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Chemico-Biological Interactions 171 (2008) 283–293

Available online at www.sciencedirect.com

Hepatoprotective effects of Solanum nigrum Linn extractagainst CCl4-iduced oxidative damage in rats

Hui-Mei Lin a, Hsien-Chun Tseng a,b, Chau-Jong Wang a,Jin-Jin Lin c, Chia-Wen Lo a, Fen-Pi Chou a,∗

a Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University,No. 110, Section 1, Chien-Kuo N. Road, Taichung 402, Taiwan

b Department of Radiation Oncology, Chung Shan Medical University Hospital,Taichung, Taiwan

c Academia Sinica Genomics Research Center, Taipei, Taiwan

Received 2 May 2007; received in revised form 7 August 2007; accepted 8 August 2007Available online 19 August 2007

bstract

Solanum nigrum L. (SN) is an herbal plant that has been used as hepatoprotective and anti-inflammation agent in Chinese medicine.n this study, the protective effects of water extract of SN (SNE) against liver damage were evaluated in carbon tetrachloride (CCl4)-nduced chronic hepatotoxicity in rats. Sprague-Dawley (SD) rats were orally fed with SNE (0.2, 0.5, and 1.0 g kg−1 bw) along withdministration of CCl4 (20% CCl4/corn oil; 0.5 mL kg−1 bw) for 6 weeks. The results showed that the treatment of SNE significantlyowered the CCl4-induced serum levels of hepatic enzyme markers (GOT, GPT, ALP, and total bilirubin), superoxide and hydroxyladical. The hepatic content of GSH, and activities and expressions of SOD, GST Al, and GST Mu that were reduced by CCl4 wererought back to control levels by the supplement of SNE. Liver histopathology showed that SNE reduced the incidence of liveresions including hepatic cells cloudy swelling, lymphocytes infiltration, hepatic necrosis, and fibrous connective tissue proliferation

nduced by CCl4 in rats. Therefore, the results of this study suggest that SNE could protect liver against the CCl4-induced oxidativeamage in rats, and this hepatoprotective effect might be contributed to its modulation on detoxification enzymes and its antioxidantnd free radical scavenger effects.

2007 Elsevier Ireland Ltd. All rights reserved.

s; Hepa

eywords: Solanum nigrum Linn; Carbon tetrachloride; Liver fibrosi

. Introduction

Numerous medicinal plants and their formulation aresed for liver disorders in ethnomedical practice as well

Abbreviations: SN, Solanum nigrum Linn; CCl4, carbon tetrachlo-ide; SOD, superoxide dismutase; GST, glutathione-S-transferase.∗ Corresponding author. Tel.: +886 4 24730022x11676;

ax: +886 4 23248195.E-mail address: fpchou@csmu.edu.tw (F.-P. Chou).

009-2797/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reservdoi:10.1016/j.cbi.2007.08.008

toprotection

as traditional medicine in Chinese. Solanum nigrum L.(SN) is a common herb that grows wildly and abundantlyin open fields. It has been used to treat inflammation,edema, mastitis and hepatic cancer for a long time inoriental medicine. A freshly prepared extract of the herbis effective in treating cirrhosis of liver, and juice of the

leaves alleviates pain in inflammation of the kidney andbladder, and internally for cardialgia.

Previous investigations showed that extracts of SNsuppressed the oxidant mediated DNA–sugar damage,

ed.

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284 H.-M. Lin et al. / Chemico-Biolo

and the plant exerted cytoprotection against gentamicin-induced toxicity on Vero cells and anti-neoplasticactivity against Sarcoma 180 in mice. More recentstudies revealed an inhibitory effect of extracts ofSN on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced tumor promotion in HCT-116 cells, and aremarkable hepatoprotective effect of the ethanol extractof dried fruits of SN against CCl4-induced liver dam-age [1]. These studies suggest that SN possesses abeneficial activity as an antioxidant, and antitumor-promoting, and hepatoprotective agent, although themechanism for the activity remains to be elucida-ted.

CCl4 is a classical hepatotoxicant that causes rapidliver damage progressing from steatosis to centrilobu-lar necrosis. Long-term administration of CCl4 causeschronic liver injury, and is a widely accepted modelto produce hepatic fibrosis [2,3]. CCl4 is known toinduce reactive oxygen formation, deplete GSH ofphase II enzyme, and reduce antioxidant enzyme andantioxidant substrates to induce oxidative stress that isan important factor in acute and chronic liver injury.The liver injury induced by CCl4 is resulted fromfree radicals and lipid peroxidation that cause hep-atic cell damage. CCl4 requires bioactivation by phaseI cytochrome P450 system in liver to form reactivemetabolic trichloromethyl radical (CCl•3) and proxytrichloromethyl radical (•OOCCl3). These free radicalscan bind with polyunsaturated fatty acid (PUFA) to pro-duce alkoxy (R•) and peroxy radicals (ROO•), that, inturn, generate lipid peroxide, cause damage in cell mem-brane, change enzyme activity and finally induce hepaticinjury or necrosis [4–6].

Recently, we have demonstrated that the water extractof SN (SNE) contains several antioxidants, such as gallicacid, PCA, catechin, caffeic acid, epicatechin, rutin andnarigenin, and possesses strong antioxidative activity invitro [1]. In this study, we investigated the antioxida-tive activity and potential protective effects of SNE inthe CCl4-induced hepatic damage in rats. The protectionactivity of SNE was compared with silymarin, which hasbeen used for over 20 years in clinical practice for thetreatment of toxic liver disease [7]. It has been describedto be an antioxidant and exhibits anti-inflammatory, anti-carcinogenic, growth-modulatory and hepatoprotectioneffects [8]. The effect of SNE on the expression of phaseII detoxifying and antioxidative enzymes was also exam-ined. The results of this study showed that SNE could be

used as a protective agent against the chemical-inducedoxidative damage in rats, and the hepatoprotective effectmight be correlated with its antioxidative and free radicalscavenger effect.

teractions 171 (2008) 283–293

2. Materials and methods

2.1. Preparation of water extracts of SN (SNE)

The whole plant of SN was collected from the moun-tain in Miaoli, Taiwan. The plants were washed, cutinto small pieces, shade dried for 3 days, and then driedovernight in an oven. The dried SN (800 g) was mixedwith water (5000 mL) for 30 min, and subjected to con-tinuous hot extraction (100 ◦C, 40 min). The resultingwater extract was filtered and subsequently concentratedwith a water bath (90 ◦C) until it became creamy, andwas then dried in an oven (70 ◦C) that finally gave 185 g(23.125% of initial amount) of powder (SNE, waterextract of SN). The concentration used in the experimentwas based on the dry weight of the extract.

2.2. Animals and treatment

SD rats weighing 150–180 g were housed in conven-tional cages with free access to water and rodent chowat 20–22 ◦C with a 12-h light–dark cycle. All proceduresinvolving laboratory animal use were in accordance withthe guidelines of the Instituted Animal Care and UseCommittee of Chung Shan Medical University (IACUC,CSMU) for the care and the use of laboratory animals.Rats were treated orally with CCl4 (20% CCl4/corn oil;0.5 mL kg−1 body weight twice a week; Monday andThursday) for 6 weeks to cause chronic reversible cir-rhosis as described previously with some modification[9]. At the same time, the rats were fed with a dietcontaining SNE (0.2, 0.5 and 1.0 g kg−1 body weight),or given silymarin orally (0.2 g kg−1 body weight; fourtimes a week; Tuesday, Wednesday, Friday and Satur-day) [10,11]. The control rats were treated with corn oiland fed with a normal diet. Blood samples (0.2 ml) withheparin (10 U mL−1) were collected from the tail veinbefore the first CCl4 treatment and at the end of the thirdand sixth weeks. At the end of the experiment, blood andlivers were immediately obtained after the animals weresacrificed. Liver tissue samples were taken from the leftliver lobe, and cut into two pieces. One piece was fixed informalin for pathological examination. The other piecewas utilized for the following biological analyses. Liverhomogenates (10%, w/v) were obtained in 50 mM phos-phate buffer (pH 7.0) and stored at −80 ◦C for analysiswithin 2 weeks.

2.3. Histological examinations

A portion of the liver was fixed in 10% formalin,processed by routine histology procedures, embedded in

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araffin, and cut into 5 �m sections. The samples weretained with hematoxylin and eosin for histopathologi-al examination and stained with Masson’s trichrome forssessment of fibrosis [12]. Liver steatosis was gradedn a three-point scale: 1+, hepatocytes in the area of onehird of the lobules showed fatty accumulation, 2+ forwo thirds and 3+, for all hepatocytes. The criteria usedor scoring fibrosis severity were as follows: 0, normal;+, fibrosis present (collagen fiber present that extendsrom portal triad or central vein to peripheral region); 2+,ild fibrosis (the collagen fiber present with extensionithout compartment formation); 3+, moderate fibrosis

the collagen fiber present with some pseudo lobe forma-ion); and 4+, severe fibrosis (the collagen fiber presentith thickening of the partial compartments and frequentseudo lobe formation).

.4. Determination of biochemical markers ofepatic injury

Blood were placed at room temperature for 1 h,nd then centrifuged at 1000 × g for 10 min to obtainerum. Serum biochemical parameters (GOT, GPT, ALP,nd total-bilirubin) were assayed by the Boehingerannheim reagents (International Federation of Clinicalhemistry (IFCC) Scientific Committee recommended)

13,14].

.5. Determination of glutathione (GSH) content

The procedure used for determining GSH with-phtalaldehyde (OPA) was performed as previousescribed [15]. Liver GSH levels were determined using–10 mL of deproteinized homogenate. OPA-deriveduorescence was measured at 365 nm excitation and30 nm emissions in a F4500 fluorescence spectropho-ometer (Hitachi, Japan).

.6. Assay of superoxide dismutase (SOD) activity

The SOD activity was measured according to theethod of Beauchamp and Fridovich [16]. An adequate

mount of the liver supernatant was mixed with the reac-ion mixtures, which contained 0.1 mM EDTA, 25 mMBT, 0.1 mM xanthine, 50 mM sodium carbonate buffer

pH 10.2), and the final volume of the reaction mixtureas brought up to 3 mL with distilled water. The reac-

ion was initiated by the addition of 2 mU mL−1 xanthine

xidase and maintained under two 40 W lamps at 25 ◦C.fter 15 min, the inhibition rate of NBT reduction was

pectrophotometrically determined at 560 nm. One unitf SOD is defined as the amount of enzyme required

teractions 171 (2008) 283–293 285

to reduce NBT by 50%. The specific activity of SODwas expressed as a unit mg−1 protein in each superna-tant.

2.7. Assay of glutathion-S-transferase (GST)activity

Total GST activity and the activities for specificGST isoform were measured according to the methodof Habig et al. [17] using 1-chloro-2,4-dinitrobenzene(CDNB; Sigma Chemical, St. Louis, MO) as sub-strate for total activity, 4-chloro-7-nitrobenzofurazan(NBD-Cl) for GST Al, ethacrynic acid (EA) for GSTPi, and 1,2-dichloro-4-nitrobenzene (DCNB) for GSTMu. Enzyme activity was expressed as nanomoles ofsubstrate–GSH conjugate produced per minute per mil-ligram of cytosolic protein. The change in absorbance oftotal GST, GST Al, GST Pi and GST Mu was obtained at340, 419, 270, and 345 nm, respectively, and the enzymeactivity was calculated as nmol of CDNB, NBD-Cl, EA,and DCNB conjugate formed min−1 mg−1 protein usinga molar extinction coefficient of 9.6 × 103 M−1 cm−1,respectively. Protein concentration was determinedwith a standard commercial kit (Bio-Rad Lab. Ltd.,Watford, England) with bovine serum albumin as astandard.

2.8. Immunohistochemistry

Liver sections were deparaffinized in xylene, rehy-drated in graded alcohol series, and blocked for theendogenous peroxidase by incubating in a solutionof 3% hydrogen peroxide (H2O2) in methanol for10 min. Tissue sections were washed with PBS, andthen immunostained with primary antibodies for GSTAl, GST Pi and GST Mu (1:300, Oxford Biomed-ical Research, USA). The antigen–antibody complexwas visualized by a labeled streptavidin–biotin methodusing a Histostatin-PLUS Bulk Kit (Zymed Laborato-ries Inc., USA) followed by diaminobenzidine (DAB)as a chromogen. After washing, sections were counter-stained with Meyer’s hematoxylin and washed with tapwater.

2.9. Determination of superoxide and hydroxyl freeradical in blood samples

For the measurement of superoxide and hydroxyl rad-

ical in whole-blood, blood samples from the carotidartery were obtained immediately after sacrifice usingheparin as anti-coagulator, wrapped with aluminum foiland kept on ice until Chemiluminescence measure-

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greater than grade 3+, and seven out of eight had liver

286 H.-M. Lin et al. / Chemico-Biolo

ment within 2 h [18]. Right before Chemiluminescencemeasurement, 0.1 mL of phosphate-buffered saline (pH7.4) was added to 0.2 mL of blood sample. Chemilu-minescence was measured in the dark chamber of aChemiluminescence Analyzing System. After a 100 sbackground level determination, 1.0 mL of and 0.1 mMlucigenin and lucigenin in phosphate-buffered saline(pH 7.4) were injected into the sample, respectively.The chemiluminescence was monitored continuouslyfor additional 600 s. The total amount of chemilumi-nescence was calculated by integrating the area underthe curve and subtracting the background level from it.The assay was performed in duplicate for each sampleand expressed as chemiluminescence counts/10 s. Themean ± S.E. of chemiluminescence level of each samplewas calculated.

2.10. Statistical analysis

The experimental results were expressed asmean ± S.D. Statistical significance was analyzed byone-way analysis of variance (ANOVA). Duncan’s mul-tiple tests were used to determine the difference amonggroups. Student’s t-test was used in the two-groupcomparison. A p-value <0.05 were considered statisti-cally significant (Sigma-Stat 2.0, Jandel Scientific, SanRafael, CA).

3. Results

3.1. Effect of SNE on the body and organ weight of

rats

In the present design of animal experiment asdescribed in Section 2, the body weights, and rela-

Table 1Body weight, relative liver and spleen weight of CC14-treated rats with or wi

Group Body weight

Control 314 ± 22CC14 (20%) 221 ± 18##

Silymarin0.2 g kg−1 of bw + CC14 270 ± 17*

SNE0.2 g kg−1 of bw + CCU 259 ± 19*

0.5 g kg−1 of bw + CCl4 272 ± 24*

1.0 g kg−1 of bw + CCl4 282 ± 26*

*Significantly different from the group treated with CC14, #p < 0.05, **p < 0.0a Valued are mean ± S.D., n = 8.

teractions 171 (2008) 283–293

tive liver and spleen weights of the CCl4-treated ratswere significantly higher than those of control group(*P < 0.05, **P < 0.01, Table 1). The supplement of sily-marin or SNE (0.5 and 1 g kg−1 of body weight) for 6weeks reversed significantly the CCl4-induced body andorgan weights.

3.2. Effect of SNE on CCl4-Induced livermorphological change and fibrosis in rats

The general liver morphological changes and fibro-sis induced by CCl4 administration were evidenced byboth qualitative and quantitative histopathological exam-ination. As compared to normal rat liver, the tissuesection in Fig. 1A, b showed that the CCl4-inducedchronic liver injury revealed concave on the liver sur-face and lymphocytes infiltration in the central vein.The hepatic cells were found to be cloudy swelling,and necrosis, cytoplasmic vacuolization and fatty degen-eration were observed in central zone and mid-zone.Masson trichrome stain clearly demonstrated that theCCl4-intoxicated liver become fibroic with fiber exten-sion and collagen accumulation (Fig. 1B, b). Thesepathological changes (such as fibrosis and inflammation)were ameliorated by the co-treatment of silymarin andSNE that, especially, showed a dose-dependent effect(Fig. 1A and B). The severity of the liver morphologi-cal changes and fibrosis induced by CCl4 treatment werescored and summarized in Table 2. As shown, six out ofeight CCl4-exposed rats had liver morphological change

fibrosis and necrosis greater than grade 2. SNE exposuregreatly improved liver morphological changes, fibrosisand necrosis. The average severity scores of SNE-treatedrats were markedly reduced in a dose-dependent manner.

thout the gavage of extract of Solanum nigrum L. (SNE) for 6 weeks

Relative organ weighta (g 100 g−1 of bw)

Liver Spleen

3.5 ± 0.2 0.20 ± 0.015.1 ± 0.6## 0.50 ± 0.04##

3.8 ± 0.7* 0.38 ± 0.05*

4.2 ± 0.3 0.39 ± 0.033.9 ± 0.8* 0.33 ± 0.03*

3.8 ± 0.4* 0.27 ± 0.01**

1. #Significantly different from control group, #p < 0.05, ##p < 0.01.

H.-M. Lin et al. / Chemico-Biological Interactions 171 (2008) 283–293 287

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ig. 1. Effects of SNE on CCl4-induced liver damage in SD rats: (a) Coil), (c) animals treated with CCl4 and 0.2 g silymarin kg−1 bw, (d) anind 0.5 g SNE kg−1 bw, and (f) animals treated with CCl4 and 1.0 g S00×.

.3. Effect of SNE on serum biochemicalarameters of liver function

The serum levels of hepatic enzymes, such as GOT,PT, ALP, and total bilirubin, used as biochemicalarkers for evaluation of early hepatic injury were sig-

ificantly elevated in the CCL -treated animals (Fig. 2).

4NE administration at the dose of 0.2, 0.5 or 1.0 g kg−1

ody weight during CCl4 treatment significantly low-red, in a dose-dependent manner, the serum GOT, GPT,LP, and total bilirubin activities as compared with those

oup (n = 8), (b) animals treated with CCl4 (0.5 ml of 20% CCl4 in cornated with CCl4 and 0.2 g SNE kg−1 bw, (e) animals treated with CCl41 bw. (A) Hematoxylin/eosin stain, (B) Masson stain; magnification

of CCl4 treatment group. The supplement of silymarin(0.2 g kg−1 body weight) showed a similar effect as thatof high dose SNE.

3.4. Effect of SNE on GSH content and SOD level

GSH constitutes the first line of defense against free

radicals. The toxicity of CCl4 significantly decreasedthe hepatic GSH levels (�mol mg−1 protein) from3.3 ± 0.46 in the control group to 2.3 ± 0.37 (Fig. 3).An addition of silymarin and SNE at doses of 0.5 and

288 H.-M. Lin et al. / Chemico-Biological InTa

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teractions 171 (2008) 283–293

1.0 g kg−1 body weight in diet reversed the GSH level.Especially both high dose of SNE (1.0 g kg−1 bodyweight) and silymarin restored almost completely theGSH content to the normal level. The hepatic level ofSOD was also significantly reduced in the CCl4 group.Silymarin effectively, however only partially, lessenedthe influence of CCl4. A significant dose dependent andpartial reversal of the SOD level was observed by SNEat 0.5 and 1.0 g kg−1 body weight.

3.5. Effect of SNE on hepatic GSTs activity

GSTs are a family of dimeric enzymes responsible forthe metabolism of a broad range of xenobiotics and car-cinogens. As shown in Fig. 3, CCl4 caused a decrease inthe activity of total GST, GST Al, and GST Mu to 48%,58% and 57% of control level, along with a two-foldincrease in GST Pi activity in liver tissue. SNE treat-ment at doses of 0.5 and 1.0 g kg−1 body weight for 6weeks diminished the CCl4-caused reduction in hepatictotal GST, GST Al and GST Mu enzyme activities andinduction in GST Pi activity. Silymarin showed a similareffect as SNE.

3.6. Immunohistochemical examination of hepaticGST subunits

In order to confirm the effect of SNE on the hep-atic GST system under CCl4 toxicity, the protein levelsof GST subunits were examined by immunohistochem-istry. The results demonstrated that the expression ofGST Al and GST Mu was repressed in the hepatocytesof CCl4-induced liver chronic damage and, on the otherhand, GST Pi was induced (Fig. 4). The supplementof SNE at high dose in diet reversed the expressionsof GST subunits comparable to the control levels, thatwere increasing the levels of GST Al and GST Mu andreducing GST Pi, even though the animals were toxi-cated by CCl4. These effects were also demonstrated bythe co-treatment of silymarin.

3.7. Effect of SNE on serum free radicals

The serum levels of superoxide and hydroxyl radicalwere increased to 13-fold and 8-fold of those of controlin the CCl4-treated animals (Fig. 5). This inductions in

free radicals caused by CCl4 were diminished by oraladdition of SNE dose-dependently. Silymarin was alsoable to reduce the CCl4-induced free radicals with aneffect comparable to high dose of SNE.

H.-M. Lin et al. / Chemico-Biological Interactions 171 (2008) 283–293 289

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ig. 2. Effects of SNE on serum GOT, GPT, ALP and total bilirubin in Cs compared with the control group. *P < 0.05, **P < 0.01, and ***P <

. Discussion

Hepatic fibrosis represents the wound healingesponse of the liver to repeated liver injuries, and isssociated with increased inflammatory cell infiltrationnd may involve the interplay of different inflammatoryediators, which is a common stage in most chronic

iver disease. It is well know that constant fibrosis canead to the development of hepatocellular carcinoma19]. If treated properly in this stage, hepatic fibrosis cane reversed and its progression to irreversible cirrhosishat often leads to lethal complications and high mortal-ty may be prevented. Interrupting or reversing hepaticbrosis may well be a new approach to avoiding its pro-ression to hepatocellular carcinoma [20]. However, therevention for liver fibrosis is not yet well established.

In this study, we demonstrated that SN, a traditionalhinese medicine, had preventive effects on hepaticbrosis induced by CCl4 exposure in rats. Our studyossesses some unique features as compared to thether published literature including, the use of the waterxtract of whole plant, long-term (6 weeks) animal expo-ures, induction of liver fibrosis, and identification of

he mechanisms of action of SN on detoxification andntioxidative ability, especially the effect on the activitynd expression of GST subunits. CCl4 has been widelysed for inducing experimental hepatic damage due to

ated rats. The data were presented as mean ± S.D., n = 8. ###P < 0.005as compared with 20% CCl4-treated group.

free radical formation during its metabolism by hepaticmicrosome, which, in turn, to causes lipid peroxida-tion of the cellular membrane leading to the necrosisof hepatocyte. Rats treated with CCl4 developed signif-icant hepatic damage and oxidative stress as evidencedby substantial increases in the serum activities of GOP,GPT, ALP, and total bilirubin that are indicators of cel-lular leakage and loss of functional integrity of cellmembrane in liver [21,22]. The reduction in the lev-els of these parameters toward the respective normalvalues by SNE at two doses (0.2 and 0.5 g kg−1 bodyweight) is an indication of the stabilization of plasmamembranes as well as repair of hepatic tissue damagecaused by CCl4. This effect is in agreement with thecommonly accepted view that serum levels of transami-nases would return to normal after the healing of hepaticparenchyma and regeneration of hepatocytes. This indi-cates that the anti-lipid peroxidation and/or adaptivenature of the systems brought about by SNE acted againstthe damaging effects of free radicals produced by CCl4.During hepatic injury, superoxide and hydroxyl radicalgenerate at the site of damage, and SOD is exhaustedas a result of oxidative stress caused by CCl that fur-

4ther leads to accumulation of free radicals. The SODactivity was brought to increase and free radicals weredecreased by the co-treatment of SNE in the CCl4-treatedrats. These results evidently demonstrate the antioxidant

290 H.-M. Lin et al. / Chemico-Biological Interactions 171 (2008) 283–293

, GST Ared wi

Fig. 3. Effects of SNE on hepatic GSH content, and SOD, total GSTpresented as mean ± S.D., n = 8. #P < 0.05, and ##P < 0.01, as compagroup.

property of SNE against superoxide and hydroxyl freeradical.

To avoid redox imbalance and oxidative damage, aer-obic organisms possess an efficient biochemical defensesystem including enzymes, such as SOD and GSTs, andGSH, which, however, could not provide complete pro-tection against the attack of ROS in conditions of severeoxidative stress [23,24]. Therefore, scientists have beentrying to find bioactive substances possessing cytopro-tective ability against cellular oxidative damage, as wellas enhancing ability on antioxidant enzymes activities[25,26]. GSTs are a family of dimeric enzymes respon-

sible for the metabolism of a broad range of xenobioticsand carcinogens [27]. Numerous studies showed thatthe regulation of different GST isoforms was associatedwith various pathological processes. For example, the

l, GST Pi, and GST Mu activity in CCl4-treated rats. The data wereth the control group. *P < 0.05, as compared with 20% CCl4-treated

up-regulation of GST Pi, which is absent from normal ratand human hepatocytes, is associated with carcinogene-sis [28,29] and drug resistance of tumor tissue [30,31].The absent expression of GST Mu and GST Theta, on theother hand, is related to cancer susceptibility in humanstudies [32,33]. Chemicals like chloroform and CCl4alter the hepatic GST activity [34]. In this study, CCl4treatment decreased the activity and expression of GSTAl, and GST Mu and increased those of GST Pi, indi-cating that the hepatic GST system had been shifted to adirection that may lead to tumorgenesis during chronicliver damage. The GSH content and the expression of

GST subunits were brought back to control levels by theuse of SNE. Whether these effects of SNE are attributedto its direct action on GST system or its detoxificationof CCl4 remains to be elucidated. Anyhow, the ability of

H.-M. Lin et al. / Chemico-Biological Interactions 171 (2008) 283–293 291

Fig. 4. Effects of SNE on GSTs expression in rats with hepatic fibro-sis. Immunohistochemistry (400×) of GST Al (A), Pi (B), and Mu(C) in rat liver of (a) control group, (b) CCl4 group, (c) CCl4 + 0.2 gsilymarin kg−1 bw group, (d) CCl4 + 0.2 g SNE kg−1 bw group, (e)Cg

Sitac

afiG

Fig. 5. Effects of SNE on superoxide (A) and hydroxyl radical (B) in

Cl4 + 0.5 g SNE kg−1 bw group, and (f) CCl4 + 1.0 g SNE kg−1 bwroup.

NE to sustain hepatic GSTs expression at normal phys-ological condition under the toxicity of CCl4 suggestshat it is effective in protecting liver from chemical dam-ge and preventing further progression of pathologicalondition.

In conclusion, our results demonstrate that SNE wasble to reverse the pathological parameters, such asbrotic histological feature and serum levels of GOT,PT, ALP, and total bilirubin, of chronic liver damage

blood of CCl4-treated rats. The data were presented as mean ± S.D.,n = 8. #P < 0.05 and ##P < 0.01, as compared with the control group.*P < 0.05 and **P < 0.01, as compared with 20% CCl4-treated group.

induced by CCl4. This protecting ability of SNE wasdue to, at least partially, its modulation on detoxifica-tion enzymes (GSTs) and antioxidative enzyme (SOD)that, in turn, repressed the production of free radicalsand the subsequent liver damage. Furthermore, the highcontent of polyphenols, alkaloids and saponins in SNE[11] contributes free radical scavenging and antioxida-tion activities. Although it seems difficult to entirelyrecuperate the chronic hepatic damage-induced by CCl4through the supplement of phytochemicals, SNE indeedretarded the liver injury by blocking the oxidative stress.Therefore, dietary SNE may be useful as a hepatoprotec-tive agent against chemical-induced chronic liver fibrosisin vivo.

Acknowledgement

This work was supported by the grants from NationalScience Council of Taiwan (NSC 95-2320-B-040-022).

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