Toxicology 232 (2007) 50–56

Protective effects of echinacoside on carbontetrachloride-induced hepatotoxicity in rats

Yu Wu a, Lin Li a, Tao Wen b, Yue-Qi Li a,∗a Institute of Liver Diseases, The 302th Hospital of PLA, Beijing 100039, PR China

b Research Center of Digestive Diseases, The Third Hospital of Peking University, Beijing 100083, PR China

Received 7 November 2006; received in revised form 3 December 2006; accepted 7 December 2006Available online 23 December 2006

Abstract

The aim of this study was to investigate the possible protective effects of echinacoside, one of the phenylethanoids isolated fromthe stems of Cistanches salsa, a Chinese herbal medicine, on the free radical damage of liver caused by carbon tetrachloride inrats. Treatment of rats with carbon tetrachloride produced severe liver injury, as demonstrated by dramatic elevation of serum ALT,AST levels and typical histopathological changes including hepatocyte necrosis or apoptosis, haemorrhage, fatty degeneration, etc.In addition, carbon tetrachloride administration caused oxidative stress in rats, as evidenced by increased reactive oxygen species(ROS) production and MDA concentrations in the liver of rats, along with a remarkable reduction in hepatic SOD activity andGSH content. However, simultaneous treatment with echinacoside (50 mg/kg, intraperitoneally) significantly attenuated carbontetrachloride-induced hepatotoxicity. The results showed that serum ALT, AST levels and hepatic MDA content as well as ROSproduction were reduced dramatically, and hepatic SOD activity and GSH content were restored remarkably by echinacoside

administration, as compared to the carbon tetrachloride-treated rats. Moreover, the histopathological damage of liver and the numberof apoptotic hepatocytes were also significantly ameliorated by echinacoside treatment. It is therefore suggested that echinacosidecan provide a definite protective effect against acute hepatic injury caused by CCl4 in rats, which may mainly be associated with itsantioxidative effect.© 2006 Elsevier Ireland Ltd. All rights reserved.

ROS; E

Keywords: Carbon tetrachloride; Oxidative stress; Acute liver injury;

1. Introduction

Drug/chemicals-induced liver injury, also known astoxic hepatopathy, is a major clinical problem. It is

showed by epidemiological studies that the prevalenceof drug/chemicals-induced hepatopathy went up overthe past decades especially in the developing countries

∗ Corresponding author at: Xisihuan Zhonglu 100, District of Feng-tai, Beijing 100039, PR China. Tel.: +86 10 66933391;fax: +86 10 63053484.

E-mail address: springmaze@gmail.com (Y.-Q. Li).

0300-483X/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reservdoi:10.1016/j.tox.2006.12.013

chinacoside

(Bissel et al., 2001; Larrey, 2000; Lee, 2003). Despitethat extensive studies have been made for decades, theprecise mechanisms underlying drug/chemicals-inducedliver injury are still unclear and remain controversial.And there is still lack of effective therapeutic strate-gies or specific medicines for such liver diseases. Recentstudies indicated that oxidative stress might be a piv-otal originating factor in the pathogenesis of the liverdiseases including drug-induced hepatic damage, alco-

holic hepatitis, and viral hepatitis or ischemic liver injury(Albano, 2002; Amin and Hamza, 2005; Jaeschke etal., 2002). Over production of free radicals are toxic tohepatocytes and initiate reactive oxygen species (ROS)-

ed.

Y. Wu et al. / Toxicology 2

maPtv2eds

ihb2bt(seitflioi

2

2

UMm(DSo

2

2

Fig. 1. Chemical structure of echinacoside.

ediated cascade causing hepatocyte death, leading tocute hepatic damage (Jaeschke, 2000; Gutteridge, 1993;essayre, 1995; Sies, 1991). Therefore, antioxidative

reatment was proposed to be a potential means of pre-enting or attenuating toxic liver injury (Hensley et al.,000; Higuchi and Gores, 2003; Kaplowitz, 2002). How-ver, the effects of most antioxidants against acute liveramage are not satisfactory enough to apply into clinicalituations.

Echinacoside (Fig. 1) is one of the phenylethanoidssolated from the stems of Cistanches salsa, a Chineseerbal medicine, which is an important crude drug usedoth as an antisenium and antifatigue agent (Deng et al.,004; Xiong et al., 1996). Several phenylethanoids haveeen shown to possess free radical scavenging proper-ies and protect oxidative stress-induced toxic injuriesDeng et al., 2004; Gao et al., 1999). In the presenttudy we aimed to investigate the potential effects ofchinacoside in reducing oxidative stress, and improv-ng histopathological abnormality in the liver of ratsreated with CCl4 that not only could provide some help-ul information for the therapy or prevention of suchiver disease, but might promote the understanding ofts mechanisms, especially the role of free radicals orxidative stress in the pathogenesis of acute toxic livernjury.

. Materials and methods

.1. Chemicals and reagents

Echinacoside from C. salsa was kindly supplied by Pekingniversity Modern Research Center for Traditional Chineseedicine. The purity of the compounds was shown to beore than 95% on high-performance liquid chromatography

HPLC). The TUNEL assay kit was obtained from Rocheiagnostics Company (Germany). PBN was purchased fromigma Chemical Co., USA. All other reagents or drugs weref analytical grade.

.2. Animals and treatments

Twenty-four adult male Sprague–Dawley rats weighing00–250 g obtained from Peking University Animal Breed-

32 (2007) 50–56 51

ing Unit were used in this study. All animals were kept underthe same laboratory conditions of temperature (25 ± 2 ◦C) andlighting (12:12 h light:dark cycle) and were given free access tostandard laboratory chow and tap water. All rats were allowedto acclimatize for 1 week prior to experimentation. All exper-imental procedures involving animals were approved by theethical animal committee of Peking University, Beijing, PRChina.

The animals were randomly divided into three groups con-taining eight rats in each. Group 1 served as controls andreceived an injection of vehicle (olive oil) alone; group 2 wasinjected intraperitoneally (i.p.) with CCl4 dissolved in an equalvolume of olive oil at a dose of 3 ml/kg, which is well docu-mented to induce hepatotoxicity. Echinacoside was dissolvedin physiological saline and then administered to group 3 at adose of 50 mg/kg body weight, followed by CCl4 treatmentto induce hepatic damage in rats. The dose of echinacosideadopted in this study was based on the preliminary studies inour research group.

2.3. Sample collection and hepatic function assays

Before the rats were killed, they were fasted for 24 h afteradministration of CCl4. At the end of the experiment, all ani-mals were sacrificed to collect blood samples, which wereallowed to clot and centrifuged at 3000 × g for 10 min to obtainserum. All serum samples were sterile, haemolysis-free andwere kept at −70 ◦C before determination of the biochemicalparameters. Serum levels of alanine aminotransferase (ALT),aspartate aminotransferase (AST) as markers of hepatic func-tion were measured by using a multiparameteric analyzer (AU5400, Olympus, Japan).

2.4. Measurement of MDA, SOD and GSH in the liver ofrats

Part of the liver tissue was prepared to make homog-enization with a buffer containing 0.15 M KCl to obtain1:10 (w/v) homogenates. Homogenates were then centrifugedat 12,000 × g (4 ◦C) for 20 min to collect the supernatantfor determination of MDA, GSH concentrations and SODactivity (Wen et al., 2006). Protein concentrations weremeasured according to the Bradford method (Bradford,1976).

MDA, the last product of lipid breakdown caused by oxida-tive stress, was evaluated by the thiobarbituric acid reactivesubstances method (TBARS) and was expressed as nmol/mgprotein (Draper and Hadley, 1990).

Glutathione (GSH) concentration was measured by akinetic assay using a dithio-nitrobenzoic acid recyclingmethod and was expressed as �mol/g protein (Gutteridge andHalliwell, 1990).

Superoxide dismutase (SOD) activity was estimated by amethod based on the production of H2O2 from xanthine by xan-thine oxidase and reduction of nitroblue tetrazolium (Sarban etal., 2005). The product was evaluated spectrophotometricallyat 560 nm and expressed as U/mg protein.

ology 2

ESR specta of the spin trapping adduct of oxygenradicals with PBN extracted from the liver tissue areshown in Fig. 2. The peak height of spectra represents

Fig. 2. Effect of echinacoside on reactive oxygen species (ROS) pro-duction in the liver of rats treated with CCl4. The peak height of

52 Y. Wu et al. / Toxic

2.5. Measurement of ROS production in the liver of rats

Part of the liver tissue was homogenized with spin trap agentthat was prepared as follows: N-tert-butyl-a-phenylnitrone(PBN) and diethtylonethiaminepentaacetic acid (DETAPAC)were dissolved in PBS (pH 7.4) at a final concentration of100 and 2 mM, respectively. Liver homogenates were thencentrifuged at 14,000 × g for 10 min after adding 0.4 ml ethylacetate and the supernatants were finally collected for ESRmeasurement. An X-band ESR spectrometer (Varian E-109E;Varian, Inc., Palo Alto, CA) was used to detect the levelsof reactive oxygen species (ROS) directly with measure-ment conditions as: X-band, central magnetic field (3445 G),scan width (200 G), microwave power (20 mW), frequency(100 kHz), modulation amplitude (2.5 G), and time constant(0.128 s) at 37 ◦C (Capani et al., 2001; Liu et al., 2004). Beforemeasurement, the ESR spectrometer was standardized withdiphenyl-picri-hydrazyl to make sure that the equipment wasin the same condition in every experiment.

2.6. Histological examination

Liver tissues were removed immediately and fixed in 10%neutral formalin solution for at least 24 h, then embedded inparaffin wax and sectioned (4 �m thickness) for histopatholog-ical evaluation. Liver sections were stained with haematoxylinand eosin (H&E) using a standard protocol, and then analyzedby light microscopy.

2.7. Measurement of hepatocyte apoptosis

Apoptotic hepatocytes were detected by terminal deoxynu-cleotidyl transferase-mediated dUTP nick end-labeling(TUNEL) staining using a commercial ready-to-use kit (RocheDiagnostics Company, Germany). Following deparaffinizationand rehydration, 4 �m liver tissue sections were treated withproteinase K in PBS (10 �g/ml) for 30 min, and after washingwith PBS, these sections were incubated with TUNEL reactionmixture for 60 min at 37 ◦C. Following converter-POD additionand diaminobenzidine (DAB) substrate reaction, these sectionswere eventually examined under light microscope.

2.8. Statistical analysis

Data were expressed as mean ± S.E.M. and were analyzedwith SPSS, Version 12.0 software. Differences between groupmeans were calculated by a one-way analysis of variance(ANOVA). Results were considered statistically significantwhen P < 0.05.

3. Results

3.1. Effect of echinacoside on serum ALT, ASTlevels

It showed that serum ALT and AST levels weresignificantly elevated at 24 h following CCl4 admin-

32 (2007) 50–56

istration to rats while simultaneous echinacosidetreatment decreased remarkably the levels of serumALT, AST in the liver of CCl4-treated rats, althoughthe enzyme levels were still higher than in the controlrats.

3.2. Effect of echinacoside on hepatic SOD, MDAand GSH contents

The results indicated that CCl4 exposure increasedMDA concentrations but decreased SOD activities andGSH contents markedly in the liver of rats, as comparedwith the control rats, suggesting that lipid peroxidationresulted from CCl4 poisoning. Echinacoside treatmentwas able to decrease MDA concentrations and restoreSOD activities and GSH contents effectively in contrastto the CCl4-treated rats, thus reducing lipid peroxidationto some extent.

The results are summarized in Table 1.

3.3. Effects of echinacoside on ROS production inthe rat liver

spectrum represented relative contentration of oxygen radicals. (A)The control rats; weak signal of ROS in the liver tissue of normal rat.(B) The significant generation of ROS in the liver tissue of rat at 24 hfollowing CCl4 treatment. (C) The decreased generation of ROS at24 h in the liver tissue of rat treated with echinacoside plus CCl4.

Y. Wu et al. / Toxicology 232 (2007) 50–56 53

Table 1Effects of echinacoside on serum ALT, AST levels and hepatic SOD activity, MDA and GSH content in rats treated with CCl4

Group ALT (U) AST (U) SOD (U/mg) MDA (nmol/mg) GSH (umol/g)

Control 78.4 ± 25.3 86.3 ± 32.1 84.6 ± 7.8 2.71 ± 0.67 65.7 ± 13.4CCl4 only 1874.4 ± 213.6* 1637.2 ± 195.2* 52.7 ± 17.8* 6.12 ± 1.24* 41.6 ± 9.4*

E # .7# # # #

D using o#

trcsapC

3c

c

Fhiib

FrpT

chinacoside + CCl4 412.6 ± 108.2 537.6 ± 91

ata are presented as mean ± S.E.M. (n = 8). Means were comparedP < 0.01 vs. CCl4 alone group.

he relative concentration of oxygen-free radicals. Theesult showed that only a weak signal of oxygen radicalsould be detected in the liver tissue of control rats. Theignificant release of oxygen-free radicals was measuredt 24 h after CCl4 injection, while ROS in echinacosidelus CCl4-treated group were distinctly lower than in theCl4-treated rats at the same time point.

.4. Effect of echinacoside on histopathological

hanges and hepatocyte apoptosis

There was no abnormal appearance or histologicalhanges in the liver of control rats, which received

ig. 3. Effect of echinacoside on histopathological damages induced byematoxylin–eosin method. CV denotes central vein in the liver. (A) The cng CCl4 treatment, liver sections with massive hepatocyte degeneration, andicated. (C) Echinacoside + CCl4, the liver sections showed reduced signs oe infrequently observed, as arrow indicated. Original magnifications 200×.

ig. 4. Effect of echinacoside on hepatocyte apoptosis in CCl4-treated rats. Lats, apoptotic hepatocytes can hardly be observed in the liver sections. (B) Atositive hepatocytes could be observed, as arrows indicated. (C) EchinacosidUNEL positive hepatocytes could be seen, as arrow indicated. Original mag

71.8 ± 12.7 3.94 ± 1.13 54.6 ± 15.7

ne-way analysis of variance (ANOVA). *P < 0.01 vs. control group;

vehicle (olive oil) only. CCl4 administration caused clas-sical damage in the rat liver at 24 h, as demonstratedby severe hepatocyte necrosis, inflammatory cells infil-tration, fatty degeneration, haemorrhage, and hydropicdegeneration (Fig. 3). Fibrosis or vacuolar degener-ation, and microvesicular steatosis were occasionallyobserved. Additionally, the number of apoptotic hepato-cytes stained with the TUNEL method was significantlygreater in the liver of CCl -treated rats than in the con-

4trol group, suggesting that massive hepatocyte apoptosistook place as a result of CCl4 intoxication (Fig. 4).Treatment with echinacoside was able to amelioratethe CCl4-induced liver injuries and typical histological

CCl4 in the liver of rats. Liver sections were stained using theontrol rats, without any signs of liver damage. (B) At 24 h follow-poptosis or necrosis, and inflammatory cells infiltration, as arrowsf liver damage; only mild hydropic degeneration of hepatocytes could

iver sections were stained using the TUNEL method. (A) The control24 h following CCl4 treatment, extensive and large number of TUNELe + CCl4, hepatocyte apoptosis had been attenuated a lot; only a fewnifications 400×.

ology 2

54 Y. Wu et al. / Toxic

changes were markedly absent in the liver sections.Meanwhile, echinacoside also reduced dramatically thenumber of TUNEL-positive cells in the liver as comparedwith CCl4-treated group.

4. Discussion

The aim of the present study was to investigatethe potential hepatoprotective effects of echinacosideon the free radical damage of liver caused by carbontetrachloride in rats. Liver is well known to be themajor organ responsible for the metabolism of drugsand toxic chemicals, and therefore is the primary tar-get organ for nearly all the toxic chemicals (Bissel et al.,2001; Larrey, 2000; Lee, 2003). Various pharmacologi-cal or chemical substances are known to cause hepaticinjuries, such as acetaminophen, CC14, d-galactosamine(GalN) and dimethylnitrosamine (DMN). Excessivedose exposure to these hepatotoxins may induce acuteliver injury characterized by abnormality of hepaticfunction, and degeneration, necrosis or apoptosis ofhepatocytes (Basu, 2003; Higuchi and Gores, 2003;Kaplowitz, 2002; Nelson, 1990). With the increasingingestion of drugs or exogenous chemicals, the possi-bility of toxic liver injury will rise undoubtedly (Lee,2003). More severely, because of the clinical manifes-tations of toxic hepatopathy are so similar to those ofviral hepatitis or other hepatic diseases, that the differ-ential diagnosis will nearly be impossible. So it should beattached much importance to the mechanisms involvingdrug/chemicals-induced liver injury. At the same time,trying to find some effective therapeutical methods forthe treatment of toxic liver injuries are considered veryimportant in clinical situations.

It was widely believed that the occurrence ofdrug/chemicals-induced liver injury was especially asso-ciated with oxidative stress, a cellular imbalance betweenthe production and elimination of free radicals (Aminand Hamza, 2005; Castro and Freeman, 2001; Jaeschke,2000); the overproduction of free radicals resultingfrom oxidative stress could directly injure hepatocel-lular membrane by lipid peroxidation or other means,followed by a series of cascades of cellular eventssuch as the massive release of inflammatory media-tors or cytokines, which eventually lead to liver injuries(Dizdaroglu et al., 2002; Higuchi and Gores, 2003;Pessayre, 1995). Therefore, it is valuable to identifynatural drugs or compounds that can antagonize the dele-

terious action of free radicals and act as an antioxidant toprotect hepatocyte from damage. It is also reported thatantioxidative treatment appears to be an effective meansof preventing or attenuating liver injury induced by var-

32 (2007) 50–56

ious causes such as hepatotoxins, drugs, alcohol abuse,ischemia or reperfusion, which all supported the key roleof oxidative damage in the pathogenesis of liver injury,and would contribute greatly to the prevention and treat-ment of liver injury with natural or artificial antioxidantsin the future (Castro and Freeman, 2001; El-Sokkary etal., 2005; Hensley et al., 2000; Jaeschke, 2000). Someantioxidants such as vitamin E, vitamin C, melatonin orfree radical scavengers including superoxide dismutase(SOD), catalase have already demonstrated their poten-tial efficacy to ameliorate various hepatotoxins-inducedhepatocellular damage in previous studies. In the presentstudy, we adopted the rat model of CCl4-induced liverinjury to investigate the possible hepatoprotective effectsof echinacoside, a novel antioxidant, and to explore theunderlying mechanisms behind CCl4-induced hepato-toxicity.

Our results showed that CCl4 administration causedsevere acute liver damage in rats, demonstrated by sig-nificant elevation of serum AST, ALT levels and classichistopathological changes, which was consistent withthe findings of other investigators (Basu, 2003; Brattinet al., 1985; Williams and Burk, 1990). CCl4 injectioncould also induce oxidative stress, as indicated by ele-vated MDA concentrations and decreased SOD activitiesand GSH contents in the liver of rats, which would furtherinduce lipid peroxidation, initiate free radicals damage tohepatocelluar membrane, and lead to liver injury eventu-ally (Taniguchi et al., 2004; Sun et al., 2001). In addition,we employed electron spin resonance (ESR) to directlymeasure reactive oxygen species (ROS) production inthe liver of CCl4-treated rats, with an aim to furtherexamine the role of oxidative stress damage in acuteliver injury. Because of the high reactivity and shortlife, ROS has been generally analyzed by measuringthe changes in antioxidases such as SOD, GSH-Px orthe products of lipid peroxidation such as MDA, butthere is still an absence of detailed and direct data ofROS changes in studies of acute liver injury. In ourinvestigation, ESR spin trapping technique provides asensitive, direct and accurate means of monitoring ROSand it has been considered as one of the most effectivetechniques for detecting short life-span free radicals andhas been applied in basic research and clinical studies(Finkelstein et al., 1980; Liu et al., 2004). The resultsindicated that ROS production was highly elevated inthe liver of rats at 24 h after CCl4 administration, thusfurther confirming that free radicals and oxidative dam-

age certainly play a vital role in the pathogenesis ofacute liver injury, which also provides strong evidencefor natural antioxidants applied in the treatment of toxichepatopathy.

ology 2

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B

B

Y. Wu et al. / Toxic

C. salsa (C.A. Mey) G. Beck, one species of Cis-anches which belongs to Orobanchaceae family, is aarasitic plant native in the northwest of China. The stemf this plant is an important traditional Chinese medicinend used for kidney deficiency, female infertility, mor-id leucorrhea, neurasthenia and senile constipation dueo colonic inertia (Deng et al., 2004). The major activeonstituents of this herb are phenylethanoid glycosides.everal phenylethanoids have been shown to possessree radical scavenging properties and protect oxidativetress-induced toxic injuries. Among which, echinaco-ide is one of the phenylethanoids isolated from thetems of C. salsa, whose pharmacological and biologicalctivities are believed to be more powerful and multi-unctional (Deng et al., 2004; Xiong et al., 1996). In ourtudy, we indicated for the first time that echinacosidereatment has a potent protective effect against oxida-ive stress and acute liver damage induced by CCl4 inats, as revealed by remarkable decrease in ROS pro-uction, MDA content, together with elevation of SODctivity, GSH concentration in the liver. Additionally,chinacoside could ameliorate acute liver damage to aigh degree, as demonstrated by reduction of serum ALT,ST levels and histopathological changes improvement.part from mild hydropic degeneration of hepatocytes,

he liver had a nearly normal appearance in CCl4-reated rats treated with echinacoside simultaneously.he mechanisms underlying hepatoprotection of echi-acoside may be related to both its radical scavengingroperties and indirect effects as a regulator of antiox-dative systems, but more studies should be carried outn the future. In conclusion, our investigation providedonvincing data that echinacoside has an impressive hep-toprotective effects on acute liver injuries induced byCl4, which might be considered to be therapeutic inlinical situations.

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