resveratrol prevents fibrosis, nf-κb activation and tgf-β increases induced by chronic ccl4...

RESVERATROL PREVENTS LIVER FIBROSIS 35

Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2008; 28: 35–43

DOI: 10.1002/jat

JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 2008; 28: 35–43Published online 11 April 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/jat.1249

Resveratrol prevents fibrosis, NF-κκκκκB activation and TGF-βββββincreases induced by chronic CCl4 treatment in rats

Enrique Chávez,1 Karina Reyes-Gordillo,1 José Segovia,2 Mineko Shibayama,3 Victor Tsutsumi,3

Paula Vergara,2 Mario G. Moreno1 and Pablo Muriel1,*

1 Sección Externa de Farmacología, Cinvestav-IPN, Apdo Postal 14-740, México 07000, D.F. México2 Departamento de Fisiología Biofísica y Neurociencias, Cinvestav-IPN, Apdo Postal 14-740, México 07000, D.F. México3 Departamento de Patología Experimental, Cinvestav-IPN, Apdo Postal 14-740, México 07000, D.F. México

Received 8 January 2007; Revised 19 February 2007; Accepted 21 February 2007

ABSTRACT: Resveratrol is a nonflavonoid polyphenol with antioxidant, anticancer and antiinflammatory properties.

Moreover, it has been reported that this compound inhibits NF-κκκκκB, which regulates the transcription of several genes

including cytokines such as the profibrogenic TGF-βββββ . The aim of this work was to evaluate the pharmacological effects

of resveratrol on CCl4-induced cirrhosis in the rat. Four groups were formed: the control group that received the vehicles

only; the CCl4 group that received the toxin (0.4 g kg−−−−−1, i.p., three times a week, for 8 weeks); the CCl4 plus resveratrol

(10 mg kg−−−−−1, daily) group; and the resveratrol alone group. Alanine aminotransferase, alkaline phosphatase and bilirubins

were increased by CCl4, but resveratrol afforded some degree of protection. Glycogen was decreased markedly by CCl4

and resveratrol prevented almost completely this effect. No antioxidant effect of resveratrol was observed. One of the most

prominent effects was on fibrosis which increased near 5-fold (hydroxyproline) in the CCl4 group; resveratrol preserved

the content of collagen. These results were corroborated by histopathology. To elucidate the antifibrogenic mechanism of

resveratrol, the activation of NF-κκκκκ B and the production of TGF-βββββ were measured; in both cases CCl4 increased them and

resveratrol abolished them; however, changes in NF-κκκκκ B were modest and did not reach statistical significance, while the

increase in TGF-βββββ was about three fold and resveratrol decreased it under control values. Together, the present results

indicate that resveratrol possesses a strong antifibrogenic effect at least in the CCl4 model of cirrhosis. Moreover, the

action mechanism is probably associated with its ability to reduce NF-κκκκκB activation and TGF-βββββ content. Copyright © 2007

John Wiley & Sons, Ltd.

KEY WORDS: resveratrol; cytokines; liver injury; carbon tetrachloride; necrosis; inflammation; fibrosis; cirrhosis; NF-κB;

TGF-β

* Correspondence to: Dr Pablo Muriel, Sección Externa de Farmacología,

Cinvestav-IPN, Apdo Postal 14-740, México 07000, D.F., México.

E-mail: [email protected]

Contract/grant sponsor: Conacyt; contract/grant number: 42721.

particular transforming growth factor-β (TGF-β) has been

associated with the fibrotic process and thus it has been

the aim of some studies to interfere with the disease

(Muriel, 2007b). Nuclear factor κB (NF-κB) is an induc-

ible factor that functions to enhance the transcription

of a variety of genes including those for cytokines and it

is well known for its involvement in inflammatory

responses (Ghosh et al., 1998). Therefore, NF-κB is an-

other interesting target in the treatment of liver diseases.

On the other hand, resveratrol (3,5,4′-trans-trihydro-

xystilbene), a natural phytoalexin present in grapes,

peanuts and red wine, has various pharmacological

effects including antiinflammatory and antioxidant prop-

erties, modulation of lipid metabolism and prevention

of cancer (Fremont, 2000; Dong, 2003; Aggarwal et al.,

2004). In addition, recently it was demonstrated that

resveratrol protects against acetaminophen-induced toxi-

city in mice (Sener et al., 2006a). Importantly, it has been

reported that resveratrol inhibits the activation of NF-κB;

some authors described a reduced nuclear appearance

of NF-κB factor (Tsai et al., 1999) and others showed

an interference with their transcription activity (Pendurthi

Introduction

Experimental CCl4-induced cirrhosis seems to reproduce

the main characteristics of human cirrhosis: the liver is

grossly nodular; there is portal hypertension in most of

the animals, and the normal architecture is replaced by

nodules of regenerating liver surrounded by fibrotic septa

with proliferating bile ducts. Therefore, the CCl4-induced

cirrhosis is the most widely used model to reproduce

human cirrhosis in experimental animals to study patho-

physiological mechanisms of the disease, and many

possible antifibrotic drugs are tested in this model before

using them in humans (Muriel, 2007a). Hepatic stellate

cells (HSC) are activated as a result of liver injury. Once

activated, HSC increase their production of extracellular

matrix (ECM) proteins finally leading to fibrosis and

cirrhosis. Activation of HSC is dependent on cytokines, in

36 E. CHAVEZ ET AL.


DOI: 10.1002/jat

et al., 1999). Furthermore, it has been shown that

resveratrol decreases proinflammatory cytokine expres-

sion in vitro (Wadsworth and Koop, 1999). Therefore this

work investigated whether resveratrol was capable of

preventing CCl4-induced liver cirrhosis. It was found

that CCl4 produced fibrosis, activation of NF-κB and

production of TGF-β; resveratrol prevented fibrosis

completely and this effect was accompanied by preven-

tion of the translocation of NF-κB to the nucleus and

attenuation of the profibrotic cytokine TGF-β.

Material and Methods

Chemicals

Resveratrol, chloramine-T, methyl cellosolve sodium

thiosulphate, p-dimethylaminobenzaldehyde, antrone,

carboxymethylcellulose, thiobarbarbituric acid, γ-

glutamyl-p-nitroanilide, L-γ-glutamyl-p-nitroaniline,

p-nitrophenyl phosphate and bovine serum albumin

were purchased from the Sigma Chemical Company

(St Louis, MO, USA). Carbon tetrachloride, sodium

acetate, sodium hydroxide, glacial acetic acid, hydro-

chloric acid, sulfuric acid, ethanol, methanol, potassium

hydroxide and formaldehyde were obtained from J.T.

Backer (Xalostoc, Mexico).

Study Design

Wistar male rats weighing initially 90–100 g and fed

a Purina chow rat diet ad libitum were used. Cirrhosis

was produced by i.p. administration of CCl4 (0.4 g kg−1

of body weight) dissolved in mineral oil three times

per week for 8 weeks. Four groups were performed.

Group 1 (n = 8), consisted of control animals receiving

the vehicle only (oil). In group 2 (n = 15), CCl4 was

administered for 8 weeks. Animals in group 3 (n = 15)

received resveratrol (10 mg kg−1 suspended in 0.7%

carboxymethyl-cellulose (CMC) p.o., for 8 weeks) plus

CCl4. The rats in group 4 (n = 8) received resveratrol

only. All the animals were killed under light ether

anesthesia 72 h after the last dose of CCl4 or mineral oil.

All determinations with the exception of NF-κB (see

below) were measured at this time. In order to determine

the activation of NF-κB another set of animals weighing

around 200 g received the same treatments, but CCl4 was

given only once and resveratrol administered 15 min

before CCl4; in this particular case the animals were

killed 30 min after CCl4 or mineral oil because this is

approximately the time in which NF-κB is activated

and translocated to the nucleus. Blood was collected

by cardiac puncture and the liver was rapidly removed.

All samples were kept on ice until analysis. All animals

received humane care according to the institution’s

guidelines and the Mexican Official Norm (NOM-

062-ZOO-1999) regarding technical specifications for

production, care and use of laboratory animals.

Serum Enzyme Activities and BilirubinDetermination

Serum was obtained for determination of liver damage

by measuring the activities of alanine aminotransferase

(ALT) (Reitman and Frankel, 1957), alkaline phosphatase

(ALP) (Bergmeyer et al., 1983) and bilirubin content (kit

Bioxon-Mexico).

Assessment of Lipid Peroxidation

The extent of lipid peroxidation was estimated in liver

homogenates by measurement of malondialdehyde

(MDA) formation using the thiobarbituric acid method

(Okawa et al., 1979). Protein was determined according

to Bradford (1976) using bovine serum albumin as

standard.

Reduced and Oxidized GlutathioneDeterminations in Liver

GSH and GSSG were prepared on the day of the experi-

ment in 0.1 M sodium phosphate 0.005 M EDTA buffer

(pH 8.0) and kept on ice until used. The OPT solution

was prepared in reagent-grade absolute methanol just

prior to use.

Liver samples were homogenized on ice using a

polytron homogenizer. The solution used for homogeni-

zation consisted of 3.75 ml of the phosphate-EDTA

buffer and 1 ml of 25% H3PO4, which was used as a pro-

tein precipitant. The total homogenate was centrifuged

at 4 °C at 100 000 g for 30 min to obtain the supernatant

for the assay of GSH and GSSG.

Determination of GSH was performed according to

Hissin and Hilf (1976). To 0.01 ml of the 100 000 g

supernatant, 4.9 ml of the phosphate-EDTA buffer

pH 8.0 was added. The final assay mixture (2.0 ml) con-

tained 100 μl of the diluted tissue supernatant, 1.8 ml of

phosphate-EDTA buffer, and 100 μl of the OPT solution,

containing 100 μg of OPT. After through mixing and

incubation at room temperature for 15 min, the solu-

tion was transferred to a quartz cuvette. Fluorescence at

420 nm was determined with the activation at 350 nm.

For the GSSG assay, a 0.5 ml portion of the original

100 000 g supernatant was incubated at room temperature

with 200 μl of 0.04 M N-ethylmaleimide (NEM) for

30 min to interact with the GSH present in the tissue. To

this mixture, 4.3 ml of 0.1 N NaOH was added. A 100 μl

portion of this mixture was taken for the measurement of



DOI: 10.1002/jat

GSSG, as outlined above for the GSH assay, except

that 0.1 N NaOH was employed as a diluent rather than

phosphate-EDTA buffer.

Glycogen Determination

Small pieces of liver (0.5 g) were separated for glycogen

determination using the anthrone reagent according to

Seifter et al. (1950).

Histology

Samples of liver were taken from all the animals and

fixed with 10% formaldehyde in phosphate buffered

saline for 24 h. Those tissue pieces were washed with tap

water, dehydrated in alcohol and embedded in paraffin.

Sections of 6–7 μm were mounted on glass slides and

covered with silane. Masson’s trichromic stains were

performed on each slide.

Western Blot Assays

The TriPure reagent (Roche Diagnostics) was used to

isolate total protein from samples of liver tissue. Fresh

tissue was homogenized in 1 ml of TriPure reagent, then,

0.2 ml of chloroform was added to homogenates, and the

lower phase was treated with isopropanol to precipitate

total protein. Samples were centrifuged at 12 000 rpm

for 10 min at 4 °C, then, 3 washes were performed with

0.3 M guanidine hydrochloride in 95% ethanol. A final

wash was performed with 100% ethanol, samples were

centrifuged as previously described (Pérez-Severiano

et al., 2002), and the pellet resuspended in 1% SDS.

Volumes equivalent to 50 μg of proteins (determined by

the bis-cinconinic acid method) were transferred onto

12% polyacrylamide gel; separated proteins were trans-

ferred onto nitrocellulose paper (Amersham). Next, blots

were blocked with 5% skim milk and 0.05% Tween-20

for 30 min at room temperature and independently

incubated overnight at 4 °C with antibodies selective

against TGF-β (MAB 1032 from Chemicon Int. Inc.).

The following day, the membranes were washed and then

exposed to a secondary peroxidase-labeled antibody

(Zymed) diluted 1:4000 in the blocking solution for 1 h

at room temperature. Blots were washed and protein

developed using the ECL detection system (NEN Life

Sciences Products). Blots were stripped and incubated

with a monoclonal antibody directed against β-actin

(García-Tovar et al., 2001), which was used as a control

to normalize cytokine protein expression levels. The pro-

cedure to strip membranes was as follows. First, blots

were washed four times with phosphate-saline buffer

pH 7.4 (0.015 M, 0.9% NaCl), then immersed in stripping

buffer (2-mercaptoethanol 100 mM, sodium dodecyl

sulfate 2% and Tris-HCl 62.5 mM, pH 6.7) for 30 min at

60 °C with gentle shaking, membranes were then washed

five times with 0.05% Tween-20 in phosphate-saline

buffer.

Images were digitalized using the BioDoc-It System

(UVP) and then analysed densitometrically using the Lab

Works 4.0 Image Acquisition and Analysis software

(UVP), as previously described (Pérez-Severiano et al.,

2002).

Nuclear Protein Isolation and Shift Assays forNF-κκκκκB

The isolation of intact and stable nuclei consisted of the

iso-osmotic lysis procedure as reported by Dyer and

Hersog (1995). With this lysis procedure, the nuclear

envelope remains intact even during the further manipu-

lations of washing, freezing and ultracentrifugation and

provides nuclear extract for use in NF-κB assay.

NF-κB was estimated using a commercial kit (Non-

Radioactive NF-κB p50/p65 Transcription Factor Assay,

SGT510 Chemicon, Mexico City). This kit combines

the principle of the electrophoretic mobility shift assay

(EMSA) with the enzyme-linked immunosorbent assay

(ELISA).

Statistical Analysis

Data are expressed as mean value ± SEM. Comparisons

were carried out by analysis of variance followed by

Dunnett’s or Tukey’s test, as appropriate, using Sigma

Stat for Windows (2.0 Version). Differences were consid-

ered statistically significant when P < 0.05.

Results

The activity of ALT in serum is shown in Fig. 1. ALT is

a cytosolic enzyme of the hepatocyte and an increase of

enzyme activity reflects an increase in plasma membrane

permeability, which, in turn, is associated with cell death

(Rosen and Keeffe, 2000). Chronic CCl4 treatment in-

creased significantly ALT activity; resveratrol administra-

tion prevented significantly this effect. Resveratrol by

itself did not modify ALT activity.

Serum enzyme activity of ALP is an indicator

of cholestasis. Carbon tetrachloride induced ALP activity

by about 3-fold (Fig. 2). Resveratrol partially prevented

the increase of this enzyme; however, the statistical

analysis shows that this effect was not significant.

The concentration of total bilirubins in serum is

depicted in Fig. 3. Chronic CCl4 treatment increased

bilirubins significantly, while co-treatment with resveratrol

38 E. CHAVEZ ET AL.


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Figure 1. Alanine aminotransferase activity deter-mined in serum from control rats, CCl4 treated rats(CCl4), CCl4 plus resveratrol treated rats (CCl4 + RSV) andrats administered with resveratrol alone (RSV). Eachbar represents the mean value of experiments per-formed in duplicate assays ± SEM (n = 6). ‘a’ Meanssignificantly different from control, P < 0.05. ‘b’ Meanssignificantly different from CCl4 group, P < 0.05

preserved the normal values; the resveratrol control group

showed no alterations of serum bilirubins.

Glycogen (Fig. 4) is the main source of energy in the

organism; the liver content of this carbohydrate indicates

metabolism and functionality. Liver glycogen was re-

duced markedly by the administration of CCl4; resveratrol

prevented partially, but significantly, the liver content of

glycogen. Again, the resveratrol control group showed no

alteration of this parameter.

Lipid peroxidation is known to play a critical role in

CCl4 induced liver injury (Muriel, 1997) and malondial-

dehyde (MDA) is one of its end products. Thus, measure-

ment of MDA can be used to assess lipid peroxidation. A

very small and non significant increment of MDA was

Figure 2. Alkaline phosphatase activity determinedin serum from control rats, CCl4 treated rats (CCl4), CCl4plus resveratrol treated rats (CCl4 + RSV) and ratsadministered with resveratrol alone (RSV). Each barrepresents the mean value of experiments performedin duplicate assays ± SEM (n = 6). ‘a’ Means signifi-cantly different from control, P < 0.05

Figure 3. Total bilirubins determined in serum fromcontrol rats, CCl4 treated rats (CCl4), CCl4 plusresveratrol treated rats (CCl4 + RSV) and rats adminis-tered with resveratrol alone (RSV). Each bar representsthe mean value of experiments performed in duplicateassays ± SEM (n = 6). ‘a’ Means significantly differentfrom control, P < 0.05. ‘b’ Means significantly differentfrom CCl4 group, P < 0.05

Figure 4. Liver glycogen content determined in con-trol rats, CCl4 treated rats (CCl4), CCl4 plus resveratroltreated rats (CCl4 + RSV) and rats administered withresveratrol alone (RSV). Each bar represents the meanvalue of experiments performed in duplicate assays ±SEM (n = 6). ‘a’ Means significantly different from con-trol, P < 0.05. ‘b’ Means significantly different fromCCl4 group, P < 0.05

observed in the groups receiving CCl4. Resveratrol pro-

duced no effect in the groups receiving CCl4 or vehicle

(Fig. 5).

As an indicator of oxidative stress at the hydrophilic

level, glutathione was measured in the liver. Reduced

(GSH), oxidized (GSSG), GSH/GSSG ratio and total

(GSH + GSSG) liver glutathione are shown in Fig. 6.

Reduced glutathione showed no significant alterations in

any group; however, GSSG was increased significantly

by CCl4 intoxication; resveratrol practically produced

no effect on this parameter. As a consequence of the



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Figure 5. Liver lipid peroxidation determined asmalondialdehyde (MDA) content in control rats, CCl4treated rats (CCl4), CCl4 plus resveratrol treated rats(CCl4 + RSV) and rats administered with resveratrolalone (RSV). Each bar represents the mean value ofexperiments performed in duplicate assays ± SE (n = 6)

increase in GSSG, the GSH/GSSG ratio decreased

significantly in the groups that received CCl4; the total

glutathione showed no significant alterations in any group.

Fibrosis is the main characteristic of any chronic liver

damage and collagen is the main protein accumulating in

fibrotic tissue. Therefore, fibrosis was analysed from two

points of view: biochemically by measuring hydroxypro-

line (an amino acid exclusive of collagen) (Fig. 7),

and histologically by visualizing the blue color of the

collagen fibers in the trichromic stained liver sections

(Fig. 8). Hydroxyproline was increased by nearly 5-fold

by chronic CCl4 intoxication and, importantly, resveratrol

prevented completely the increase in collagen (Fig. 7).

The histopathological analysis revealed that chronic

CCl4 intoxication produced a marked increase in collagen

deposition around the portal triad, also nodules of

hepatocytes surrounded by variable size collagen bands

(blue stained) were frequently observed, the normal

architecture was lost and extended necrotic areas were

present. In agreement with the biochemical analysis, the

group treated simultaneously with resveratrol and CCl4

showed no accumulation of collagen and the parenchyma

Figure 6. Reduced (GSH) and oxidized (GSSG) glutathione, GSH/GSSG ratio and total glutathione (GSH + GSSG)determined in livers from control rats, CCl4 treated rats (CCl4), CCl4 plus resveratrol treated rats (CCl4 + RSV) andrats administered with resveratrol alone (RSV). Each bar represents the mean value of experiments performed induplicate assays ± SEM (n = 6). ‘a’ Means significantly different from control, P < 0.05. ‘b’ Means significantly dif-ferent from CCl4 group, P < 0.05

40 E. CHAVEZ ET AL.


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Figure 7. Collagen content determined in livers fromcontrol rats, CCl4 treated rats (CCl4), CCl4 plusresveratrol treated rats (CCl4 + RSV) and rats adminis-tered with resveratrol alone (RSV). Each bar representsthe mean value of experiments performed in duplicateassays ± SEM (n = 6). ‘a’ Means significantly differentfrom control, P < 0.05. ‘b’ Means significantly differentfrom CCl4 group, P < 0.05

was better preserved than in the group receiving CCl4

alone.

Because NF-κB upregulates the profibrogenic cytokine

TGF-β its activation was determined by measuring it in

the nucleus. Chronic CCl4 administration increased the

presence of this transcription factor in the nucleus, while

resveratrol attenuated this effect; unfortunately, these

effects did not reach statistical difference (Fig. 9).

TGF-β protein was investigated using western blot

analysis (Fig. 10). TGF-β was elevated significantly by

CCl4 intoxication but inhibited markedly by treatment

with resveratrol. In fact, the group receiving resveratrol

plus CCl4 showed lower values than the control, and

resveratrol by itself decreased TGF-β significantly.

Discussion

Resveratrol completely prevented the fibrosis induced

by chronic CCl4 intoxication. This was demonstrated

Figure 8. Trichromic of Masson staining of liver sections from: (A) control rats (vehicle); (B) CCl4-treated rats; (C)CCl4 treated with resveratrol and (D) rats administered with resveratrol alone



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Figure 9. Analysis of NF-κB DNA-binding activity insamples of liver tissue from control rats, CCl4-treatedrats (CCl4), CCl4 treated with resveratrol (CCl4 + RSV)and rats administered with resveratrol alone (RSV).See the conditions of animal treatment to determinethis factor. NF-κB was estimated using a commercial kit(Non-Radioactive NF-κB p50/p65 Transcription FactorAssay) that combines the principle of the electrophore-tic mobility shift assay (EMSA) with the enzyme-linkedimmunosorbent assay (ELISA). HeLa whole cell (TNF-αtreated) was used as a positive control. Results are ex-pressed as relative optical density (OD) values obtainedusing spectrophometric analysis. Each bar representsthe mean value of six rats ± SEM. ‘a’ Means signifi-cantly different from control, P < 0.05. ‘b’ Means sig-nificantly different from CCl4 group, P < 0.05

both biochemically and histologically. To investigate the

action mechanism of the compound its effect on NF-κB

activation and the levels of one of its products, the

profibrotic cytokine TGF-β, which is involved in HSC

Figure 10. Resveratrol blockade of TGF-β proteinin samples of liver tissue determined by westernblot analysis from CCl4 treated rats (CCl4), CCl4 plusresveratrol treated rats (CCl4 + RSV), and rats adminis-tered with resveratrol alone (RSV). β-actin was usedas an internal control. Signal intensities were deter-mined by densitometric analysis of treated blots andvalues calculated as the ratio of TNF-α /β-actin. Eachbar represents the mean value of six rats ± SEM

activation and production of extracellular matrix proteins

were measured. It was found that CCl4 effectively in-

creased both NF-κB activation and TGF-β production;

resveratrol prevented TGF-β elevation providing a satis-

factory antifibrotic mechanism. However, other actions

of resveratrol cannot be discarded. The antifibrotic effects

of resveratrol have been observed in other organs and

in vitro (Godichaud et al., 2000; Olson et al., 2005;

Sener et al., 2006b; Abergel et al., 2006; Godichaud

et al., 2006). However, this is the first report on the

antifibrogenic effect of resveratrol on liver cirrhosis.

Liver cirrhosis is irreversible and a worldwide leading

cause of death. Today, there is no effective pharmaco-

logical treatment for this disease and fibrosis is the main

feature of the pathology. Liver fibrosis is characterized

by an excessive deposition of extracellular matrix com-

ponents. It is well known that most extracellular matrix

proteins are deposited by liver myofibroblasts. Myofibro-

blasts are very rare in the normal liver. Most studies

suggest that myofibroblast derive from HSC (Friedman,

1993). These precursor cells undergo phenotypic modifi-

cations that are collectively referred to as activation. The

principal phenotypic modifications are morphological

changes, de novo expression of α smooth muscle actin,

increased cellular proliferation and extracellular matrix

components synthesis (Bataller and Brenner, 2005).

The main factor involved in stellate cell activation is

the cytokine TGF-β (Gressner et al., 2002), making

this cytokine a suitable target to interfere with the fibro-

tic process (Gressner and Weiskirchen, 2006). In this

sense, the diminution of TGF-β protein afforded by

resveratrol observed herein is an outstanding effect of this

phytoalexin.

The TGF-β superfamily consists of multiple family

members, including the highly homologous TGF-β (TGF-

β1 TGF-β2 and TGF-β3) isoforms. TGF-β1 has shown

the most prominent fibrogenic properties (Border and

Ruoslahti, 1992; Massague et al., 1992; Muriel 2007b)

and is the isoform measured in this work. TGF-β1 seems

to lead to fibrogenesis through autocrine and paracrine

effects on HSC (Hellerbrand et al., 1999; Gressner and

Weiskirchen, 2006), the cells responsible for fibrogenesis.

Given the prominent role of TGF-β in hepatic fibrogene-

sis, a number of approaches to abrogate the effect of this

cytokine have been advanced. For example, neutralizing

antibodies (Jester et al., 1997; Yamamoto et al., 1999),

dominant negative TGF-β receptors (Qi et al., 1999) and

chimeric soluble TGF-β receptor (Yata et al., 2002) have

been shown to reduce fibrosis in a variety of models. In

this work a different approach was utilized to interfere

with TGF-β signaling; resveratrol, a non toxic and natu-

rally occurring compound, decreased TGF-β in animals

treated with CCl4 or vehicle accompanied by a complete

abrogation of CCl4-fibrosis. Resveratrol is a safer, easier

and cheaper way to interfere with the fibrogenic process,

at least in this model of liver cirrhosis.

42 E. CHAVEZ ET AL.


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The NF-κB proteins are latent gene regulatory proteins

that lie at the heart of most inflammatory responses.

There are five NF-κB proteins in mammals (RelA, RelB,

c-Rel, NF-κB1 and NF-κB2), and they form a variety of

homodimers and heterodimers, each of which activates its

own characteristic set of genes (Karin et al., 2004). RelA

is a heterodimer of P50 and P65 and is responsible for

the production of many cytokines including TGF-β(Gressner and Weiskirchen, 2006). It is widely accepted

that resveratrol inhibits NF-κB activity (Tsai et al., 1999;

Pendurthi et al., 1999; Wadsworth and Koop, 1999).

Since NF-κB upregulates TGF-β it is possible that the

effects observed on TGF-β were mediated by this factor

and therefore account for the antifibrogenic properties of

resveratrol. Glycogen is the main source of energy in

liver and is also utilized to maintain blood glucose levels.

Chronic administration of CCl4 decreased importantly and

significantly the liver glycogen content. It is worth noting

that resveratrol preserved almost completely this para-

meter. Probably, resveratrol preserved glycogen by its

antifibrotic mechanism because fibrosis disrupts the nor-

mal architecture and blood flow into the liver therefore

not allowing nutrients to be absorbed by the hepatocytes.

In addition, necrosis was elevated by CCl4 and prevented

partially by resveratrol (as glycogen content), obviously

necrotic cells do not store glycogen.

Resveratrol has been reported to prevent oxidative

stress and lipid peroxidation processes (Frankel and

Waterhouse, 1993; Chanvitayapongs et al., 1997;

Ignatowicz and Baer-Dubowska, 2001). Therefore, lipid

peroxidation and glutathione were studied in our groups.

Unexpectedly, lipid peroxidation was not increased sig-

nificantly by CCl4, despite it being well known that this

agent is converted to trichloromethyl radical (CCl3•) that

reacts with the fatty acids of membranes producing lipid

peroxidation (Muriel, 1997; Muriel, 2007b). The most

likely explanation is that the dose used in chronic liver

damage is relatively small (0.4 g kg−1) compared with

the dose administered to produce acute liver damage

(4 g kg−1) (Muriel, 2007a); more importantly, after acute

liver damage, lipid peroxidation is increased after a few

hours of intoxication, while in this work it was measured

72 h after the last dose. Therefore, it is possible that

MDA was produced in small amounts and that it was

depurated within 72 h. These circumstances did not allow

the effect of resveratrol on lipid peroxidation to be tested.

Glutathione is a useful parameter for studying oxida-

tive stress in the cytosol of hepatocytes. The GSH/GSSG

ratio decreased by CCl4 but resveratrol was not able to

prevent this effect. These results are in apparent contrast

to those obtained by Sener et al. (2006c) who found that

resveratrol preserved GSH in ischemia/reperfusion-

induced oxidative injury in rats. The main difference is

that Sener et al. (2006c) utilized 30 mg of resveratrol

and this work used only 10 mg kg−1. In addition, no sig-

nificant changes in GSH were observed, and the diminu-

tion in the ratio GSH/GSSG was due to an increased

concentration of GSSG, which was slightly prevented by

resveratrol.

Leiro et al. (2004) investigated the effects of the cis

isomer of resveratrol on the responses of inflamma-

tory murine peritoneal macrophages. The results that

they obtained were similar to those generated by trans

resveratrol (the isoform used here) in different sys-

tems. Cis resveratrol at 100 μM decreased inducible nitric

oxide synthase and COX-2 in lipopolysaccharide and

interferon-γ treated macrophages. The Leiro et al. (2004)

results show that cis resveratrol significantly attenuates

several components of the inflammatory response to

proinflammatory stimuli. Furthermore, Leiro et al. (2005)

found that cis resveratrol has a significant modulatory

effect on the NF-κB signaling pathway. These results

suggest that the isomeric form of resveratrol may be not

relevant for their biological activity, although compara-

tive studies are necessary between both isomers.

Quercetin, a flavonoid has shown similar results to

those observed by resveratrol in the same model of liver

damage (Pavanato et al., 2003); however, the mechanism

of action seems to be different. Quercetin acts by increas-

ing total peroxyl radical-trapping antioxidant capacity of

liver, while resveratrol failed to prevent oxidative stress

but decreased the profibrogenic cytokine TGF-β. Anyway

both substances afforded beneficial effects on fibrosis and

other markers of liver damage.

In general we were unable to observe the antioxidant

properties of resveratrol in this model and at the dose

employed. However, resveratrol completely prevented

fibrosis and totally abolished the increase in TGF-β and

NF-κB (although this latter effect was not significant).

Therefore, in this model of fibrosis and at the dose

employed, resveratrol is an excellent antifibrogenic drug.

Furthermore, it seems that the protective effects of the

compound is more associated with its ability to reduce

the profibrogenic cytokine TGF-β, maybe by inactivating

NF-κB, than to its antioxidant properties.

Acknowledgements—The authors express their gratitude to Mr BenjamínSalinas Hernández, Mr Ramón Hernández and MVZ Ricardo Gaxiolafor their excellent technical assistance. IF Enrique Chavez was a fellowof Conacyt. This work was supported in part by Conacyt grant 42721(JS).

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