liver fibrosis in mice induced by carbon tetra chloride

8/2/2019 Liver Fibrosis in Mice Induced by Carbon Tetra Chloride

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Liver fibrosis in mice induced by carbon tetrachloride and its reversion by luteolin

Robert Domitrovi a,, Hrvoje Jakovac b, Jelena Tomac c, Ivana ain 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

a b s t r a c ta r t i c l e i n f o

Article history:

Received 22 June 2009Revised 26 August 2009

Accepted 2 September 2009

Available online 8 September 2009

Keywords:

Carbon tetrachloride

Liver fibrosis

Luteolin

-Smooth muscle actin

Glial fibrillary acidic protein

Matrix metalloproteinase

Metallothionein

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 investigated

the effects of luteolin on carbon tetrachloride (CCl4)-induced hepatic fibrosis. 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 CCl 4 control group has been

observed for spontaneous reversion of fibrosis. 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 fibrosis was observed

histologically. 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 -smooth muscle

actin indicated deactivation of hepatic stellate cells. Our results suggest the therapeutic effects of luteolin on

CCl4-induced liver fibrosis by promoting extracellular matrix degradation in the fibrotic 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.

Introduction

Liver fibrosis is a frequent event which follows a repeated or

chronic insult of sufficient 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 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 contribute

to both onset and progression of fibrosis (Poli, 2000). Antioxidant

treatment in vivo seems to be effective in preventing or reducing

chronic liver damage and fibrosis (Parola and Robino, 2001).

Polyphenols, naturally occurring antioxidants in fruits, vegetables,

andplant-derived beverages such as tea andwine, have been associated

with a variety of beneficial properties (Havsteen, 2002). The flavone

luteolin (3,4,5,7-tetrahydroxyflavone) is an important member of the

flavonoid family, present in glycosylated forms and as aglycone in

various plants (Shimoi et al., 1998). Luteolin is reported to have anti-

inflammatory (Ziyanet al., 2007;Veda et al., 2002), antioxidant (Perez-

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 liverinjury, HSCs become activated, converting into

myofibroblast-like cells. Activated HSCs proliferate and produce

extracellular matrix (ECM), playing a major role in hepatic fibrosis

and regeneration (Friedman, 2000). ECM, which consists of collagens

and other matrix components such as proteoglycans,fibronectins, 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

fibrogenesis and fibrinolysis (Knittel et al., 2000).

The metallothioneins (MTs), small cysteine-rich heavy metal-

binding proteins, participate in an array of protectivestress responses.

In mice, among the fourknown MTgenes, the MTI and MTII genes are

Toxicology and Applied Pharmacology 241 (2009) 311321

Corresponding author. Fax: +385 51 651 135.

E-mail address: [email protected] (R. Domitrovi).

0041-008X/$ see front matter 2009 Elsevier Inc. All rights reserved.

doi:10.1016/j.taap.2009.09.001

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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 inflammation (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 specific treatments to stop progressive fibrosis of the

liver are not available, the objective of the present study was to

investigate the therapeutic effect and mechanisms of action of luteolinin chemically induced liver fibrosis 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 commer-

cially 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% fiber, 11.1% water, 54.6% carbo-

hydrates, 6.7% ash, and 2.6% by weight of lipids (native soya oil) to

prevent essential fatty acid deficiency. Total energy of the diet was

16.4 MJ/kg. The animals were maintained at 12 h light/dark cycle, at

constant temperature (201 C) and humidity (505%). 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 fibrosis 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 wereselected 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 final concentrations. The final 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 markersof 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 andGlutathioneAssayKit (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 filtered through a 0.45-m filter, 2 ml of samples was extracted

and analyzed according to the procedure of Bergman and Loxley

(1963). Briefly, 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 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),

accordingto the method previouslydescribed (Domitroviet 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 fixed in 4% phosphate-

buffered formalin, embedded in paraffin, 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 paraffin 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). Briefly, slides were incubated with peroxidase block toeliminate 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

nonspecific 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 specificity of the reaction

was confirmed by substitution of primary antibodies with irrelevant

immunoglobulins of matched isotype, used in the same conditionsand 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

modifications. After tissue homogenization in radioimmuno-

precipitation 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 (SDS-

PAGE) gels containing0.1%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 briefly

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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, Bio-

Rad 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 KruskalWallistests. Values in

the text are means standard deviation (SD). Differences with

pb0.05 were considered to be statistically significant.

Results

Hepatotoxicity

Serum AST, ALT, and ALP activities were changed significantly in

mice receiving CCl4 twice a week for 6 weeks and terminated 72 h

later (Table 1). The relative liver weight significantly increased in the

CCl4 group when compared to controls. In the CCl4 control group,

observed for the spontaneous regression offibrosis for 2 weeks, therelative 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 CCl 4and 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 significantly increased compared to the control group

and 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 control

group (Table 1). Luteolin therapy significantly decreased the hepatichydroxyproline 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 stressin 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

significantly 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 significantly 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 hepaticarchitecture.Liver specimensfrom

the CCl4 group showed remnants of degenerated and balooned/necrotic hepatocytes containing acidophilic hyaline inclusions

(Fig. 1B). Mild mononuclear cell infiltration 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.

Distendedsinusoidal spacesfilled witherythrocytes as a morphological

hallmark of congestion (disturbed blood flow) 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 significantly reduced in extent and were less

frequent compared to the CCl4 control group, whereas macrovesicular

steatosis was still present in the surrounding liver parenchyma, withnoticeable signs of congestion. The livers of mice receiving 25 mg/kg of

luteolin showed only sporadic, markedly small hepatic lesions, hyaline

Table 1

Relative 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 6.940.23b 6.170.19b 5.830.65a 5.340.20a 4.950.72a

Hydroxyproline (g/g liver) 189 37a 846159 b 1188205c 44382d 29434e 23131a

AST (U/l) 22.7 2.5a 276.54.1b 20.41.6a 18.63.3a 23.34.9a 27.010.3a

ALT (U/l) 16.0 3.7a 226.120.2b 13.12.1a 15.12.9a 14.02.3a 12.01.2a

ALP (U/l) 77.4 1.1a 114.04.0b 87.97.9b 79.84.1a 66.73.4c 60.35.0c

Each value represents the meanSD for 5 mice. Values not sharing a common letter superscript (a, b, c) are significantly different (pb0.05). Statistical difference was determined

using nonparametric ManWhitney and KruskalWallis tests.

Table 2

Hepatic indicators of oxidative stress, liver retinol, and metal content.

Control CCl4 CCl4 contro l Luteo lin 10 mg/kg Luteolin 25 mg/kg Luteolin 50 mg/kg

Cu/Zn SOD (U/mg protein) 0.950.04a 1.500.10b 0.910.23a 0.860.08a 0.880.08a 0.890.15a

GSH (M/g liver) 9.71 0.51a 9.640.21a 9.510.63a 9.560.79a 10.570.60a 11.370.65b

Retinol (M/g liver) 1.51 0.13a 0.740.09b 1.220.11c 1.020.19c 1.190.12c 1.320.23a,c

Zn (ppm) 75.3 2.6 85.3 9.2 84.8 10.4 79.3 6.1 81.3 9.1 85.3 11.6

Cu (ppm) 22.5 1.7a 14.01.3b 16.72.1b,c 16.30.7c 16.10.8c 15.80.4c

Each value represents the meanSD for 5 mice. Values not sharing a common letter superscript (a, b, c) are significantly different (pb0.05). Statistical difference was determined

using nonparametric Man

Whitney and Kruskal

Wallis tests.

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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 hepato-

cytes (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 fibrotic nodules and extensive fibrosis

predominantly in the periportal areas (Fig. 2B). The pericellular

collagen deposition and the extent of fibrotic changes were even

more pronounced in the CCl4 control group (Fig. 2C). Fibrotic lesions

were still present in thelivers of micetreated with 10 mg/kg of luteolin

(Fig. 2D), but they were significantly reduced in extent and were less

frequent compared to the CCl4 control group, whereas mild pericellular

fibrosis wasobserved in thesurrounding liver parenchyma. Thelivers of

mice receiving 25 mg/kg of luteolin showed only sporadic, markedly

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 infiltration (arrowheads). (C) In the CCl4 control group, observed for spontaneous regression offibrosis 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 signi ficantly 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

magnification 100, insets 1000. H&E stain.



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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 luteolin

showed only minor and sporadic pericellularfibrotic changes (Fig. 2F).

Immunohistochemistry of GFAP, -SMA, and MT I/II

Activated HSCs could be distinguished from the other myofibro-

blasts of the liver (such as portal myofibroblasts) by their specific

positionin the liver parenchyma (Cassiman et al.,2002). With regard tothe distribution of -SMA-positive fibrogenic cells, in the livers of

controlanimals,-SMA immunopositivity was restrictedto the smooth

musculature belongingto thearterial tunica media, as well as to thewall

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 immuno-

positivity (Fig. 3B). The HSCsvaried inshape and size. In the CCl4 control

group, perisinusoidal immunopositivity was withdrawn, but emerged

in hepatocytes located at the scar-parenchyma interface (Fig. 3C). Thelivers of mice receiving 10 and 25 mg/kg of luteolin showed only

Fig. 2. The histopathologic detection of collagen in the livers. Collagen fibers of theconnective tissues areidentified by their blue color.(A) Liver section from a control mice. (B)Mice

receiving CCl4 twice a week for6 weeks,terminated 72h after thelast CCl4 injection, had developed extensivefibrosisin theperiportal areas.(C) TheCCl4 control group, observed for

spontaneousregression offibrosisfor additional 2 weeks,had more intense collagen staining.(D) Thecollagendeposits in mice receiving10 mg/kg luteolin for2 weeks were greatly

reduced in size. (E)The livers of mice receiving25 mg/kg of luteolin showedsporadic, smallfibroticlesions in theperiportal zone. (F)In mice treated with 50mg/kgof luteolin there

were observed only minor pericellular fibrotic changes (arrow). Original magnification 100, insets 1000. Mallory trichrome stain.



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sporadic -SMA immunopositivity in the hepatocytes in the vicinity offibroticlesions (Figs. 3D and E). The livers ofmicereceiving50 mg/kg of

luteolin showed staining pattern similar to control animals.

Immunohistochemical staining using monoclonal anti GFAP anti-

body 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 ofluteolin decreased GFAP expression in the fibrotic 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 hepa-

tocytes surrounding the fibrotic areas, but strong immunopositivity

within balooned hepatocytes (Fig. 5B). In the CCl4 control group, MTI/II immunopositivity was found to be only sporadicand restricted to

Fig. 3. Theexpression andspecific tissue distributionof-SMA. (A)In thelivers of control animals,-SMAimmunopositivitywas restricted to smooth musculature(arrow), whileother

livercells remain negative.(B) In micereceiving CCl4 twicea week for6 weeks,terminated72 h afterthe last CCl4 injection,many-SMA-containingmyofibroblastswere strongly and

diffuselystainedin affectedlobuli, connectedbetweenthemselves withthin, bridging immunopositivity (arrows).(C) In the CCl4 control group, observed for spontaneous regression

offibrosis foradditional2 weeks, clustersof moderately-SMA-immunopositive hepatocytes locatedat thescar-parenchymainterfacewere found(arrows).The livers ofmice receiving

10 mg/kg (D) and 25 mg/kg (E) of luteolin showed only sporadic -SMA immunopositivity in the hepatocytes in vicinity to fibrotic lesions (arrows). (F) The livers of mice receiving

50 mg/kg of luteolin showed staining pattern similar to control animals. Original magnification 400, inset 1000. Immunohistochemical stain for -SMA.



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hyaline bodies in the context offibrous lesions, while in the remnant

liver tissuewas 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 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 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 increasein MMP-9 expressionin a dose-dependentmanner, 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.

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) 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 offibrosisfor additional 2 weeks,strong GFAP expression were limited to thescarareas. Mice receiving10 mg/kg (D)and (E)25 mg/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 showed

staining pattern similar to the control animals. Original magnification 1000. Immunohistochemical stain for GFAP.



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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 tissue

necrosis of variable entity. Activation of either resident or blood-

derived phagocytes increases the steady-state concentration of

fibrogenic cytokines, which recruit a large amount offibroblasts and

fibroblast-like cells for excess production of ECM. The terminal

outcome of liver fibrosis is the formation of nodules encapsulated

by fibrillar scar matrix (Tsukamoto, 1999). Irreversible fibrosis

develops when the appropriate cellular mediators as the source of

MMPs are absent (Issa et al., 2004). The progression of fibrosis

observed in the CCl4 control group suggests development of an

irreversible fibrosis. Additionally, numerous eosinophilic hyaline

globules of varying sizes have been found in fibrotic lesions. The

amount of hyaline bodies has correlated with abundance of collagendeposits and the degree of liver fibrosis. 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 normalstate by molecularchaperones

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 signifi

cant oxidative stress or hepatocellular damage. Thepossible reasonfor 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

reflect the degree of tissue damage (Murayama et al., 2007). These

results are of the significant 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 fibrosis and cirrhosis is

considered to be the result of activation offibroblasts, which acquire a

myofibroblastic phenotype. Myofibroblasts are produced by the

activation of precursor cells, such as HSCs and portal fibroblasts.

Significant differences between these two fibrogenic cell populations

have been reported, in the mechanisms leading to myofibroblast

differentiation, their activation and deactivation (Guyot et al., 2006).

However, the pathophysiological mechanisms of hepatic fibrogenesis

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 fibrotic lesions and

take part in the repair process (Kinnman et al., 2000). Stimulation of

HSCs with various cytokinesor 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 theirtransformation into myofibroblast-like cells (Campbell et al., 2005).In

this study, deposition of collagen in damaged hepatic areas, associated

with increased -SMA and decreased GFAP perisinusoidal immuno-

positivity, indicatesthat activated HSCs are responsible for thefibrosis

seen in the CCl4-intoxicated mice. Additionally, increased GFAP

immunopositivity in fibrotic scars suggests the accumulation of

quiescent HSCs at the places of active fibrogenesis. Changes in the

activation marker expression in the CCl4 control group, however,

indicate deactivationof HSCs, which is critical for resolution offibrosis

(Iredale, 2001). Nevertheless, -SMA immunopositive hepatocytes

located at the scar-parenchyma interface in the CCl4 control group

suggest that hepatocytes have acquired myofibroblast-like pheno-

type. Indeed, Zeisberg et al. (2007) have recently shown that adult

hepatocytes could be actively engaged in the epithelial to mesenchy-mal transition mediated by TGF-1, by expressing fibroblast-specific

protein-1 (FSP1), suggesting that hepatocyte-derived fibroblasts

could be an additional lineage of mesenchymal cells that contribute

to the progression of liver fibrosis. Furthermore, Dooley et al. (2008)

have observed hepatocyte transdifferentiation immediately adjacent

to the fibrotic lesions, which is in agreement to ourfindings. However,

to our knowledge, this is for the first time that the -SMA expression,

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 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 theactive 86 kDaform ofMMP-9was noticeable onlyin lanes 5 and6. No

significant 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 (meanSD, n =5). Values not sharing a common letter

superscript (a, b, c, d, e) are significantly different (pb0.05). MMPs were identified by

their molecular weights.

Fig. 5. The expression and specific 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 for6 weeks,terminated 72 h after thelast CCl4 injection, showedweak cytoplasmicimmunostainingof hepatocytes located at the scar-parenchymainterface, with

strong immunopositivity in the fibrotic lesions. (C) In the CCl4 control group, observed for spontaneous regression offibrosis for additional 2 weeks, immunopositivity was only

sporadic and restricted to the fibrotic scars. (D) Mice receiving 10 mg/kg luteolin therapy for 2 weeks had weak cytoplasmic MT I/II expression with strong immunopositivity in the

fibroticlesions.(E) Thelivers ofmice receiving25 mg/kg of luteolin showedmoderateperilobularMT I/II immunopositivity. (F)Strong both cytoplasmicand 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 (meanSD, n =5). Values not sharing a

common letter superscript (a, b, c, d, e) are significantly different (pb

0.05). Original magnification 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 fibrotic lesion of the liver

also typically involves, beside an accumulation of extracellular

collagen matrix and a transformation of stellate cells to actively

dividing myofi

broblasts, a reduction in stellate cell lipid droplets andhepatic 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 fibrogenesis. The reduction of retinoid levels in HSC by an

enhanced secretionof retinol from theliver into thecirculationduring

CCl4 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 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 significantly increases in the case

of acute liver injury (Arthur, 1994). In chronic liver injury sustained

tissue 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 IV

collagen in sinusoid basement, produced by HSCs. MMP-9, the 92-kD

collagenase 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 of

liver fibrosis (Iredale, 2001). Liver injury which results in HSC

activation, particularly if chronic, leads to an increase in overall

numbers of myofibroblast-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 requiredfor decrease in MMP activity (Perez-Tamayo et al., 1987, Benyon and

Iredale, 2000). The key events in the reversion of liver fibrosis include

apoptosis of HSCs, increased MMP activity, decreased expression of

TIMPs, and increased degradation of collagen (Woessner, 1991). In

the liver, myofibroblasts derived from hepatic stellate cells undergo

apoptosis during the spontaneous resolution of liver fibrosis induced

by CCl4 treatment (Iredale et al., 1998). During spontaneous recovery

from liver fibrosis, TIMP-1 level decreases, and collagenase activity

and the apoptosis of hepatic stellate cells increase, which results in an

increasedfibrolysis 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 fibrotic tissues are removed in vivo

and 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 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 significant role in this model, which was confirmed by

immunohistochemistry (data not shown).

The mechanisms by which MT participates in the pathology and

the reversion of liver fibrosis 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 detoxification (Andrews, 2000).

Recently, we have shown dose- and time-dependent up-regulation

of MTs expression by luteolin in acute hepatotoxicity induced by CCl4and 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 thisstudy, disturbance in antioxidative status in the CCl4 control groupand

groupstreated 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). Immunohisto-

chemical 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 replica-

tion, 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 fibrosis. The same

authors have shown that mice which have developed a reversible

liver fibrosis upon removal of CCl4, had a high level of hepatic MT,

but mice which developed an irreversible fibrosis, had low

expression of MT. Therefore, hepatic MT expression could reflect

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 fibrosis and a

complete down-regulation of MT I/II in the CCl4 control group

indicates development of an irreversible fibrosis. 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 fibrotic scar tissue, suggesting the pro-nounced 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 findings, there are

some limitations of the present study. The mechanistic insights into

the luteolin-induced recovery of irreversible liver fibrosis need to be

further approached. Although the important parameters of fibrosis

and liver regeneration were determined, other factors that are

possibly involved in the liver fibrinolysis need to be explored.

In conclusion, the luteolin treatment has induced a complete

reversion of established liver fibrosis by decreasing hepatic fibrogenic

potential, and stimulated MT I/II expression in the liver in a dose-

dependent manner, suggesting the strong enhancement of hepatic

regenerative capability. Importantly, the results obtained from thisstudy suggest the therapeutic application of luteolin in patients with

hepatic fibrosis, although further studies, in a more chronic model of

liver fibrosis, are required.

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgments

This research was supported by grants from the Ministry of

Science, Education and Sport, Republic of Croatia (project no. 062-

0000000-3554). 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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