Toxicology and Applied Pharmacology 241 (2009) 311–321
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Toxicology and Applied Pharmacology
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Liver fibrosis in mice induced by carbon tetrachloride and its reversion by luteolin
Robert Domitrović a,⁎, Hrvoje Jakovac b, Jelena Tomac c, Ivana Šain d
a Department of Chemistry and Biochemistry, School of Medicine, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatiab Department of Physiology and Immunology, School of Medicine, University of Rijeka, Rijeka, Croatiac Department of Histology and Embriology, School of Medicine, University of Rijeka, Rijeka, Croatiad School of Medicine, University of Rijeka, Rijeka, Croatia
Hepatic fibrosis is effusive wound healing process in which excessive connective tissue builds up in the liver.Because specific treatments to stop progressive fibrosis of the liver are not available, we have investigatedthe effects of luteolin on carbon tetrachloride (CCl4)-induced hepatic fibrosis. Male Balb/C mice were treatedwith CCl4 (0.4 ml/kg) intraperitoneally (i.p.), twice a week for 6 weeks. Luteolin was administered i.p. oncedaily for next 2 weeks, in doses of 10, 25, and 50 mg/kg of body weight. The CCl4 control group has beenobserved for spontaneous reversion of fibrosis. CCl4-intoxication increased serum aminotransferase andalkaline phosphatase levels and disturbed hepatic antioxidative status. Most of these parameters werespontaneously normalized in the CCl4 control group, although the progression of liver fibrosis was observedhistologically. Luteolin treatment has increased hepatic matrix metalloproteinase-9 levels and metallothio-nein (MT) I/II expression, eliminated fibrinous deposits and restored architecture of the liver in a dose-dependent manner. Concomitantly, the expression of glial fibrillary acidic protein and α-smooth muscleactin indicated deactivation of hepatic stellate cells. Our results suggest the therapeutic effects of luteolin onCCl4-induced liver fibrosis by promoting extracellular matrix degradation in the fibrotic liver tissue and thestrong enhancement of hepatic regenerative capability, with MTs as a critical mediator of liver regeneration.
Liver fibrosis is a frequent event which follows a repeated orchronic insult of sufficient intensity to trigger a “wound healing”-likereaction, characterized by excessive connective tissue deposition inextracellular matrix (ECM). Chronic carbon tetrachloride (CCl4)intoxication is a well-known model for producing oxidative stressand chemical hepatic injury. Its biotransformation produces hepato-toxic metabolites, the highly reactive trichloromethyl free radical,which are further converted to the peroxytrichloromethyl radical(Williams and Burk, 1990). Reactive oxidant species likely contributeto both onset and progression of fibrosis (Poli, 2000). Antioxidanttreatment in vivo seems to be effective in preventing or reducingchronic liver damage and fibrosis (Parola and Robino, 2001).Polyphenols, naturally occurring antioxidants in fruits, vegetables,andplant-derived beverages suchas tea andwine, havebeenassociatedwith a variety of beneficial properties (Havsteen, 2002). The flavoneluteolin (3′,4′,5,7-tetrahydroxyflavone) is an important member of theflavonoid family, present in glycosylated forms and as aglycone invarious plants (Shimoi et al., 1998). Luteolin is reported to have anti-inflammatory (Ziyan et al., 2007; Veda et al., 2002), antioxidant (Perez-
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Garcia et al., 2000), antiallergic (Veda et al., 2002), antitumorigenic (Juet al., 2007), anxiolytic-like (Coleta et al., 2008), and vasorelaxativeproperties (Woodman and Chan, 2004). Previously, we have shown ahepatoprotective activity of luteolin in acute liver damage in mice(Domitrović et al., 2008a, Domitrović et al., 2009).
Hepatic stellate cells (HSCs) are a minor cell type most commonlyfound in the space of Disse, intercalated between hepatocytes andcells lining the sinusoid, projecting their dendritic processes to nearbyhepatocytes and endothelial cells (Blouin et al., 1977, Mermelsteinet al., 2001). Upon liver injury, HSCs become activated, converting intomyofibroblast-like cells. Activated HSCs proliferate and produceextracellular matrix (ECM), playing a major role in hepatic fibrosisand regeneration (Friedman, 2000). ECM, which consists of collagensand othermatrix components such as proteoglycans, fibronectins, andhyaluronic acid (Arthur, 1994), is regulated by a balance of synthesisand enzymatic degradation of ECM. The key enzymes responsible fordegradation of all the protein components of ECM and basementmembrane are matrix metalloproteinases (MMPs), a zinc-dependentfamily of endopeptidases. Previous studies have demonstrated thatthe activity of these enzymes is altered during the processes offibrogenesis and fibrinolysis (Knittel et al., 2000).
The metallothioneins (MTs), small cysteine-rich heavy metal-binding proteins, participate in an array of protective stress responses.Inmice, among the four knownMT genes, theMT I andMT II genes are
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most widely expressed. Transcription of these genes is rapidly anddramatically up-regulated in response to agents which causeoxidative stress and/or inflammation (Andrews, 2000). The inductionof MT synthesis can protect animals from hepatotoxicity induced byvarious toxins including CCl4, but also play a role in repair andregeneration of injured liver (Cherian and Kang, 2006).
Because the specific treatments to stop progressive fibrosis of theliver are not available, the objective of the present study was toinvestigate the therapeutic effect andmechanisms of action of luteolinin chemically induced liver fibrosis in mice.
Materials and methods
Materials. Luteolin, carbon tetrachloride (CCl4), olive oil, dimethylsulfoxide (DMSO), nitric acid (HNO3), hydrogen peroxide (H2O2), andgelatin were obtained from Sigma Chemical Co. (St. Louis, MO, USA).All other chemicals and solvents were of the highest grade commer-cially available.
Animals. Male Balb/c mice from our breeding colony, 2–3 monthsold, were divided into 6 groups with 5 animals per group. Mice werefed a standard rodent diet (pellet, type 4RF21 GLP, Mucedola, Italy)containing 19.4% protein, 5.5% fiber, 11.1% water, 54.6% carbo-hydrates, 6.7% ash, and 2.6% by weight of lipids (native soya oil) toprevent essential fatty acid deficiency. Total energy of the diet was16.4 MJ/kg. The animals were maintained at 12 h light/dark cycle, atconstant temperature (20±1 °C) and humidity (50±5%). Allexperimental procedures were approved by the Ethical Committeeof the Medical Faculty, University of Rijeka.
Experimental design. Mice were given CCl4 intraperitoneally (i.p.) ata 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 vehicleonly. Seventy-two hours after the last CCl4 injection, the CCl4 groupwas killed. The CCl4 control group was observed for spontaneousresolution of hepatic fibrosis for next 2 weeks. In the luteolin-treatedgroups, the polyphenolic compound was administered i.p. at a dose of10, 25, or 50 mg/kg daily for 2 weeks, respectively. These doses wereselected on the basis of preliminary studies (Domitrović et al., 2008a,Domitrović et al., 2009). Luteolin was dissolved in DMSO and dilutedin saline to the final concentrations. The final concentration of DMSOwas less than 1%. Mice from the control and CCl4 control groupsreceived diluted DMSO solution daily for 2 weeks. Animals wereterminated 24 h after the last dose of luteolin or diluted solvent bycervical dislocation. The blood was taken from orbital sinus of etheranesthetized mice. The abdomen of terminated animal was cut openquickly and the liver perfused with isotonic saline, excised, blotteddry, weighed, and divided into samples. The samples were used toassess 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 markersof hepatic function, were measured by using a Bio-Tek EL808 UltraMicroplate Reader (BioTek Instruments,Winooski, VT, USA) accordingto the manufacturer's instructions.
Determination of Cu/Zn SOD activity and GSH concentration. Cu/ZnSOD activity and total GSH, indicators of oxidative stress, weremeasured spectrophotometrically, using Superoxide Dismutase AssayKit and Glutathione Assay Kit (Cayman Chemical, Ann Arbor, MI, USA),according to the manufacturer's instructions. Protein content insupernatants was estimated by Bradford's method, with bovineserum 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 filtered through a 0.45-μm filter, 2 ml of samples was extractedand analyzed according to the procedure of Bergman and Loxley(1963). Briefly, sample neutralization was obtained with 10 M NaOHand 3 M HCl. After neutralization, subsequent steps were made induplicate for each sample. To a 200 μl of the above solution, 400 μl ofisopropanol in citrate-acetate-buffered Chloramine Twas added. After4 min, 2.5 ml of Ehrlich reagent was added. Tubes were wrapped inaluminum foil and incubated for 25 min in a water-bath at 60 °C,cooling each sample in tap water, and measuring the absorbance ofeach 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 themethodpreviously described (Domitrović et al., 2008a).
Determination of retinol. The hepatic levels of retinol were analyzedby high-performance liquid chromatography (HPLC) according toHosotani and Kitagawa (2003), as described previously (Domitrovićet al., 2008b).
Histopathology. Liver specimens were fixed in 4% phosphate-buffered formalin, embedded in paraffin, and cut into 4 μm thicksections. Sections for histopathological examination were stainedwith hematoxylin and eosin (H&E) and Mallory trichrome stain usingstandard procedure.
Immunohistochemical determination of GFAP, α-SMA, and MT I/II.Immunohistochemical studies were performed on paraffin embeddedliver tissues using mouse monoclonal anti-MT I+II antibody diluted1:50 (clone E9; DakoCytomation, Carpinteria, CA, USA), mousemonoclonal anti-GFAP antibody diluted 1:100 (clone 1B4; BDPharmingen, San Diego, CA, USA), and mouse monoclonal antibodyto α-SMA diluted 1:100 (SPM332; Abcam, Cambridge, UK),employing DAKO EnVision+ System, Peroxidase/DAB kit accordingto the manufacturer's instructions (DAKO Corporation, Carpinteria,CA, USA). Briefly, slides were incubated with peroxidase block toeliminate endogenous peroxidase activity. After washing, monoclonalantibodies diluted in phosphate-buffered saline supplemented withbovine serum albumin were added to tissue samples and incubatedovernight at 4 °C in a humid environment, followed by incubationwith peroxidase labeled polymer conjugated to secondary antibodiescontaining carrier protein linked to Fc fragments to preventnonspecific binding. The immunoreaction product was visualized byadding substrate-chromogen diaminobenzidine (DAB) solution,resulting with brownish coloration at antigen sites. Tissues werecounterstained with hematoxylin, dehydrated in gradient of alcohol,and mounted with mounting medium. The intensity of staining wasgraded as weak, moderate, and intense. The specificity of the reactionwas confirmed by substitution of primary antibodies with irrelevantimmunoglobulins of matched isotype, used in the same conditionsand dilutions as primary antibodies. Stained slides were analyzed bylight microscopy (Olympus BX51, Tokyo, Japan).
MMP zymography. MMP-2 and MMP-9 activities were analyzed bygelatin zymography assays as described (Kuo et al., 2003), withmodifications. After tissue homogenization in radioimmuno-precipitation assay buffer (4 ml of buffer per gram of tissue)containing 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5%sodium deoxycholate, 0.1% SDS, 2 mM PMSF, 1 mM sodiumorthovanadate, and 2 μg/ml of each aprotinin, leupeptin andpepstatin. 10 μg of liver tissue protein lysates were separated by a10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels containing 0.1% gelatin, at 4 °C and 150 V for 4 h. Gels werewashed for 30min in 2.5% Triton X-100 to remove the SDS, and briefly
Table 1Relative liver weight, hydroxyproline content, and serum markers of liver damage.
Control CCl4 CCl4 control Luteolin 10 mg/kg Luteolin 25 mg/kg Luteolin 50 mg/kg
Relative liver weight (g/100 g body weight) 5.31±0.40a 6.94±0.23b 6.17±0.19b 5.83±0.65a 5.34±0.20a 4.95±0.72a
Hydroxyproline (μg/g liver) 189±37a 846±159 b 1188±205c 443±82d 294±34e 231±31a
ALT (U/l) 16.0±3.7a 226.1±20.2b 13.1±2.1a 15.1±2.9a 14.0±2.3a 12.0±1.2a
ALP (U/l) 77.4±1.1a 114.0±4.0b 87.9±7.9b 79.8±4.1a 66.7±3.4c 60.3±5.0c
Each value represents the mean±SD for 5 mice. Values not sharing a common letter superscript (a, b, c) are significantly different (pb0.05). Statistical difference was determinedusing nonparametric Man–Whitney and Kruskal–Wallis tests.
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washed in the reaction buffer containing 50 mM Tris–HCl, pH 7.5,5 mM CaCl2, 1 μM ZnCl2, and 0.02% NaN3. The reaction buffer waschanged 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 thebackground of blue staining indicate the presence of gelatinaseactivities. The intensity of the bands was assayed by scanning videodensitometry (Bio-Rad GS-710 Calibrated Imaging Densitometer, Bio-Rad Laboratories, UK).
Statistical analysis. Data were analyzed using StatSoft STATISTICAversion 7.1 software. Differences between the groups were assessedby a nonparametric Man–Whitney and Kruskal–Wallis tests. Values inthe text are means±standard deviation (SD). Differences withpb0.05 were considered to be statistically significant.
Results
Hepatotoxicity
Serum AST, ALT, and ALP activities were changed significantly inmice receiving CCl4 twice a week for 6 weeks and terminated 72 hlater (Table 1). The relative liver weight significantly increased in theCCl4 group when compared to controls. In the CCl4 control group,observed for the spontaneous regression of fibrosis for 2 weeks, therelative liver weight and ALP activity were still increased; however,AST and ALT activities were normalized. Luteolin administrationattenuated the elevation of ALP activity in the mice treated with CCl4and decreased relative liver weight to control values. Higher doses ofluteolin, 25 and 50 mg/kg, decreased ALP activity below controlvalues. However, AST activity in mice treated with 50 mg/kg ofluteolin was significantly increased compared to the control groupand groups treated with luteolin.
Hepatic hydroxyproline
The liver hydroxyproline content was fivefold higher in the CCl4group than in controls and progressively increased in the CCl4 controlgroup (Table 1). Luteolin therapy significantly decreased the hepatichydroxyproline level, which was returned to normal values in thegroup receiving 50 mg/kg of luteolin.
Table 2Hepatic indicators of oxidative stress, liver retinol, and metal content.
Each value represents the mean±SD for 5 mice. Values not sharing a common letter supersusing nonparametric Man–Whitney and Kruskal–Wallis tests.
Cu/Zn SOD activity and GSH concentration
There was no evidence of oxidative stress in the CCl4 control groupwhen compared to the CCl4 group. The hepatic total GSH levelremained at normal values throughout the treatment period, exceptin the group treated with 50 mg/kg of luteolin, where it wassignificantly increased (Table 2). Cu/Zn SOD activity was increased inthe CCl4 group; however, its level was normalized in the CCl4 controlgroup and groups treated with luteolin. The liver total retinolconcentration was markedly decreased in the CCl4 group, thenincreased in the CCl4 control group, returning to normal values inthe 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 notbeen significantly changed by CCl4 intoxication or during luteolintherapy. The liver trace element content was expressed as μg/g dryliver weight (ppm).
Histopathology
Liver sections from control mice stained with hematoxylin andeosin (H&E) showednormal hepatic architecture. Liver specimens fromthe CCl4 group showed remnants of degenerated and balooned/necrotic hepatocytes containing acidophilic hyaline inclusions(Fig. 1B). Mild mononuclear cell infiltration in these areas was alsopresent. Hepatocytes in the vicinity of hepatic lesions, particularlythose situated immediately alongside the lesion border, showedfeatures of pronounced macrovesicular or microvesicular steatosis.Distended sinusoidal spacesfilledwith erythrocytes as amorphologicalhallmark of congestion (disturbed blood flow) were also observed.Hepatic lesions, fatty degeneration, and hyaline deposits as well asmorphological signatures of congestion were more pronounced in theCCl4 control group (Fig. 1C). Hepatic lesions and hyaline deposits werealso present in the livers of mice treated with 10 mg/kg of luteolin(Fig. 1D), but they were significantly reduced in extent and were lessfrequent compared to the CCl4 control group, whereas macrovesicularsteatosis was still present in the surrounding liver parenchyma, withnoticeable signs of congestion. The livers ofmice receiving 25mg/kg ofluteolin showed only sporadic, markedly small hepatic lesions, hyaline
l Luteolin 10 mg/kg Luteolin 25 mg/kg Luteolin 50 mg/kg
a 0.86±0.08a 0.88±0.08a 0.89±0.15aa 9.56±0.79a 10.57±0.60a 11.37±0.65bc 1.02±0.19c 1.19±0.12c 1.32±0.23a,c
cript (a, b, c) are significantly different (pb0.05). Statistical difference was determined
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 aweek for 6 weeks, terminated 72 h after the last CCl4 injection, showed severe hepatic lesions, degenerated and balooned/necrotic hepatocytes with cellular hyaline inclusions, andmild mononuclear cell infiltration (arrowheads). (C) In the CCl4 control group, observed for spontaneous regression of fibrosis for additional 2 weeks, hepatic lesions, baloonedhepatocytes, and hyaline bodies were more frequent. (D) Hepatic lesions and hyaline deposits in mice receiving 10 mg/kg luteolin for 2 weeks were significantly reduced in extentand 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 treatedwith 50 mg/kg of luteolin showed maintained hepatic architecture, with only weak microvesicular steatosis. Arrows show balooned hepatocytes with hyaline inclusions. Originalmagnification 100×, insets 1000×. H&E stain.
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changes, and microvesicular steatosis in the periportal areas, butwithout morphological signs of hemodynamic disturbance (Fig. 1E). Inanimals treated with 50 mg/kg of luteolin, the livers showedmaintained histoarchitecture, almost similar to controls, with onlyweak microvesicular steatosis of periportal and perilobular hepato-cytes (Fig. 1F). There were no changes which could be attributed tohepatic hemodynamic disturbances in these animals.
The livers from control mice stained with Mallory trichrome stainshowed traces of collagen only in the vascular walls. Liver section from
the CCl4 group showed multiple fibrotic nodules and extensive fibrosispredominantly in the periportal areas (Fig. 2B). The pericellularcollagen deposition and the extent of fibrotic changes were evenmore pronounced in the CCl4 control group (Fig. 2C). Fibrotic lesionswere still present in the livers ofmice treatedwith 10mg/kg of luteolin(Fig. 2D), but they were significantly reduced in extent and were lessfrequent compared to the CCl4 control group, whereasmild pericellularfibrosiswasobserved in the surrounding liver parenchyma. The livers ofmice receiving 25 mg/kg of luteolin showed only sporadic, markedly
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small fibrotic deposits in the periportal areas, with a discrete peri-cellular fibrosis (Fig. 2E). Animals treated with 50 mg/kg of luteolinshowed only minor and sporadic pericellular fibrotic changes (Fig. 2F).
Immunohistochemistry of GFAP, α-SMA, and MT I/II
Activated HSCs could be distinguished from the other myofibro-blasts of the liver (such as portal myofibroblasts) by their specificposition in the liver parenchyma (Cassiman et al., 2002).With regard tothe distribution of α-SMA-positive fibrogenic cells, in the livers of
Fig. 2. The histopathologic detection of collagen in the livers. Collagen fibers of the connectivereceiving CCl4 twice a week for 6 weeks, terminated 72 h after the last CCl4 injection, had devspontaneous regression of fibrosis for additional 2 weeks, had more intense collagen stainingreduced in size. (E) The livers of mice receiving 25mg/kg of luteolin showed sporadic, small fiwere observed only minor pericellular fibrotic changes (arrow). Original magnification 100
control animals,α-SMA immunopositivitywas restricted to the smoothmusculature belonging to the arterial tunicamedia, aswell as to thewallof majority of portal and central veins, while other liver cells remainnegative (Fig. 3A). CCl4 strongly induced perisinusoidal α-SMAexpression, which was recognized as activated HSCs, through affectedlobuli, connected between themselves with thin, “bridging” immuno-positivity (Fig. 3B). TheHSCs varied in shape and size. In the CCl4 controlgroup, perisinusoidal immunopositivity was withdrawn, but emergedin hepatocytes located at the scar-parenchyma interface (Fig. 3C). Thelivers of mice receiving 10 and 25 mg/kg of luteolin showed only
tissues are identified by their blue color. (A) Liver section from a control mice. (B) Miceeloped extensive fibrosis in the periportal areas. (C) The CCl4 control group, observed for. (D) The collagen deposits inmice receiving 10 mg/kg luteolin for 2 weeks were greatlybrotic lesions in the periportal zone. (F) In mice treated with 50mg/kg of luteolin there×, insets 1000×. Mallory trichrome stain.
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sporadic α-SMA immunopositivity in the hepatocytes in the vicinity offibrotic lesions (Figs. 3D and E). The livers ofmice receiving 50mg/kg ofluteolin showed staining pattern similar to control animals.
Immunohistochemical staining using monoclonal anti GFAP anti-body showed thin, irregular immunopositive bands lining the hepaticsinusoids of control animals (staining pattern considered as acharacteristic for HSC) (Fig. 4A). In the CCl4 and CCl4 control groups,moderate and strong GFAP expressions, respectively, were limited tothe scar areas (Figs. 4B and C). Treatment with 10 and 25 mg/kg ofluteolin decreased GFAP expression in the fibrotic areas, but induced
Fig. 3. The expression and specific tissue distribution ofα-SMA. (A) In the livers of control animliver cells remain negative. (B) Inmice receiving CCl4 twice a week for 6 weeks, terminated 72diffusely stained in affected lobuli, connected between themselves with thin, “bridging” immuoffibrosis for additional 2weeks, clusters ofmoderatelyα-SMA-immunopositivehepatocytes l10 mg/kg (D) and 25 mg/kg (E) of luteolin showed only sporadic α-SMA immunopositivity50 mg/kg of luteolin showed staining pattern similar to control animals. Original magnificati
sporadic assembling of GFAP positive cells in the surrounding tissue(Figs. 4D and E). The livers of mice receiving 50 mg/kg of luteolinshowed staining pattern similar to control animals, characterized bymoderate GFAP expression alongside the sinusoidal border (Fig. 4F).
Control animals had moderate cytoplasmic MT I/II expression inthe perilobular areas (Fig. 5A). Liver tissues from the CCl4 groupshowed weak cytoplasmic immunostaining of fatty changed hepa-tocytes surrounding the fibrotic areas, but strong immunopositivitywithin balooned hepatocytes (Fig. 5B). In the CCl4 control group, MTI/II immunopositivity was found to be only sporadic and restricted to
als,α-SMA immunopositivitywas restricted to smoothmusculature (arrow),while otherh after the last CCl4 injection, manyα-SMA-containingmyofibroblasts were strongly andnopositivity (arrows). (C) In the CCl4 control group, observed for spontaneous regressionocatedat the scar-parenchyma interfacewere found (arrows). The livers ofmice receivingin the hepatocytes in vicinity to fibrotic lesions (arrows). (F) The livers of mice receivingon 400×, inset 1000×. Immunohistochemical stain for α-SMA.
Fig. 4. The expression and specific tissue distribution of GFAP. (A) The liver sections from control mice showed thin GFAP immunopositive bands lining the hepatic sinusoids. (B) Inthe 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 fibrosis for additional 2 weeks, strong GFAP expression were limited to the scar areas. Mice receiving 10mg/kg (D) and (E) 25mg/kg luteolinfor 2 weeks had decreased GFAP expression in the fibrotic areas, with sporadic expression in the surrounding tissue. (F) The livers of mice receiving 50 mg/kg of luteolin showedstaining pattern similar to the control animals. Original magnification 1000×. Immunohistochemical stain for GFAP.
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hyaline bodies in the context of fibrous lesions, while in the remnantliver tissue was not detected (Fig. 5C). The livers of animals receiving10 mg/kg of luteolin showed weak cytoplasmic MT I/II expression inthe remaining hepatic parenchyma andmore frequent appearance ofMT I/II immunopositive hyaline deposits (Fig. 5D). Concomitantlywith the histoarchitecture improvement, moderate perilobularMT I/II immunopositivity was observed in the livers of animalstreated with 25 mg/kg of luteolin (Fig. 5E). Liver sections from micetreatedwith 50mg/kg of luteolin have shown strong cytoplasmic andnuclear immunopositivity, remarkably in the periportal and perilob-ular areas (Fig. 5F). Fig. 5G shows the extent of MT I/II expression.
MMP zymography
MMPs were identified by their molecular weights. Liver extractscontained mainly the latent form of MMP-9 at about 92 kDa (Fig. 6).CCl4-intoxication decreased MMP-9 expression, which was even morepronounced in the CCl4 control group. Treatment with lutolin resultedin an increase inMMP-9expression in a dose-dependentmanner, beingthe 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 25and 50 mg/kg of luteolin. In contrast to MMP-9, both latent and activeforms 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 thecontrol and treated animals. (A) Expression of latent form of 92 kDa MMP-9 in equalprotein quantities of hepatic homogenates from control mice (lane 1), mice treatedwith CCl4 (lane 2), mice left for spontaneous recovery (lane 3), and from mice treatedwith luteolin 10 mg/kg (lane 4), 25 mg/kg (lane 5), and 50 mg/kg (lane 6). Weakexpression of the active 86 kDa form of MMP-9 was noticeable only in lanes 5 and 6. Nosignificant effect of CCl4-intoxication or the treatment with luteolin on the MMP-2expression or proMMP-2 activation has been detected. (B) Densitometric analysis ofbands detected in gelatin zymograms. The bar graph shows averages of band intensityof proMMP-9 expression (mean±SD, n=5). Values not sharing a common lettersuperscript (a, b, c, d, e) are significantly different (pb0.05). MMPs were identified bytheir molecular weights.
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Discussion
Liver fibrosis represents chronic wound repair following liverinjury (Friedman, 1999). Independently from the nature of the injury,the usual initiating event of fibrogenesis is represented by tissuenecrosis of variable entity. Activation of either resident or blood-derived phagocytes increases the steady-state concentration offibrogenic cytokines, which recruit a large amount of fibroblasts andfibroblast-like cells for excess production of ECM. The terminaloutcome of liver fibrosis is the formation of nodules encapsulatedby fibrillar scar matrix (Tsukamoto, 1999). Irreversible fibrosisdevelops when the appropriate cellular mediators as the source ofMMPs are absent (Issa et al., 2004). The progression of fibrosisobserved in the CCl4 control group suggests development of anirreversible fibrosis. Additionally, numerous eosinophilic hyalineglobules of varying sizes have been found in fibrotic lesions. Theamount of hyaline bodies has correlated with abundance of collagendeposits and the degree of liver fibrosis. Hyaline deposits, such asMallory bodies, could be occasionally found in the hepatocytes inalcoholic hepatitis but also in different nonalcoholic liver disorders(Denk et al., 2000). These inclusions consist of aggregates ofabnormally phosphorylated, ubiquitinated, and cross-linked keratinsand nonkeratin components (Stumptner et al., 2002). Proteinaggregates could be reverted to normal state bymolecular chaperones
Fig. 5. The expression and specific tissue distribution of MT I/II. (A) The livers from control mCCl4 twice a week for 6 weeks, terminated 72 h after the last CCl4 injection, showedweak cytostrong immunopositivity in the fibrotic lesions. (C) In the CCl4 control group, observed forsporadic and restricted to the fibrotic scars. (D) Mice receiving 10 mg/kg luteolin therapy forfibrotic lesions. (E) The livers of mice receiving 25mg/kg of luteolin showedmoderate periloperiportal staining in the group treated with 50 mg/kg of luteolin. (G) The bar graph showscommon letter superscript (a, b, c, d, e) are significantly different (pb0.05). Original magni
or degradated by proteasomes (Kopito, 2000). However, if reparationor degradation fails, abnormal proteins became segregated in thecytoplasm as inclusion bodies.
Hepatic indicators of oxidative stress and hepatic damage in thisstudy were markedly changed in CCl4-intoxicated group, 72 h afterdiscontinuation of the toxin. Two weeks later, in the CCl4 controlgroup and the groups treated with luteolin, most of the biochemicalparameters, Cu/Zn SOD activity, GSH, and aminotransferase levels didnot indicate significant oxidative stress or hepatocellular damage. Thepossible reason for this is that mammalian cells respond quickly to theinitial oxidative damage by means of a characteristic adaptativephenomenon (Radice et al., 1998). Acquisition of adaptation to tissuedamage is a common response to insults in order to reduce thevulnerability of the cell or tissue, which depends on the activity ofvarious stress sensors, signal transduction and effectors of protection(Hagberg et al., 2004). Therefore, biochemical parameters may notreflect the degree of tissue damage (Murayama et al., 2007). Theseresults are of the significant clinical importance, suggesting that theclassical biochemical parameters such as serum aminotransferaseactivities are not a reliable indicators of chronic liver damage.
The accumulation of ECM observed in fibrosis and cirrhosis isconsidered to be the result of activation of fibroblasts, which acquire amyofibroblastic phenotype. Myofibroblasts are produced by theactivation of precursor cells, such as HSCs and portal fibroblasts.Significant differences between these two fibrogenic cell populationshave been reported, in the mechanisms leading to myofibroblastdifferentiation, their activation and deactivation (Guyot et al., 2006).However, the pathophysiological mechanisms of hepatic fibrogenesisare not yet fully understood. In particular, the role of HSCs remainsunclear. HSCs have been also found to migrate in vitro (Marra et al.,1999), suggesting that they may migrate to the fibrotic lesions andtake part in the repair process (Kinnman et al., 2000). Stimulation ofHSCswith various cytokines or type I collagen, results in an increase intheir migratory capacity, which could be accompanied by orindependent of HCS proliferation (Yang et al., 2003). In the quiescentstate, HSCs express desmin and GFAP (Yokoi et al., 1984, Neubaueret al., 1996). Upon HSCs activation, GFAP expression decreases,whereas the levels of desmin and α-SMA increase, due to theirtransformation into myofibroblast-like cells (Campbell et al., 2005). Inthis study, deposition of collagen in damaged hepatic areas, associatedwith increased α-SMA and decreased GFAP perisinusoidal immuno-positivity, indicates that activated HSCs are responsible for the fibrosisseen in the CCl4-intoxicated mice. Additionally, increased GFAPimmunopositivity in fibrotic scars suggests the accumulation ofquiescent HSCs at the places of active fibrogenesis. Changes in theactivation marker expression in the CCl4 control group, however,indicate deactivation of HSCs, which is critical for resolution of fibrosis(Iredale, 2001). Nevertheless, α-SMA immunopositive hepatocyteslocated at the scar-parenchyma interface in the CCl4 control groupsuggest that hepatocytes have acquired myofibroblast-like pheno-type. Indeed, Zeisberg et al. (2007) have recently shown that adulthepatocytes could be actively engaged in the epithelial to mesenchy-mal transition mediated by TGF-β1, by expressing fibroblast-specificprotein-1 (FSP1), suggesting that hepatocyte-derived fibroblastscould be an additional lineage of mesenchymal cells that contributeto the progression of liver fibrosis. Furthermore, Dooley et al. (2008)have observed hepatocyte transdifferentiation immediately adjacentto the fibrotic lesions, which is in agreement to our findings. However,to our knowledge, this is for the first time that the α-SMA expression,
ice showed weak perilobular MT I/II immunopositivity. (B) The livers of mice receivingplasmic immunostaining of hepatocytes located at the scar-parenchyma interface, withspontaneous regression of fibrosis for additional 2 weeks, immunopositivity was only2 weeks had weak cytoplasmic MT I/II expression with strong immunopositivity in thebular MT I/II immunopositivity. (F) Strong both cytoplasmic and nuclear perilobular andthe extent of hepatocellular MT I/II expression (mean±SD, n=5). Values not sharing afication 100×. Immunohistochemical stain for MT I/II.
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characteristic for activated HSCs, is found in hepatocytes. Thissubpopulation of hepatocytes could contribute to collagen depositionobserved in the CCl4 control group. Perisinusoidal GFAP expression inthe group treated with 50 mg/kg of luteolin indicates a completereturn of HSCs into a quiescent state.
Morphological studies have shown that fibrotic lesion of the liveralso typically involves, beside an accumulation of extracellularcollagen matrix and a transformation of stellate cells to activelydividing myofibroblasts, a reduction in stellate cell lipid droplets andhepatic storage of total retinol (Blomhoff andWake, 1991). Decreasedhepatic total retinol level in the CCl4 group observed in this study is inagreement with previous studies (Seifert et al., 1994, Natarajan et al.,2005). The vitamin A status of the liver plays an important role inhepatic fibrogenesis. The reduction of retinoid levels in HSC by anenhanced secretion of retinol from the liver into the circulation duringCCl4 treatment may stimulate the transformation of these cells intofibroblasts and contribute to fibrogenesis (Seifert et al., 1994).Increased liver retinol observed in the CCl4 control group, as well asin mice receiving luteolin therapy, also indicates a decrease in thenumber of activated HSCs.
In normal tissues, matrix protein degradation is accomplished by afamily of MMPs, whose expression significantly increases in the caseof acute liver injury (Arthur, 1994). In chronic liver injury sustainedtissue damage and inflammation 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 IVcollagen in sinusoid basement, produced by HSCs. MMP-9, the 92-kDcollagenase IV/gelatinase B, a major MMP in basement membrane-like ECM remodeling, is secreted by neutrophil granulocytes, macro-phages 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 ofliver fibrosis (Iredale, 2001). Liver injury which results in HSCactivation, particularly if chronic, leads to an increase in overallnumbers of myofibroblast-like activated HSCs that are activelyproducing matrix, while simultaneously preventing degradation ofthe matrix through expression of TIMPs. It has been consensuallyaccepted that increased production and activity of TIMPs are requiredfor decrease in MMP activity (Perez-Tamayo et al., 1987, Benyon andIredale, 2000). The key events in the reversion of liver fibrosis includeapoptosis of HSCs, increased MMP activity, decreased expression ofTIMPs, and increased degradation of collagen (Woessner, 1991). Inthe liver, myofibroblasts derived from hepatic stellate cells undergoapoptosis during the spontaneous resolution of liver fibrosis inducedby CCl4 treatment (Iredale et al., 1998). During spontaneous recoveryfrom liver fibrosis, TIMP-1 level decreases, and collagenase activityand the apoptosis of hepatic stellate cells increase, which results in anincreased fibrolysis and removal of excessively deposited ECM. MMPscould be derived either from activated HSCs prior to or duringapoptosis, from quiescent HSCs, or from other liver cells (Iredale,2001). It is still unknown how the fibrotic tissues are removed in vivoand how MMPs, which are usually down-regulated in establishedfibrotic tissues, are expressed again during the resolution process(Han, 2006). Our result showed that CCl4-intoxication decreasedMMP-9 expression in CCl4 group 72 h after toxin discontinuation, withfurther decrease in the CCl4 control group 2 weeks later. Luteolintherapy has induced the expression of MMP-9, with the highestincrease in the group treated with 50 mg/kg. The MMP-9 productionby other cells distinct from HSCs could explain its increasedexpression despite HSC deactivation. In contrast, MMP-2 did notplay a significant role in this model, which was confirmed byimmunohistochemistry (data not shown).
The mechanisms by which MT participates in the pathology andthe reversion of liver fibrosis are still largely unknown. Although anumber of biological functions have been proposed for MTs, most ofthem are related to its metal-binding property, mainly Zn and Cu
homeostasis and heavy metal detoxification (Andrews, 2000).Recently, we have shown dose- and time-dependent up-regulationof MTs expression by luteolin in acute hepatotoxicity induced by CCl4and the enhancement of hepatic regenerative capability (Domitrovićet al., 2008a). Hepatic MT I/II expression in necrotic areas wascorrelated with the Zn and Cu content. However, MTs expression inchronic exposure to CCl4 in this study was not correlated with eitherthe Zn or Cu hepatic content. Increased MT synthesis also occurs inresponse to oxidative stress (Chubatsu and Meneghini, 1993). In thisstudy, disturbance in antioxidative status in the CCl4 control group andgroups treatedwith luteolin was not observed, therefore we could notexplain the down-regulation of MT I/II as a consequence of oxidativestress. Furthermore, MT has the essential role in cell proliferation andliver regeneration (Apostolova and Cherian, 2000). Immunohisto-chemical studies have demonstrated increased MT expression both inthe cytoplasm and nucleus of rapidly proliferating cells (Miles et al.,2000). MT may donate Zn and Cu to certain metalloenzymes andtranscription factors associated with gene transcription, cell replica-tion, differentiation, and growth (Włostowski, 1993, Coyle et al.,2002). Jiang and Kang (2004) have suggested that gene therapy withMT could improve the recovery from liver fibrosis. The sameauthors have shown that mice which have developed a reversibleliver fibrosis upon removal of CCl4, had a high level of hepatic MT,but mice which developed an irreversible fibrosis, had lowexpression of MT. Therefore, hepatic MT expression could reflectthe severity of chronic liver damage, showing down-regulation ininjured tissue (Carrera et al., 2003). The results of this study showthat the MT I/II expression was progressively down-regulated fromthe CCl4 group to the CCl4 control group. The level of MTs in liverswas related to the progression of CCl4-induced fibrosis and acomplete down-regulation of MT I/II in the CCl4 control groupindicates development of an irreversible fibrosis. On the other hand,the positive correlation of MT I/II expression with luteolin dosageand liver tissue repairment suggests its direct involvement in theregeneration of liver tissue. The liver sections from mice treatedwith luteolin 50 mg/kg have shown strong up-regulation of MT I/II,including nuclear expression, which was accompanied with acomplete resolution of fibrotic scar tissue, suggesting the pro-nounced therapeutic effect of luteolin. Our results support the viewthat MT I/II could be the factors associated with a favorable clinicaloutcome in the response to therapy (Carrera et al., 2003).
Regardless of these interesting and important findings, there aresome limitations of the present study. The mechanistic insights intothe luteolin-induced recovery of irreversible liver fibrosis need to befurther approached. Although the important parameters of fibrosisand liver regeneration were determined, other factors that arepossibly involved in the liver fibrinolysis need to be explored.
In conclusion, the luteolin treatment has induced a completereversion of established liver fibrosis by decreasing hepatic fibrogenicpotential, and stimulated MT I/II expression in the liver in a dose-dependent manner, suggesting the strong enhancement of hepaticregenerative capability. Importantly, the results obtained from thisstudy suggest the therapeutic application of luteolin in patients withhepatic fibrosis, although further studies, in a more chronic model ofliver fibrosis, are required.
Conflict of interest statementThe authors declare no conflict of interest.
Acknowledgments
This research was supported by grants from the Ministry ofScience, Education and Sport, Republic of Croatia (project no. 062-0000000-3554). The authors thank Ing. Hrvoje Križan, Ljubica Črnac,and Jadranka Eškinja for their excellent technical support and Dipl.Ing. Sunčica Ostojić for her assistance with SDS-PAGE.
321R. Domitrović et al. / Toxicology and Applied Pharmacology 241 (2009) 311–321
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