl4 injection, showed weak cytoplasmic immunostaining of hepatocytes located at the scar-parenchyma interface, with strong immunopositivity in the brotic lesions. (C) In the CCl4 control group, observed for spontaneous regression of brosis for additional 2 weeks, immunopositivity was only sporadic and restricted to the brotic scars. (D) Mice receiving 10 mg/kg luteolin therapy for 2 weeks had weak cytoplasmic MT I/II expression with strong immunopositivity in the brotic lesions. (E) The livers of mice receiving 25 mg/kg of luteolin showed moderate perilobular MT I/II immunopositivity. (F) Strong both cytoplasmic and nuclear perilobular and periportal staining in the group treated with 50 mg/kg of luteolin. (G) The bar graph shows the extent of hepatocellular MT I/II expression (mean SD, n = 5). Values not sharing a common letter superscript (a, b, c, d, e) are signicantly different (p b 0.05). Original magnication 100. Immunohistochemical stain for MT I/II.

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characteristic for activated HSCs, is found in hepatocytes. This subpopulation of hepatocytes could contribute to collagen deposition observed in the CCl4 control group. Perisinusoidal GFAP expression in the group treated with 50 mg/kg of luteolin indicates a complete return of HSCs into a quiescent state. Morphological studies have shown that brotic lesion of the liver also typically involves, beside an accumulation of extracellular collagen matrix and a transformation of stellate cells to actively dividing myobroblasts, a reduction in stellate cell lipid droplets and hepatic storage of total retinol (Blomhoff and Wake, 1991). Decreased hepatic total retinol level in the CCl4 group observed in this study is in agreement with previous studies (Seifert et al., 1994, Natarajan et al., 2005). The vitamin A status of the liver plays an important role in hepatic brogenesis. The reduction of retinoid levels in HSC by an enhanced secretion of retinol from the liver into the circulation during CCl4 treatment may stimulate the transformation of these cells into broblasts and contribute to brogenesis (Seifert et al., 1994). Increased liver retinol observed in the CCl4 control group, as well as in mice receiving luteolin therapy, also indicates a decrease in the number of activated HSCs. In normal tissues, matrix protein degradation is accomplished by a family of MMPs, whose expression signicantly increases in the case of acute liver injury (Arthur, 1994). In chronic liver injury sustained tissue damage and inammation also induce MMPs overexpression (Kossakowska et al., 1998). MMP-2, the 72-kD collagenase IV/ gelatinase A, is an important factor in the metabolism of type IV collagen in sinusoid basement, produced by HSCs. MMP-9, the 92-kD collagenase IV/gelatinase B, a major MMP in basement membranelike ECM remodeling, is secreted by neutrophil granulocytes, macrophages and leukocytes as a major source for MMP-9 expression (Salguero Palacios et al., 2008), with HSCs as an additional source (Knittel et al., 1999). MMP activity decreases with the progression of liver brosis (Iredale, 2001). Liver injury which results in HSC activation, particularly if chronic, leads to an increase in overall numbers of myobroblast-like activated HSCs that are actively producing matrix, while simultaneously preventing degradation of the matrix through expression of TIMPs. It has been consensually accepted that increased production and activity of TIMPs are required for decrease in MMP activity (Perez-Tamayo et al., 1987, Benyon and Iredale, 2000). The key events in the reversion of liver brosis include apoptosis of HSCs, increased MMP activity, decreased expression of TIMPs, and increased degradation of collagen (Woessner, 1991). In the liver, myobroblasts derived from hepatic stellate cells undergo apoptosis during the spontaneous resolution of liver brosis induced by CCl4 treatment (Iredale et al., 1998). During spontaneous recovery from liver brosis, TIMP-1 level decreases, and collagenase activity and the apoptosis of hepatic stellate cells increase, which results in an increased brolysis and removal of excessively deposited ECM. MMPs could be derived either from activated HSCs prior to or during apoptosis, from quiescent HSCs, or from other liver cells (Iredale, 2001). It is still unknown how the brotic tissues are removed in vivo and how MMPs, which are usually down-regulated in established brotic tissues, are expressed again during the resolution process (Han, 2006). Our result showed that CCl4-intoxication decreased MMP-9 expression in CCl4 group 72 h after toxin discontinuation, with further decrease in the CCl4 control group 2 weeks later. Luteolin therapy has induced the expression of MMP-9, with the highest increase in the group treated with 50 mg/kg. The MMP-9 production by other cells distinct from HSCs could explain its increased expression despite HSC deactivation. In contrast, MMP-2 did not play a signicant role in this model, which was conrmed by immunohistochemistry (data not shown). The mechanisms by which MT participates in the pathology and the reversion of liver brosis are still largely unknown. Although a number of biological functions have been proposed for MTs, most of them are related to its metal-binding property, mainly Zn and Cu

homeostasis and heavy metal detoxication (Andrews, 2000). Recently, we have shown dose- and time-dependent up-regulation of MTs expression by luteolin in acute hepatotoxicity induced by CCl4 and the enhancement of hepatic regenerative capability (Domitrovi et al., 2008a). Hepatic MT I/II expression in necrotic areas was correlated with the Zn and Cu content. However, MTs expression in chronic exposure to CCl4 in this study was not correlated with either the Zn or Cu hepatic content. Increased MT synthesis also occurs in response to oxidative stress (Chubatsu and Meneghini, 1993). In this study, disturbance in antioxidative status in the CCl4 control group and groups treated with luteolin was not observed, therefore we could not explain the down-regulation of MT I/II as a consequence of oxidative stress. Furthermore, MT has the essential role in cell proliferation and liver regeneration (Apostolova and Cherian, 2000). Immunohistochemical studies have demonstrated increased MT expression both in the cytoplasm and nucleus of rapidly proliferating cells (Miles et al., 2000). MT may donate Zn and Cu to certain metalloenzymes and transcription factors associated with gene transcription, cell replication, differentiation, and growth (Wostowski, 1993, Coyle et al., 2002). Jiang and Kang (2004) have suggested that gene therapy with MT could improve the recovery from liver brosis. The same authors have shown that mice which have developed a reversible liver brosis upon removal of CCl4, had a high level of hepatic MT, but mice which developed an irreversible brosis, had low expression of MT. Therefore, hepatic MT expression could reect the severity of chronic liver damage, showing down-regulation in injured tissue (Carrera et al., 2003). The results of this study show that the MT I/II expression was progressively down-regulated from the CCl4 group to the CCl4 control group. The level of MTs in livers was related to the progression of CCl4-induced brosis and a complete down-regulation of MT I/II in the CCl4 control group indicates development of an irreversible brosis. On the other hand, the positive correlation of MT I/II expression with luteolin dosage and liver tissue repairment suggests its direct involvement in the regeneration of liver tissue. The liver sections from mice treated with luteolin 50 mg/kg have shown strong up-regulation of MT I/II, including nuclear expression, which was accompanied with a complete resolution of brotic scar tissue, suggesting the pronounced therapeutic effect of luteolin. Our results support the view that MT I/II could be the factors associated with a favorable clinical outcome in the response to therapy (Carrera et al., 2003). Regardless of these interesting and important ndings, there are some limitations of the present study. The mechanistic insights into the luteolin-induced recovery of irreversible liver brosis need to be further approached. Although the important parameters of brosis and liver regeneration were determined, other factors that are possibly involved in the liver brinolysis need to be explored. In conclusion, the luteolin treatment has induced a complete reversion of established liver brosis by decreasing hepatic brogenic potential, and stimulated MT I/II expression in the liver in a dosedependent manner, suggesting the strong enhancement of hepatic regenerative capability. Importantly, the results obtained from this study suggest the therapeutic application of luteolin in patients with hepatic brosis, although further studies, in a more chronic model of liver brosis, are required.
Conict of interest statement The authors declare no conict of interest.

Acknowledgments This research was supported by grants from the Ministry of Science, Education and Sport, Republic of Croatia (project no. 0620000000-3554). The authors thank Ing. Hrvoje Krian, Ljubica rnac, and Jadranka Ekinja for their excellent technical support and Dipl. Ing. Sunica Ostoji for her assistance with SDS-PAGE.

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