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Journal of Ethnopharmacology 112 (2007) 514–523

Effects and mechanisms of extract from Paeonia lactiflora and Astragalusmembranaceus on liver fibrosis induced by carbon tetrachloride in rats

Wu-Yi Sun a, Wei Wei a,∗, Li Wu a,b, Shuang-Ying Gui a,c, Hua Wang a,d

a Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory of Anti-inflammatory and Immunopharmacology in Anhui Province, KeyLaboratory of Research and Development of Chinese Medicine in Anhui Province, Hefei, Anhui Province 230032, China

b Institute of Materia Medica, Nanjing University of Traditional Chinese Medicine, Nanjing 210046, Chinac Anhui College of Traditional Chinese Medicine, Hefei 230031, China

d Department of Oncology, The Affiliated Anhui Provincal Hospital of Anhui Medical University, Hefei 230032, China

Received 30 January 2007; received in revised form 27 March 2007; accepted 18 April 2007Available online 24 April 2007

bstract

Paeonia lactiflora and Astragalus membranaceus are two popular traditional Chinese medicines, commonly used in Chinese herb prescriptiono treat liver disease. The extract prepared from the roots of Paeonia lactiflora and Astragalus membranaceus (PAE) demonstrated more excellentepato-protective activity than the single herbs used individually as indicated in our preliminary studies. The present study was carried out tonvestigate the effects of PAE on liver fibrosis in rats induced by carbon tetrachloride (CCl4) and to explore its possible mechanisms. Liver fibrosisas induced in male Sprague–Dawley rats by injection with 50% CCl4 subcutaneously twice a week for 8 weeks. At the same time, PAE (40,0 and 160 mg/kg) was administered intragastrically. Upon pathological examination, the PAE-treated rats significantly reduced the liver damagend the symptoms of liver fibrosis. Administration of PAE decreased CCl4-induced elevation of serum transaminase activities, hyaluronic acid,aminin and procollagen type III levels, and contents of hydroxyproline in liver tissue by approximately 30–60%. It also restored the decrease inOD and GSH-Px activites and inhibited the formation of lipid peroxidative products during CCl4 treatment. Moreover, PAE (80, 160 mg/kg, ig)ecreased the elevation of TGF-�1 by 47.7% and 53.1%, respectively. In the primary cultured hepatic stellate cells (HSCs), PAE also significantlyecreased [3H] thymidine incorporation in cells stimulated with platelet-derived growth factor-B subunit homodimer (PDGF-BB) and suppressed

3H] proline incorporation. These results suggested that PAE significantly inhibited the progression of hepatic fibrosis induced by CCl4, and thenhibitory effect of PAE on hepatic fibrosis might be associated with its ability to scavenge free radicals, decrease the level of TGF-�1 and inhibitollagen synthesis and proliferation in HSCs.

2007 Elsevier Ireland Ltd. All rights reserved.

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eywords: Paeonia lactiflora; Astragalus membranaceus; Liver fibrosis; Oxida

. Introduction

Liver fibrosis represents a major medical problem with sig-ificant morbidity. Hepatitis viral infections, including hepatitis

and hepatitis C (especially hepatitis B in China), represent

he major cause of liver fibrosis. Other stimuli for liver fibrosisnclude drug-induced, helminthic infection, autoimmune disor-ers, iron or copper overload and biliary obstruction. Fibrosisan be classified as a wound healing response to a variety of

∗ Corresponding author. Tel.: +86 551 516 1208; fax: +86 551 516 1208.E-mail address: wwei@ahmu.edu.cn (W. Wei).

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378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2007.04.005

tress; Transforming growth factor-�1; Hepatic stellate cell

hronic stimuli. It is characterized by an excessive deposition ofxtracellular matrix proteins of which type I collagen predom-nates. This excess deposition of extracellular matrix proteinsisrupts the normal architecture of the liver that alters the normalunction of the organ, resulting in pathophysiological damageo the organ (Bataller and Brenner, 2005; Tsukada et al., 2006).epatic stellate cells (HSCs) are presently regarded as one of theey cell types involved in the progression of liver fibrosis. Thectivation of HSCs to a proliferative, myofibroblastic phenotypelays a key role in hepatic fibrogenesis, since these cells are the

rincipal cellular source of the excess collagen synthesis dur-ng hepatic fibrosis (Friedman, 2004). If left untreated, fibrosisan progress to liver cirrhosis ultimately leading to organ fail-re and death. Current evidence indicates that hepatic fibrosis

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W.-Y. Sun et al. / Journal of Ethn

ven cirrhosis is dynamic and can be bidirectional (involvinghases of progression and regression) (Pinzani and Rombouts,004). Efforts have been made to search for effective anti-fibroticgents. However, no effective antifibrotic therapies are availablentil now. Therefore, the prevention of liver fibrosis has a veryreat significance both in theory and in practice.

Medicinally, natural drugs have made a significant contri-ution to the treatment of liver fibrosis. Use of herbal drugsn the treatment of liver diseases has a long tradition, espe-ially in Eastern medicine. Examples could be found in ‘Yellowmperor’s Internal Classic’, a highly systematic and comprehen-ive ancient Chinese medical text dated back to 475–221 B.C.Schuppan et al., 1999; Chor et al., 2005). Herbal medicinessed in China are now being manufactured as drugs contain-ng ingredients of standardized quality and quantity. Traditionalhinese medicine treatment is based on overall analysis of symp-

oms and signs, as well as the physical condition of the patient.owadays, practitioners of traditional Chinese medicine rou-

inely use herbs to treat chronic liver disease and cirrhosis.aeonia lactiflora pall root, a traditional Chinese herb, has beensed to relieve the pain and has been a component of effectiverescriptions for treatment of liver disease (Dai et al., 1993).otal glucosides of peony (TGP) which consist of paeoniflorin,lbiflorin, benzoylpaeoniflorin, oxypaeoniflorin, paeonin, etc.,xtracted from Paeonia lactiflora pall root, have been recog-ized as the valuable traditional herbs used in the treatment ofheumatoid arthritis, systemic lupus erythematosus and hepati-is with a long history in traditional Chinese medicine (Zhangt al., 2001). The anti-inflammatory, anti-oxidative, anti-hepaticnjury and immunoregulatory activities of TGP have been exten-ively proved in our laboratory for many years. Previous studiesemonstrated that TGP could retard the progression of exper-mental immunological hepatic fibrosis through inhibition ofollagen synthesis and decreasing oxidative stress, but hadodest effect on HSCs proliferation (Wang et al., 2005). Astra-

alus membranaceus is a popular traditional Chinese herb. Its used to replenish the vital energy for the treatment of lack-ng strength, anorexia and loose stools, spontaneous sweatingnd chronic nephritis with edema and proteinuria (Pharma-opoeia of the People’s Republic of China, 2005 edition). Theodern pharmacological studies revealed that Radix Astragali

ossessed immunostimulant and anti-fibrotic properties (Chengt al., 2000; Lee et al., 2003). In vitro studies in our laboratoryemonstrated that Radix Astragali, its extracts, and its activengredients such as astragalosides, astragalus polysaccharidesould significantly inhibit HSCs proliferation and collagen pro-uction (Wu et al., 2003). We have sought a preparation thatas the ability to reduce matrix synthesis as well as affect-ng HSCs proliferation. In traditional Oriental medicine, it isonventional to combine different herbs in order to achieve aariety of treatment purposes simultaneously, or to enhance aingle effect without causing severe side effects (Nishiyama etl., 1995). In order to obtain a more effective remedy for the

reatment of liver fibrosis other than exclusively using Paeo-ia lactiflora or Astragalus membranaceus, we combined thesewo herbs based on both traditional references and the resultsf our previous work mentioned above. As a result of prelimi-

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rmacology 112 (2007) 514–523 515

ary tests of extracts with several different combination-ratiosdata not shown), we found that the combination in the ratiof 4:1 of two plants, Paeonia lactiflora roots and Astragalusembranaceus roots, respectively, exhibited the most significantepato-protective activity among the combinations tested. Stan-ardized extract of the Chinese herb prescription composed ofaeonia lactiflora and Astragalus membranaceus (PAE) may bexpected to synergistically exert the antifibrotic effects based onndings from our previous reports. PAE was mainly composedf the total glucosides of paeony and the total astragalosides. Ourrevious studies have shown that PAE has protective effects onhemical liver injury in mice through inhibiting oxidative stressWu et al., 2006). These studies have resulted in considerablenterest in PAE as a therapeutic agent in chronic liver disease.o further evaluate the antifibrotic activity of PAE, the presenttudy was designed to investigate the effects of PAE administra-ion on carbon tetrachloride (CCl4)-induced rats liver fibrosis inivo. Furthermore, the actions of PAE on markers of oxidativetress and fibrogenesis were investigated. In addition, the effectsf PAE on proliferation and collagen synthesis in cultured ratSCs were evaluated in vitro.Colchicine is an alkaloid agent that has been widely

sed in clinical practice for the treatment of acute goutnd other immunologic diseases. Long-term colchicine treat-ent in patients with hepatic fibrosis appears to exert an

nti-inflammatory, anti-fibrotic and immunomodulatory effectNikolaidis et al., 2006). In experimental studies, colchicineeduces acute liver injury (Mourelle et al., 1988), inhibits col-agen secretion, and increases collagen degradation, thereby iteduces liver fibrosis (Poo et al., 1993). In this study, colchicineas used as a suitable positive control.

. Materials and methods

.1. Reagents

Radix Paeonia lactiflora and Radix Astragali were purchasedrom Hefei Heyitang Pharmacy, China. The production areas ofaeonia lactiflora pall and Astragalus membranaceus (Fisch.)ge. were Anhui Province and Shanxi Province(China) respec-

ively. Voucher specimen (Pan 2004002 and Pan 2004013)ere identified by Professor Lumin Pan in Department ofateria Madica, School of Pharmacy, Anhui College of Tradi-

ional Chinese Medicine and deposited in the Chinese Materiaadica Specimen Center of Anhui College of Traditionalhinese Medicine. Colchicine was obtained from Sigma Chem-

cal Co. (St. Louis, MO, USA). CCl4 was purchased fromhanghai Xinzhong Chemical Factory (China). Commercialits used for determining aspartate aminotransferase (AST),lanine aminotransferase (ALT), superoxide dismutase (SOD),lutathione peroxidase (GSH-Px) and malondialdehyde (MDA)ere obtained from Nanjing Jiancheng Institute of Biotech-ology (China). The hyaluronic acid (HA), laminin (LN) and

rocollagen type III (PC III) radioimmunoassay kits wereurchased from Shanghai Navy Medical Institute (China).LISA kits of transforming growth factor-beta1 (TGF-�1) werebtained from Sigma Chemical (St. Louis, MO). Dulbecco’s

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16 W.-Y. Sun et al. / Journal of Ethn

odified Eagle Medium (DMEM) and fetal bovine serum (FBS)ere from Gibco. Collagenase IV, Dnase and Nycodenz werebtained from Sigma Chemical Co (St. Louis, MO, USA).ronase-E was purchased from Merck (Germany). Recombinantuman TGF-�1 and platelet-derived growth factor-BB (PDGF-B) were obtained from Peprotech EC. [3H] thymidine and [3H]roline were purchased from China institute of atomic energy.ther chemicals used in the experiment were reagent grade from

ommercial sources.

.2. Preparation of PAE

The sliced dried roots of Radix Paeonia lactiflora and Radixstragali were mixed in the ratio of 4:1. The mixed roots

25 kg) were extracted two times with six times of 70% aqueousthanol, 1.5 h each time. Then, the extracts were combined andvaporated to dry powder under reduced pressure. The residuebtained from the combined extracts was dissolved with 8 L ofater. After filtration, the aqueous solution was extracted three

imes with 8 L water-saturated n-butanol successively each time,hich yielded dry powders after being combined and evapo-

ated to dryness under reduced pressure. Then, about 1 kg of the-butanol extract was chromatographed on polystryrene resinD101, 0.3–1.25 mm, Nankai Chemical Factory, Tianjin, China)ith water, 10% ethanol and 70% ethanol, respectively. Por-

ions of 70% ethanol were collected, and evaporated to drynessnder reduced pressure, which yielded 310 g of yellow dry pow-er. The dry powder contained 74.7% total glycoside of paeonyTGP) and 20.6% astragalosides (ASTs) by chromatometry.GP consist of paeoniflorin, oxy-paeoniflorin, benzoyl paeoni-orin, albiflorin and lactiflorin, etc. (Zhang et al., 2001). ASTsonsist of astragaloside I–IV, and aoyasapogenoside, etc. (Ma etl., 2002). Before administration to rats, PAE was dissolved in.5% sodium carboxymethylcellulose solutions.

.3. Animals and treatment

Male Sprague–Dawley rats, weighing 130–150 g, were pro-ided by the Shanghai BK Experimental Animal Center (GradeI, Certificate No. D-65). All animals were housed in conven-ional cages under control conditions of temperature (23 ± 3 ◦C)nd relative humidity (50 ± 20%), with light illumination for2 h/day. All experimental protocols described in this studyere approved by the Ethics Review Committee for Animalxperimentation of Institute of Clinical Pharmacology, Anhuiedical University (ER2005031201). After 1 week of acclima-

ization, the rats were randomly divided into five experimentalroups. The animals were allowed access to food and tap waterd libitum throughout the acclimatization and experimentaleriods.

The procedure for CCl4-induced model of liver fibrosis wasased on the method described previously (Iredale et al., 1998;ang et al., 2004), with some modifications. Rats were sub-

utaneously injected with 50% CCl4 mixed with vegetal oil,wice a week for 8 weeks. The amount of CCl4 adminis-ered was 1 ml/kg. The rats were randomly divided into fiveroups. Group 1 was normal control, Group 2 was CCl4 con-

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rmacology 112 (2007) 514–523

rol, Groups 3, 4 and 5 were CCl4 and PAE (40, 80 and60 mg/kg) treated rats, Groups 6 was CCl4 and colchicine0.1 mg/kg) treated rats, served as positive control. Groups 2–6eceived CCl4 subcutaneously twice weekly for 8 week. Group

was fed the basal diet throughout the experiment and wasesigned as a model group. Groups 3–5 and 6 were treatedith PAE (40, 80 and 160 mg/kg, ig) and colchicine (0.1 mg/kg,

g) per day respectively at the beginning of injection of CCl4.he control group was administered with the same volume ofehicle.

At 24 h after final injection of CCl4, all rats were anaes-hetized with diethyl ether, samples of blood were drawn fromhe abdominal aorta and collected in polyethylene tubes. Theerum samples obtained by centrifugation (3000 × g, 4 ◦C) for0 min and were kept frozen at−80 ◦C until assayed as describedelow. After the animals were killed, the liver and spleen wereromptly removed, and weighed. A portion of the liver wasxed for histopathology, and the remaining tissues were storedt −80 ◦C until required.

.4. Analysis of liver function

The serum activities of ALT and AST were evaluated bypectrophotometry using commercially available kits (Nanjingiancheng Institute of Biotechnology, China).

.5. Measurement of hydroxyproline content in liver

Liver collagen concentration was determined by measuringydroxyproline content in fresh liver samples according to theethod of Mitchell and Taylor (Mitchell and Taylor, 1970) withinor modifications. Samples of liver tissue obtained at the end

f the experiment were weighed, hydrolyzed in 6.0 M HCl,nd analyzed for total hydroxyproline content. Results werexpressed as mg/g wet tissue.

.6. Measurement of serum fibrotic markers and serumGF-β1

The levels of HA, LN and PCIII in the serum were assayedith radioimmunoassay. The operations were performed accord-

ng to the manufacturer’s instruction.TGF-�1 level in serum was determined by using rat enzyme-

inked immunoadsordent assay (ELISA) kit.

.7. Histopathological examination

Immediately after sacrifice, the liver tissue was removed andportion of the tissue was instantly fixed in 10% phosphate

uffered formalin, processed by routine histology procedures,mbedded in paraffin, cut in 5 �m pieces and mounted on thelide. The samples were stained with hematoxylin and eosinHE) for histopathological examination. Two pathologists who

ad no knowledge of their sources examined the stained slidesndependently. Histological grade of hepatic fibrosis was deter-

ined by a semi-quantitative method according to the followingcoring systerm (Li et al., 2003): 0, no fibrosis, normal liver and

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recorded in only CCl4-treated animals after 8 weeks. PAE(160 mg/kg) treatment prevented the increase of liver and spleenindices after CCl4 administration, the same result as colchicine(0.1 mg/kg, ig)(Table 1).

Table 1Effect of PAE on indices of liver and spleen of CCl4-induced liver fibrosis inrats (n = 8; mean ± S.D.)

Groups Doses (mg/kg) Liver index (%) Spleen index (%)

Normal – 3.08 ± 0.97 0.27 ± 0.40Model – 6.90 ± 1.73## 0.69 ± 0.22##

PAE 40 5.39 ± 1.70 0.55 ± 0.1580 4.76 ± 1.58* 0.46 ± 0.14

W.-Y. Sun et al. / Journal of Ethn

bsence of fibrosis; I, fibrosis present (collagen fiber present thatxtends from portal triad or central vein to peripheral region); II,ild fibrosis (mild collagen fiber present with extension with-

ut compartment formation); III, moderate fibrosis (moderateollagen fiber present with some pseudo lobe formation); IV,evere fibrosis (severe collagen fiber present with thickening ofhe partial compartments and frequent pseudo lobe formation).ach sample was observed at 100× magnification. The degreef fibrosis was expressed as the mean of 10 different fields inach slide.

.8. Estimation of antioxidant enzyme and lipideroxidation

Liver tissues were washed with normal saline to removeny red blood and clots, and then were homogenized on iceith Tris–HCl (5 mmol/L containing 2 mmol/L EDTA, pH 7.4).omogenates were centrifuged at 1000 × g for 15 min at 4 ◦C.liquots samples of the supernatants were analyzed for antioxi-ant enzymes and lipid peroxidation. The assays were performedccording to each manufacturer’s recommended protocol. Forhe determination of antioxidant enzymes, we measured thectivities of SOD and GSH-Px based on their ability to inhibithe oxidation of oxyamine by the xanthine-xanthine oxidase sys-em. The results are expressed as the units for SOD or GSH-Pxer milligram of liver tissue. Lipid peroxidation in the liver wasetermined by measuring the level of MDA, an end product ofipid peroxidation, using a thiobarbiturate method (Shimizu etl., 1999). The level of hepatic MDA was expressed as �mol/grotein.

.9. HSCs isolation and culture

HSCs were isolated from the normal rat livers by sequential initu perfusion with collagenase and pronase as introduced in pre-ious studies (Alpini et al., 1994; Benyon et al., 1999). The HSCsere separated from the resulting cell suspension by density gra-ient centrifugation through a single layer of 18% Nycodenz.fter centrifugation (1200 × g for 20 min at 4 ◦C), purified HSCsere collected from the top of the Nycodenz cushion and resus-ended in DMEM containing 20% FBS, penicillin (100 U/ml),treptomycin (100 �g/ml). The viability of HSCs prepared wasore than 95% as determined by trypan blue exclusion. HSCsere identified as more than 90% pure by their characteris-

ic lipid droplets on phase-contrast microscopy, endogenousitamin A autofluorescence (Friedman and Roll, 1987). Thensolated HSCs seeded at a density of 1.0 × 105 cells/ml in 96-ell culture plates. After cells became subconfluent (at 70–80%

onfluence), the cells were cultured with DMEM without con-aining FBS for 24 h (serum starvation) before the start of allxperiments.

.10. HSCs proliferation assay and collagen assay

To evaluate HSCs proliferation, we measured the intracel-ular uptake of [3H] thymidine (Matsuoka et al., 1989; Pinzanit al., 1989). HSCs in first passage were plated at a density

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rmacology 112 (2007) 514–523 517

f 1.0 × 105 cells/ml in 96-well culture plates. The confluentells were growth-arrested in thymidine-free medium (DMEMontaining 0.5% bovine serum albumin) for 24 h. Subsequently,ells were incubated with 10 ng/ml PDGF-BB and either vehi-le or a given concentration of PAE (10, 20, 40, 80, 160 mg/L).eanwhile, 1 �Ci/ml of [3H] thymidine was added, followed

y incubation at 37 ◦C in 5% CO2 for 24 h. Washed with PBS,he HSCs were digested with trypsin and absorbed on glassber filter paper. The cells were then washed once with 10%

richloroacetic acid and three times with saline. The uptakef [3H] thymidine was measured using a liquid scintillationounter. HSCs cultured in PDGF-BB free DMEM mixed with�Ci/ml of [3H] thymidine for 24 h were used as a control. Sixultures in each group were examined.

To evaluate the ECM production, we measured the productionf 3H-hydroxyproline 24 h after treatment with [3H] proline. Theedium was changed to DMEM containing 2 ng/ml of TGF-�1

Casini et al., 1993; Sugawara et al., 1998) containing variousoncentrations of PAE (10, 20, 40, 80, 160 mg/L) and 1 �Ci/mlf [3H] proline, followed by incubation at 37 ◦C in 5% CO2 for4 h. After 24 h of incubation, cells were washed with PBS. Theadioactivity was determined by liquid scintillation counting.SCs cultured in medium that did not contain TGF-�1 (2 ng/ml)ere used as a control.

.11. Statistical analysis

Data were expressed as means ± S.D. Statistical significancef the difference between groups was determined by one-waynalysis of variance and the t-test. The frequency data were com-ared using Ridit procedure. P < 0.05 indicated a statisticallyignificant difference.

. Results

.1. Effect of PAE on liver and spleen indices

A significant increase in liver index and spleen index was

160 3.47 ± 1.03** 0.47 ± 0.14**

olchicine 0.1 4.65 ± 1.30* 0.43 ± 0.14**

#P < 0.01 compared with normal control group; *P < 0.05, **P < 0.01 comparedith model group.

518 W.-Y. Sun et al. / Journal of Ethnopharmacology 112 (2007) 514–523

Table 2Effect of PAE on liver function, hydroxyproline content and production of TGF-�1 in hepatic fibrosis rats (n = 8; mean ± S.D.)

Groups Doses (mg/kg) ALT (U/L) AST (U/L) Hydroxyproline (mg/g protein) TGF-�1 (�g/L)

Normal – 39.94 ± 12.68 32.23 ± 9.34 1.16 ± 0.21 11.21 ± 2.84Model – 108.55 ± 32.54## 111.17 ± 28.24## 3.08 ± 0.68## 34.41 ± 8.69##

PAE 40 76.20 ± 18.42* 86.38 ± 21.98 2.23 ± 0.56* 28.12 ± 7.6580 56.99 ± 15.45** 52.92 ± 15.81** 1.76 ± 0.54** 18.01 ± 4.84**

160 56.67 ± 15.53** 53.94 ± 16.31** 1.84 ± 0.41** 16.13 ± ± 5.27**

Colchicine 0.1 71.30 ± 22.61** 67.93 ± 19.45** 1.82 ± 0.46** 18.02 ± 5.42**

##P < 0.01 compared with normal control group; *P < 0.05, **P < 0.01 compared with model group.

Table 3Effect of PAE on serum HA, LN and PCIII levels of CCl4-induced liver fibrosis in rats (n = 8; mean ± S.D.)

Groups Doses (mg/kg) HA (�g/L) LN (�g/L) PCIII (�g/L)

Normal – 107.49 ± 34.84 102.95 ± 33.76 93.53 ± 29.60Model – 267.45 ± 73.62## 225.21 ± 64.90## 252.69 ± 56.68##

PAE 40 191.68 ± 50.62* 159.46 ± 41.51* 192.15 ± 62.4780 150.79 ± 30.37** 136.87 ± 26.55** 156.01 ± 47.70**

160 152.57 ± 42.95** 138.23 ± 38.11** 153.90 ± 40.45**

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.2. Effect of PAE on liver function

Analysis of serum ALT and AST activities was carried outn order to evaluate the extent of liver injury after chronic CCl4reatment. ALT and AST activities at the end of the experimentre shown in Table 2. A significant increase in the activities ofhe two enzymes (ALT 1.7-fold and AST 2.4-fold) was observedn the model group compared with those of the normal controlroup. Treatment with PAE (80 and 160 mg/kg) or colchicine0.1 mg/kg) caused significant decrease in serum transaminasectivities.

.3. Effect of PAE on hydroxyproline content in liver

Analysis of hepatic hydroxyproline content was carried outs an index of liver fibrosis. Elevated hydroxyproline levels were

easured in CCl4-treated animals with respect to normal con-

rol group (Table 2). Treatment with PAE (80 and 160 mg/kg),ydroxyproline content in liver tissue was significantly reducedy 42.9% and 40.3%, respectively compared with CCl4 con-

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able 4ffect of PAE on the pathologic grading of CCl4-induced liver fibrosis in rats

roups Dose (mg/kg) Pathologic grading of hepatic fibr

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rol group. The same results as colchicine at dosage of 0.1g/kg.

.4. Effect of PAE on serum HA, LN and PCIII

As shown in Table 3, serum levels of HA, LN and PC III, theurrogate markers of liver fibrogenesis, increased significantlyn hepatic fibrotic rats in model group. Administration with PAEr colchicine effectively decreased the serum HA, LN and PCII levels, with the degree of protection being 40–73%.

.5. Effect of PAE on liver histological examination

CCl4 control group had a high degree of fibrosis. Treat-ent with PAE (80 and 160 mg/kg) or colchicine (0.1 mg/kg)

ignificantly improved histological scores in comparison with

odel group (Table 4). Representative photographs of the liverorphology are shown in Fig. 1. The control group showed

ormal lobular architecture with central veins and radiating hep-tic cords (Fig. 1A). In contrast, CCl4 administration elicited

osis P value

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W.-Y. Sun et al. / Journal of Ethnopha

Fig. 1. Effect of PAE on the histological morphology of fibrotic rat liver byHE staining (×100). (A) Normal control. (B) CCl4 control. (C) CCl4 + PAE80 mg/kg. (D) CCl4 + PAE 160 mg/kg.

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rmacology 112 (2007) 514–523 519

xtensive changes in liver morphology, including marked fattyegeneration, necrosis, obvious collagen deposition, hepatocyteallooning and infiltration of inflammatory cells in liver inter-titial (such as macrophages and lymphocytes) after 8 weeksf treatment (Fig. 1B). PAE (80 and 160 mg/kg) treatmentarkedly alleviated the degree of liver fibrosis indicated by

ecreased collagen deposition and lowed inflammation (Fig. 1Cnd D).

.6. Effect of PAE on hepatic lipid peroxidation and SOD,SH-Px activity

Lipid peroxidation is considered as an important factor for theepatotoxicity of CCl4. Data on lipid peroxidation in terms ofDA formation of whole liver homogenate of control and exper-

mental animals are presented in Table 5. A marked increase74%) in MDA production was found in the liver of CCl4-reated animals relative to normal control group. PAE-treatedats at doses of 80 and 160 mg/kg significantly reduced MDAevel by 33% and 33.7% compared to the CCl4 control, respec-ively. Colchicine (0.1 mg/kg) treatment decreased the MDAevel, but had no statistical significance compared with CCl4ontrol group.

At the end of the experiment the changes in SOD and GSH-Pxctivities were assayed in rat livers in order to evaluate endoge-ous antioxidant defences (Table 5). A significant decrease inOD and GSH-Px activity (54.7% and 48.2%, respectively) wasbserved in the liver of CCl4-treated rats relative to control ratsdministered with vehicle alone. Administration with PAE (80nd 160 mg/kg) or colchicine (0.1 mg/kg) significantly elevatedhe activities of both enzymes. Compared with colchicine group,he efficacy of PAE (80 and 160 mg/kg) restored the activities ofOD is more significant.

.7. Effect of PAE on production of TGF-β1 in hepaticbrosis rats

As shown in Table 2, when the rats were challenged withCl4, the level of TGF-�1 was elevated significantly (2.1-fold)ompared with normal control group. PAE (80 and 160 mg/kg,g) significantly decreased the TGF-�1 level (by 47.7% and3.1%) in serum when compared with CCl4 control group.he efficacy is similar to that of colchicine at dosage of 0.1g/kg.

.8. Effect of PAE on HSCs proliferation

PDGF-BB has a growth stimulatory effect on HSCs (Pinzani,002). The uptake of [3H] thymidine in HSCs with PDGF-BBas significantly increased compared with that in HSCs with-ut PDGF-BB (Fig. 2). Under the condition where the growth

f HSCs was stimulated by PDGF-BB, the effect of PAE wasnvestigated. When various concentrations of PAE (10, 20, 40,0 and 160 mg/L) were added to medium containing PDGF-BB,he uptake of [3H] thymidine was significantly inhibited.

520 W.-Y. Sun et al. / Journal of Ethnopharmacology 112 (2007) 514–523

Table 5Effects of PAE on MDA level, SOD and GSH-Px activities in liver homogenates of liver fibrosis rats induced by CCl4 (n = 8; mean ± S.D.)

Groups Doses (mg/kg) MDA (�mol/g protein) SOD (U/mg protein) GSH-Px (U/mg protein)

Normal – 25.26 ± 7.94 275.02 ± 65.14 436.99 ± 76.53Model – 43.94 ± 11.74## 124.60 ± 34.92## 226.25 ± 73.87##

PAE 40 36.07 ± 11.34 195.90 ± 39.87* 269.00 ± 61.1580 29.48 ± 8.69* 234.40 ± 50.50**† 371.66 ± 69.46**

160 29.55 ± 9.31* 229.93 ± 42.80**† 354.52 ± 37.46**

Colchicine 0.1 33.94 ± 7.14

##P < 0.01 compared with normal control group; *P < 0.05, **P < 0.01 compared wit

Fig. 2. Effect of PAE on HSCs proliferation and collagen synthesis in vitro.[3H] thymidine incorporation(an index of proliferation) in HSCs incubated for24 h with either DMEM alone (control), or 10 ng/ml PDGF-BB with a givenconcentration of PAE (white bars). [3H] proline incorporation (a general indexof collagen synthesis) in HSCs incubated for 24 h with either DMEM alone(control), or 2 ng/ml TGF-�1 with a given concentration of PAE (black bars).#

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#P < 0.01 compared with control group; *P < 0.05, **P < 0.01 compared withells treated with PDGF-BB or TGF-�1 alone. Values are mean ± S.D. for sixbservations.

.9. Effect of PAE on collagen synthesis in HSCs

A main constituent amino acid of collagen is proline, whichs converted to hydroxyproline. To evaluate the ECM produc-ion, we measured the production of 3H-hydroxyproline afterreatment with [3H] proline (Matsuoka and Tsukamoto, 1990).GF-�1 has been shown to stimulate the production of colla-en in HSCs (Casini et al., 1993; Sugawara et al., 1998). In theGF-�1-treated group, the incorporation of [3H] proline wasignificantly increased compared with that in the control group.AE significantly inhibited the production of 3H-hydroxyprolinen HSCs stimulated by TGF-�1 (Fig. 2).

. Discussion

Hepatic fibrosis is present in various chronic hepatic diseases.t is well known that constant fibrosis can lead to the develop-ent of hepatocellular carcinoma (Nissen and Martin, 2002;kita et al., 2002). Interrupting and/or reversing hepatic fibro-

is may be a new approach for improving its progression toepatocellular carcinoma. However, the therapy for reversingiver fibrosis is not yet well established (Bataller and Brenner,

005). Recently, research for new drugs has refocused on nat-ral products. Traditional Chinese medicine has been practicedidely in China for thousands of years and is a potential sourcef pharmaceutical remedies (Fowell and Iredale, 2006). The

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181.24 ± 42.00* 322.94 ± 76.91*

h model group; †P < 0.05 compared with colchicine (0.1 mg/kg) group.

resent study demonstrated that PAE, a standardized extractf a Chinese herb prescription, had therapeutic effects on liverbrosis induced by CCl4 exposure in rats. The animal modelf CCl4-induced liver fibrosis was established firstly. The histo-ogical results showed that the normal structure of lobules wasestroyed and pseudolobules formed. Moreover, the increasedydroxyproline content in liver and serum HA, LN and PCIIIlso confirmed the hepatic fibrogenesis in rats. Treatment withAE effectively decreased these biomarkers. The inhibitoryffect of PAE (80 mg/kg) on the content of hydroxyproline iniver tissue was appeared to be more potent than that of PAE160 mg/kg) and appeared not dose dependent. This emphasizeshe fact that herbal medicines often contain multiple active sub-tances and many active substances have regulatory activities.he synergistic effects of the potential active components on

he liver fibrosis will be further studied. Hepatocellular necro-is leads to elevation of the serum marker enzymes, whichre released from the liver into blood. The increased levelsf ALT and AST are conventional indicators of liver injuryAchliya et al., 2004). The present study revealed a signifi-ant increase in the activities of ALT and AST on exposureo CCl4, indicating considerable hepatocellular injury. Admin-stration of PAE (80 and 160 mg/kg) for 8 weeks attenuatedhe increased activities of the serum enzymes and caused aubsequent recovery towards normalization similar to that ofolchicine treatment. The hepato-protective effect of PAE wasurther concluded by marked improvement in the histopatho-ogical examinations. PAE administration not only protectedgainst hepatocyte damage and reduced collagen deposition, butlso ameliorated oxidative stress. In vitro experiments, PAE sig-ificantly inhibited HSCs proliferation and collagen synthesis.esults from these experiments clearly showed that PAE could

nhibit the progression of liver fibrosis induced by CCl4 in rats.Chronic CCl4 treatment is frequently used in rats to pro-

uce an experimental model to study hepatic fibrosis (Li et al.,003; Oh et al., 2003; Inao et al., 2004). Oxidative stress and itsonsequent lipid peroxidation have been currently consideredo be involved in the generation of CCl4-induced liver fibro-is (Hernandez-Munoz et al., 1997; Poli, 2000). When CCl4 issed to treat rats in studies of liver fibrosis, it is metabolized byytochrome P450 in liver cells to yield the trichloromethyl-free

adical, which either extracts a hydrogen atom from unsatu-ated membrane lipids to initiate lipid peroxidation or reactsith the sulfhydryl compounds, triggering a chain of lipid per-xidation. These changes lead to cell injury, and chronic liver

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njury result in liver fibrosis (Wasser and Tan, 1999). Oxida-ive stressors are also commonly detected in livers from patientsith alcohol abuse, hepatitis C virus infection, iron overload, or

hronic cholestasis (Loguercio and Federico, 2003; Aboutweratt al., 2003). Many antioxidants have been proved to have pro-ective or therapeutic effects on liver injury (Gebhardt, 2002).ur previous studies showed that PAE has protective effect onCl4-induced liver injury in mice through inhibiting oxidative

tress (Wu et al., 2006). To confirm the effect of PAE on oxidativetress in liver fibrosis, we examined the oxidative stress param-ters, including SOD, GSH-Px, and MDA. Consistent with therevious studies (Cabre et al., 2000), increased liver MDA lev-ls and depressed SOD and GSH-Px activities were observedn the CCl4-treated rats. The increase in MDA levels in liveruggests enhanced lipid peroxidation leading to tissue damagend failure of antioxidant defense mechanisms to prevent theormation of excessive free radicals (Drewa et al., 2002). Admin-stration of PAE intragastrically could significantly elevate thectivity of SOD and GSH-Px, two antioxidant enzymes, andarkedly decreased MDA level, the products of lipid peroxida-

ion, in liver fibrosis rats. These results of present study indicatehat PAE might inhibit lipid peroxidation and effectively recoverhe anti-oxidative defense system in liver fibrosis. It is possiblehat the mechanism of hepato-protective effects of PAE maye due to its antioxidant action. From the tables it is clear thathe PAE showed greater antioxidant activity which is compara-le with the positive control, colchicine. Furthermore, oxidativetress, particularly lipid peroxidation induces collagen synthesiso aggravate liver fibrosis (Lee et al., 1995). Hence, the reduc-ion of extracellular matrix in PAE-treated group, as evidencedy HA, LN, PCIII and hdroxyprline, was probably related to itsnti-oxidant activity.

Cytokines play an important role in the development ofiver fibrosis. One of the major fibrogenic cytokine whose rolen patients with cirrhosis and in animal-induced liver fibrosisas been clearly established is TGF-�1 (Gressner et al., 2002;ressner and Weiskirchen, 2006). The evidence for TGF-�1aving a central role in liver fibrosis is sizable. First, its overex-ression has been correlated with the degree of fibrosis in bothnimal models as well as in human disease. Second, TGF-�1nhibits matrix degradation by upregulation of tissue inhibitorf metalloproteinase-1 and -2, while stimulating the depositionf extracellular matrix by HSCs. Third, transgenic mice thatver-express TGF-�1 develop acute hepatic fibrosis (Rachfalnd Brigstock, 2003). Due to the pleiotropic fibrogenic effectsf TGF-�1, strategies aimed at disrupting TGF-�1 synthesisnd/or signaling pathways can markedly decrease fibrosis inxperimental models (Shek and Benyon, 2004). Therefore, theffect of PAE on the level of TGF-�1 was assessed using ELISA.he results showed that PAE treatment significantly decreased

he elevated level of TGF-�1 in fibrotic rats. These findings sug-est that the inhibitory effects of PAE on liver fibrosis might beelated to its action on the production of TGF-�1.

HSCs localized in close proximity to sinusoidal endothelialell and hepatocyte in the space of Disse, are the most patho-enetically relevant cell type for development of liver fibrosis. Inhe normal liver, the HSC is in a quiescent state where it is the pri-

B

rmacology 112 (2007) 514–523 521

ary storage site for retinoids. However, following liver injury,SCs lose vitamin A and transform to myofibroblastic cells,

ermed activated HSCs. Activation of HSCs results in two majorvents that potently promote the fibrogenic response of theseells (Beljaars et al., 2002; Reeves and Friedman, 2002). First,SCs change their pattern of gene expression which results in aramatic increase in the synthesis and deposition of extracellularatrix proteins; hence, these cells become directly fibrogenic.econdly, the proliferation rate of HSCs increases followingellular activation which effectively amplifies the number ofbrogenic cells present in the liver. Therefore, it is believed

hat effective treatment regimes aimed at reducing or inhibit-ng either biosynthesis of extracellular matrix or proliferativeesponses of HSCs would reduce the harmful effects of acti-ated HSCs in the development and progression of liver fibrosisTsukada et al., 2006). In vitro experiments, PAE markedlyuppressed [3H] thymidine incorporation of HSCs in the DNAynthesis study with PDGF-BB stimulation, clearly demonstratehat PAE has a strong antiproliferative effect in HSCs. Moreover,AE significantly suppress the production of extracelluar matrixomponents such as collagen in HSCs. Taken together, it maye speculated that the antiproliferative effect of PAE, with anfficient inhibition of collagen synthesis, may be importantlynvolved in the pharmacological mechanisms of the antifibroge-etic effect of PAE in CCl4-induced liver fibrosis.

In conclusion, results from the present study demonstratedhat PAE was effective in the prevention of CCl4-induced liverbrosis in rats. The primary mechanisms of this therapeuticffect could be due to its radical scavenging action, antioxidantctivity, decreasing the level of TGF-�1, inhibition of collagenynthesis and proliferation in HSCs.

cknowledgements

This work was supported by grants from the State Highechnology Research and Development Program of China (863rogram, No. 2002AA2Z3235). The authors acknowledge theelp of the staff members of the Institute of Clinical Pharmacol-gy, Anhui Medical University in conducting the study.

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