Asiatic acid from Potentilla chinensis attenuate ethanol ...

Biological and Pharmaceutical Bulletin Advance Publication by J-STAGE DOI:10.1248/bpb.13-00634

Ⓒ 2013 The Pharmaceutical Society of Japan

Advance Publication

September 26, 2013

Asiatic acid from Potentilla chinensis attenuate ethanol-induced

hepatic injury via suppression of oxidative stress and Kupffer cell

activation

Jinbin Weib,1

, Quanfang Huang

a,1, Renbin Huang

b, Yongxing Chen

b, Shujuan Lv

b, Ling Wei

b,

Chunhong Liangb, Shuang Liang

a, Lang Zhuo

b,c, Xing

Lin

b,*

aThe First Affiliated Hospital of Guangxi University of Chinese Medicine; Nanning 530023,

China

bGuangxi Medical University; Nanning 530021, China

cInstitute of Bioengineering and Nanotechnology; Singapore 169483, Singapore

*Corresponding author. Tel: +86-771-5358342. E-mail: [email protected] (X. Lin).

1These authors contributed equally to this work.

Biological and Pharmaceutical Bulletin Advance Publication

Abstract

This study examined the effect of asiatic acid from Potentilla chinensis (AAPC) on

chronic ethanol-induced hepatic injury. Rats underwent intragastric administration of ethanol

(5.0–9.0 g/kg) once a day for 12 weeks. A subset of rats were also intragastrically treated

with AAPC (2, 4 or 8 mg/kg) once a day. In the end, AAPC treatment significantly protected

against ethanol-induced liver injury, as evidenced by the decrease in serum alanine and

aspartate aminotransferases levels and the attenuation of histopathological changes in rats.

Additionally, AAPC significantly decreased blood alcohol and acetaldehyde concentrations

by enhancing alcohol dehydrogenase and aldehyde dehydrogenase activities. Mechanistically,

studies showed that AAPC remarkably alleviated the formations of malondialdehyde and

myeloperoxidase, restored impaired antioxidants, including superoxide dismutase,

glutathione peroxidase, glutathione reductase and catalase, and inhibited CYP2El activity.

Moreover, the over-expression of cytokines, such as TNF-α, IL-1β, iNOS and COX-2, the

elevated plasma endotoxin level and the up-regulated Toll-like receptor 4 (TLR4) , CD14 and

myeloid differentiation factor 88 (MyD88) as well as nuclear factor-κB were also suppressed

by AAPC in ethanol-intoxicated rats. In conclusion, the protective effect of AAPC on

ethanol-induced hepatotoxicity was mainly due to its ability to attenuate oxidative stress and

inhibit Kupffer cell activation by decreasing the level of plasma endotoxin and the expression

of TLR4, CD14 and MyD88.

Keywords: Asiatic acid; Potentilla chinensis; Ethanol; Hepatic injury


1. Introduction

Alcohol abuse and alcoholism are major global health, social and economic issues.

Alcoholic liver disease (ALD) is a pathological process characterized by progressive liver

damage leading to steatosis, steatohepatitis, fibrosis and cirrhosis. Cirrhosis may eventually

progress to hepatic decompensation and hepatocellular cancer 1,2)

. Most of the evidence

shows that both oxidative stress and abnormal cytokine production play an important

etiological role in the pathogenesis of ALD. Ethanol intake causes the accumulation of

reactive oxygen species (ROS), such as superoxide, hydroxyl radical, and hydrogen peroxide

3). These reactive moieties cause lipid peroxidation (LPO) of cellular membranes and protein

and DNA oxidation, ultimately leading to hepatocyte injury 4)

. Therefore, agents with

antioxidant and anti-inflammatory properties are promising therapeutic interventions for

ALD.

Thanks to thousands of years of experience, herbal medicines are considered as a rich

source of new therapeutic agents. Many compounds with new structural features and

mechanisms of actions have been isolated from herbal medicines. Natural products are

potential sources of novel anti-hepatitis drugs that may be applicable to liver disease therapy

5). An example of a traditional herb that is often used in popular folk medicine in China is

Rosaceae Potentilla chinensis Ser., which is often used for treating immune disorders and

liver diseases 6,7)

. Asiatic acid isolated from P. chinensis has several health benefits and has

thus attracted medical and research professionals. Previous studies have shown that asiatic

acid has a variety of pharmacological effects on anti-inflammation 8,9)

, anti-tumor 10)

,

neuroprotection 11)

. In particular, asiatic acid has been shown to be a hepatoprotective agent.

A number of studies demonstrated that asiatic acid can protect liver from injury via

mechanisms underlying antimitochondrial stress and cellular antioxidant system in cultured

hepatocytes and Kupffer cells, and in a mouse model induced by D-galactosamine and

lipopolysaccharides 12,13)

. It has been also reported that asiatic acid is able to inhibit liver

fibrosis by blocking TGF-beta/Smad signaling pathway 14)

. However, the role and

mechanisms by which asiatic acid inhibits liver injury induced by ethanol remain unknown.

Therefore, the present study was conducted to investigate potential protective effects of


asiatic acid from P. chinensis on ethanol-induced liver injury in rats. The study also aimed to

explore the underlying mechanisms of such effects.

2. Materials and methods

2.1 Chemicals

P. chinensis was purchased from Nanning Qianjinzi Chinese Pharmaceutical Co. Ltd

(Nanning, China). A voucher specimen (CALX12071306) was identified by Q.F. Huang in

the First Affiliated Hospital of Guangxi Traditional Chinese Medicine University, and

deposited in the herbarium of Department of Pharmacology of Guangxi Medical University.

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde

(MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione

reductase (GSH-Rd) and catalase kits were obtained from Nanjing Jiancheng Bioengineering

Research Institute (Nanjing, China). Interleukin-1β (IL-1β) and tumor necrosis factor-α

(TNF-α) kits were purchased from Wuhan Boster Bio-engineering Co. Ltd. (Wuhan, China).

2.2 Preparation of asiatic acid from P. chinensis (AAPC)

Dried P. chinensis powder (5 kg) was extracted with 40 L of 95% ethanol by filtration.

The solvent was evaporated under a vacuum to obtain 785.2 g of crude extract, which was

successively extracted with petroleum ether, CHCl3 and acetic ether. Acetic ether (267.5 g)

was dissolved in CH3OH and filtered through a syringe filter (0.45 μm). The filtrate yielded a

powder (189.3 g) after concentration, which was purified by recrystallization in CH3OH to

yield a fraction (131.5 g). The fraction was then subjected to chromatography with a silica gel

column (200–300 mesh, Yantai, PR China; 10×300 cm) and was eluted with a gradient

mixture of CHCl3/CH3OH (100:0–0:100, each 500 mL) to afford 8 fractions (Frs. 1–8). Fr. 6

(65.9 g) was further separated by silica gel column (3×80 cm, 200–300 mesh) via successive

elutions with a gradient of CHCl3/CH3OH (100:0–0:100, each 200 mL), to yield 4

sub-fractions (Frs. I–IV). Frs. III was re-crystallized with CH3OH to yield a white crystal

after concentration, which was purified by Sephadex LH-20 and preparative HPLC to

produce a compound (4.17 g). Its structure was elucidated on the basis of physicochemical

properties and spectral data, and identified as aiasic acid (Fig. 1). This compound is normally

stored at 4 °C, and it is dissolved in distilled water and diluted with physiological saline for


animal tests.

2.3 Animals and treatments

Male SPF-Wistar rats weighing 180-200 g were provided by the Experimental Animal

Center of Guangxi Medical University (Guangxi, China). The research was conducted

according to protocols approved by the institutional ethical committee of Guangxi Medical

University (approval no.: 12061726). After a period of one week, the rats were divided into

six groups consisting of 15 rats per group as follows:

Group I received the same volume of saline.

Group II received 8 mg/kg AAPC.

Group III received ethanol.

Group IV received ethanol + 2 mg/kg AAPC.

Group V received ethanol + 4 mg/kg AAPC.

Group VI received ethanol + 8 mg/kg AAPC.

Rats in Groups III-VI were given intragastric ethanol infusions to induce chronic liver

injury. The ethanol dose was increased gradually by using a method of Zhang 15)

as follows:

5.0 g/kg/day from 1 to 4 weeks, 7.0 g/kg/day from 5 to 8 weeks, 9.0 g/kg/day from 9 to 12

weeks. Group III served as the ethanol-induced liver injury model. In addition to ethanol, rats

in groups IV -VI were also given AAPC orally on a daily basis. The doses of AAPC were

adopted according to the previous study 14)

.

At the end of 24 weeks, the rats were anesthetized with ketamine hydrochloride (30

mg/kg b.w., i.v.) prior to euthanasia for 1.5 h after ethanol administration. Blood samples

were collected in heparinized tubes (50 U/mL). Liver samples were dissected and washed

immediately with ice-cold saline to remove as much blood as possible. One part of the liver

sample was immediately stored at -80 °C for future analysis. The other portion of the liver

sample was fixed in a 10% formalin solution for histopathological analysis.

2.4 Alcohol and acetaldehyde concentrations in plasma

Alcohol and acetaldehyde concentrations were determined according to the methods of

Sung et al 16)

. In brief, blood samples were centrifuged at 3000 rpm at 4 °C for 15 min. The

alcohol concentration was detected with a Roche Cobas Integra 400 analyzer (Roche


Diagnostics, Switzerland). The acetaldehyde concentration was determined by using a

commercial assay kit (Boehringer Mannheim, Germany) following the manufacturer`s

instructions.

2.5 Estimation of hepatic alcohol metabolizing enzyme activities

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) were measured in

liver homogenate according to the protocol established in previous studies 17,18)

.

2.6 CYP2E1 enzyme activity assay

CYP2E1 enzyme activity was determined by the method described previously 19)

. In brief,

the livers were perfused with ice-cold 0.15 M KCl and homogenised in a homogeniser with 4

volumes (w/v) of 10 mM Tris-HCl (pH 7.4) containing 0.15 M KCl, 0.1 mM EDTA, 1.0 mM

dithiothreitol and 0.01 mM phenylmethylsulphonyl fluoride. Hepatic microsomal fractions

were obtained by differential centrifugation. The microsomal fractions were used to

determine CYP2E1-specific oxidative activities. The aniline hydroxylase was determined by

measuring p-aminophenol formation activities, and the microsomal protein levels were

determined by using the Bradford method with bovine serum albumin as the standard 20)

. All

assays were run in triplicate. CYP2E1 was detected immunochemically, as in a previous

study 21)

.

2.7 Estimating AST and ALT activities

Serum levels of ALT and AST were measured using commercially available kits (Nanjing

Jiancheng Bioengineering Research Institute, Nanjing, China) according to the

manufacturer’s instructions.

2.8 Liver myeloperoxidase activity assays

Myeloperoxidase (MPO) activity was measured according to the method of Yoshida et al

22). Tissue was homogenized in 50 mM phosphate buffer (pH 6.0) containing 0.5%

hexadecyltrimethylammonium bromide. The supernatant remaining after centrifugation was

then mixed with 10 mM phosphate buffer (pH 6.0) and 1 mL of 1.5 mM o-dianisidine

hydrochloride containing 0.2 mM H2O2. The change in absorbance for each sample was

recorded at 450 nm. MPO activity was expressed as μmol of oxidized product


formed/min/mg protein using the extinction coefficient 10,062 M−1

cm−1

. The protein content

was determined by using bovine serum albumin as the standard.

2.9 Liver TNF-α and IL-1β assays

Liver samples were disintegrated in 4 volumes of icecold RIPA buffer (150 mM NaCl, 5

mM EDTA, 50 mM Tris [pH 7.4]), containing protease inhibitors (1 μg/mL aprotinin, 10

μg/mL leupeptin and 1 μg/mL pepstaitn), DNase (0.05 mg/mL), and detergents (0.3% Triton

X-100, 0.03% sodium dodecyl sulfate, 0.3% sodium deoxycholate). After incubating on ice

for 30 min, samples were centrifuged twice at 20,000 ×g for 15 min at 4 °C. The resulting

supernatants were harvested and stored at -80 °C until the quantification of intrahepatic

cytokines by murine ELISA kits (Minneapolis, MN). Liver lysates were adjusted to equal the

protein concentrations after being quantified with the Coomassie blue (BioRad, Hercules,

USA). The results were expressed as pg/mg protein.

2.10 Estimation of antioxidant enzyme and lipid peroxidation

Liver tissue was homogenized on ice with Tris-HCl (5 mmol/L containing 2 mmol/L

EDTA, pH7.4). Homogenates were centrifuged at 1000×g for 15 min at 4 °C. The

supernatants were used immediately for SOD, GSH-Px, GSH-Rd and catalase assays. All

enzymes were evaluated according to our previously established methods 23)

. Liver lipid

peroxidation was determined by measuring the MDA level, an end product of lipid

peroxidation, using a thiobarbituric acid method 24)

. The level of hepatic MDA was expressed

as μmol/g protein.

2.11. Determination of plasma endotoxin level

The plasma endotoxin level was measured by using a quantitative chromogenic end-point

tachypleus amebocyte lysate endotoxin detection kit (Chinese Horseshoe Crab Reagent

Manufactory, Xiamen, China) following the manufacturer's instructions. In brief, plasma

samples were diluted to 1:10 with water/Tris-HCl buffer and heated at 70 °C for 10 min to

denature endogenous endotoxin inhibitors. After being centrifuged at 1500 ×g for 10 min, the

supernatant was removed and incubated with limulus amebocyte lysate at 37 °C for 10 min,

followed by incubation with chromogenic substance for 6 min. The absorbance was measured

at 545 nm after adding the appropriate azo-reagents.


2.12. Western blot immunoassay

Liver protein was prepared and determined as previously described 25)

. In brief, whole

protein was extracted from liver tissue and analyzed with a bicinchoninic acid (BCA) protein

concentration assay kit (Shanghai Haoran Bio Technologies Co., Ltd, China). A total of 20

μg of whole protein was used to determine the content of Toll-like receptor 4 (TLR4), CD14,

myeloid differentiation factor 88 (MyD88), TIR domain-containing adapter-inducing

interferon-b (TRIF), cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS).

NE-PER reagents (Pierce Biotechnology, Rockford, IL) were used to extract the nuclear and

cytosolic fractions according to the manufacturer`s instructions. A total of 20 μg of nuclear

protein was used to determine the NF-κB/p65 subunit level. The protein samples were

separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a

polyvinylidene fluoride membrane using a semi-dry transfer process. After the transfer, the

membranes were washed with Tris-buffered saline (TBS) and blocked for 1 h at room

temperature with 5% (w/v) skim milk powder in TBS. The blots were then incubated

overnight at 4 °C with polyclonal antibodies. The following primary antibodies were used:

TLR4 (Santa Cruz Biotechnology, Inc.), CD14 (Santa Cruz Biotechnology, Inc.), MyD88

(Lab Vision, Fremont, CA, USA), COX-2 (Cayman Chem, Ann Arbor, MI), iNOS (Santa

Cruz Biotechnology, Inc.), and NF-κB/p65 (Lab Vision, Fremont, CA). The signals were

normalized to that of β-actin (Sigma Chemical Co.) or lamin B1 (Abcam, Cambridge, MA),

respectively. On the following day, the primary antibody was removed, and the blots were

washed with TBS/T (0.1% Tween 20 in TBS), followed by an incubation with the appropriate

secondary antibodies for 1 h at room temperature. The immunoreactive bands intensity was

determined by using a densitometer equipped with Image QuaNT software (Molecular

Dynamics, Sunnyvale, CA).

2.13 Pathological examination

Liver fragments were fixed in 10% formalin, processed and embedded in paraffin. Five

μm sections were cut and mounted on glass slides. The slices were stained with

hematoxylin-eosin (H&E). All histological examinations were performed by an experienced

pathologist who was blinded to the experimental groups.


2.14 Statistical analysis

Statistical analysis was performed using SPSS 11.5 for Windows. Differences between

the groups were assessed using a one-way analysis of variance (ANOVA) with a Tukey`s test

for post hoc multiple comparisons. The data are presented as the means ± SE. A p-value <

0.05 was considered to be statistically significant.

3. Results

3.1 The effects of AAPC on blood alcohol and acetaldehyde concentrations

Treatment of rats with AAPC significantly decreased both alcohol and acetaldehyde

concentrations in the plasma when compared with the ethanol-only group (model group).

Neither alcohol nor acetaldehyde was detected in the plasma from the normal control and

AAPC control groups (Fig. 2).

3.2 The effects of AAPC on hepatic alcohol metabolizing enzyme activities

As shown in Fig. 3, hepatic ADH and ALDH levels were decreased after ethanol

treatment. Treatment with 4 or 8 mg/kg of AAPC exhibited protection against

ethanol-induced hepatic ADH and ALDH depletion, as evidenced by its reverse to a nearly

normal level. AAPC did not significantly alter basal ADH and ALDH activities.

3.3 The effect of AAPC on CYP2E1 activity

As shown in Fig. 4, ethanol feeding significantly increased the levels of aniline

4-hydroxylation (the CYP2E1-specific substrate) and CYP2E1 protein, whereas treatment

with 4 or 8 mg/kg AAPC significantly decreased both levels. In addition, AAPC alone had a

negligible effect on the basal activation of CYP2E1.

3.4 The effects of AAPC on serum ALT and AST activities

To evaluate the extent of liver injury in rats, we conducted an analysis of serum ALT

and AST activities. As shown in Fig. 5, a significant increase in the activity of both enzymes

was observed in the ethanol only group (model group) when compared with the normal

control. Conversely, animals treated with AAPC exhibited a significant decrease in the

activity of these enzymes. AAPC had no effect on the basal serum AST and ALT activities.

3.5 The effect of AAPC on liver myeloperoxidase activity


The MPO activity of the model group was significantly higher when compared with that

of the normal control group. This up-regulation was normalized after AAPC treatment. There

was no significant difference in the MPO level between the AAPC only and normal control

groups (Fig. 6).

3.6 The effects of AAPC on liver TNF-α and IL-1β levels

Liver TNF-α and IL-1β expression was evaluated by ELISA. The results showed that

hepatic TNF-α and IL-1β levels were elevated after ethanol administration. AAPC

significantly alleviated hepatic TNF-α and IL-1β production in a dose-dependent manner. In

addition, the AAPC only treatment had a negligible effect on TNF-α and IL-1β basal

expression (Fig. 7).

3.7 The effects of AAPC on antioxidant enzymes and lipid peroxidation

Ethanol-induced liver injury provoked significant reductions in liver SOD, GSH-Px,

GSH-Rd and catalase activities and a remarkable promotion of liver MDA content when

compared with the normal control. The results showed that liver SOD, GSH-Px, GSH-Rd and

catalase activities were obviously increased after AAPC treatment (Fig. 8A); in addition, the

liver MDA level was markedly decreased after AAPC treatment (Fig. 8B).

3.8. The effect of AAPC on level of plasma endotoxin

As shown in Fig. 9, the plasma endotoxin level was markedly increased, and AAPC

treatment significantly inhibited plasma endotoxin level.

3.9 The effects of AAPC on expression of NF-κB

Nuclear factor kappa B (NF-κB) plays a critical role in chronic inflammatory diseases and

its activation is essential for cytokine production. As shown in Fig. 10A, after chronic ethanol

consumption, the levels of NF-κB markedly increased, and that increase was attenuated by

AAPC.

3.10. The effects of AAPC on expression of TLR4, CD14, MyD88, COX-2, and iNOS protein

Western blot analysis showed a significant increase in the expression of TLR4, CD14,

MyD88, COX-2, and iNOS in the liver of the model group when compared with the normal

control. These expressions were significantly lower in the liver tissue of rats treated with


AAPC. There was no significant difference in the expression of TLR4, CD14, MyD88,

COX-2, or iNOS between the AAPC control and the normal control groups (Fig. 10B).

3.11 Histopathological findings

The H&E staining assay results showed that the hepatocytes in normal rats exhibited an

intact cellular architecture without necrosis, inflammatory infiltration or impaired progression

(Fig. 11A). There were no pathological changes in AAPC-only control rats (Fig. 11B).

Conversely, pronounced morphological alterations occurred in ethanol-treated rats, including

fat deposition, hepatocellular degeneration, inflammatory responses and necrosis, which were

accompanied by a reduction in the number of cells (Fig. 11C). AAPC supplementation

ameliorated the deleterious effects of chronic ethanol exposure on the liver, as seen in the

diminished fatty infiltration, lipid change and necrosis (Fig. 11D-F).

4. Discussion

Chronic ethanol ingestion is known to be associated with defective gut motility that

results indirectly in an elevated level of liver endotoxin 26)

. Furthermore, the major metabolic

product of alcohol, that is, acetaldehyde, leads to serious consequences to the individual.

Increasing and repeating acetaldehyde exposure from increased alcohol consumption can

increase the risk of developing acetaldehyde-related pathologies in the patient; thus,

acetaldehyde is more toxic to the body than alcohol 16)

. Therefore, the complications of

ethanol intake can be solved by effectively decreasing the plasma acetaldehyde concentration.

In the present study, blood alcohol and acetaldehyde concentrations in the ethanol only group

(model group) were significantly higher than those of the normal control group; however, the

concentrations were significantly lower in AAPC-treated groups. These data indicate that the

effect of AAPC in decreasing hepatic damage may be mainly linked to faster rates of alcohol

and acetaldehyde elimination.

The ingestion of alcohol leads to the rapid conversion of acetaldehyde to acetate by

ADH; therefore, very low levels of acetaldehyde should remain in the liver tissue or in the

blood after alcohol consumption. ALDH also plays an important role in the elimination of

acetaldehyde via oxidative reactions. Therefore, liver damage can be proportional to the

down-regulation of the activity of ADH or ALDH 27)

. In this study, ethanol-treated rats


showed significant reductions in ADH and ALDH levels, suggesting that alcohol,

acetaldehyde and other toxic metabolites were deposited in the liver tissue, which induced

hepatotoxicity over time. This result is consistent with the previous study 28)

. Interestingly,

these metabolism-specific enzymes were effectively increased by AAPC treatment. These

data suggest that AAPC-mediated alcohol metabolism was associated with enhanced ADH

and ALDH activities.

Ethanol is also oxidized by CYP2E1, an ethanol-inducible isoform of CYP-450 enzymes.

Activation of CYP2E1 by ethanol produces hydroxyl radicals that contribute to the toxic

effects of alcohol. Further, the activity of aniline hydroxylase, another CYP2E1-dependent

enzyme, was also higher, resulting in greater cytotoxicity 29)

. Many studies reported that

CYP2E1 and aniline hydroxylase were elevated after ethanol administration 30)

. Treatment

with inhibitors of CYP2E1, such as cyclosporine A and diallyl sulphide, have been shown to

reduce ethanol-induced liver injury 31)

. Our data showed that AAPC treatment significantly

decreased CYP2E1-dependent aniline hydroxylation in rats. This result suggests that the

suppression of CYP2E1 by AAPC in rats was an important aspect of the hepatoprotective

effect of AAPC against liver injury as induced by chronic alcohol exposure.

The AST and ALT serum marker enzymes are usually cytoplasmic and they leak into

the blood upon liver injury as a consequence of altered membranes permeability 32)

. Our

results indicated a significant elevation in the AST and ALT serum levels from the ethanol

control group, and the effects were markedly reduced when the rats were treated with AAPC.

Histological liver sample observations also strongly supported the release of

aminotransferases and pro-inflammatory cytokines by damaged hepatocytes as well as the

protective effect of AAPC. Chronic ethanol administration caused pathological changes to the

liver, including micro- and macro-vesicular steatosis and neutrophil infiltration. Those

alterations were attenuated by AAPC, with livers showing only mild steatosis and

inflammatory cell infiltration. Those results suggest that ethanol-induced hepatocyte damage

was largely prevented by AAPC treatment.

Ethanol-induced liver injury has been associated with increased lipid peroxidation, lipid

radical formation and decreased hepatic antioxidant protection 33)

. Oxidative stress is caused

by an imbalance in pro-oxidants and antioxidants, which cause a depletion or inactivation of


the antioxidant in hepatocytes, ultimately leading to necrosis and/or apoptosis 34,35)

. MDA is

an indicator of oxidative stress and a major by-product resulting from lipid peroxidation. In

addition, SOD, GSH-Px, GSH-Rd and catalase are the enzymes for which reduced activity is

associated with the accumulation of reactive free radicals, leading to deleterious effects 32)

.

Therefore, these oxidative stress parameters, including SOD, GSH-Px, GSH-Rd, catalase and

MDA, were examined in this study. Our results revealed a decreased tendency in the former

four and an increase in the last one from the model group. AAPC significantly increased the

SOD, GSH-Px, GSH-Rd and catalase activities and markedly decreased the MDA level,

indicating that AAPC inhibits lipid peroxidation and effectively recruits the anti-oxidative

defense system during ethanol-induced liver injury.

In addition to oxidative injury, abnormal cytokine metabolism is also a major feature of

alcoholic liver disease 36)

. The expression of TNF-α and IL-1β were found to be enhanced in

both animal models and patients with alcoholic liver disease 37)

. The critical role of TNF-α in

alcoholic liver disease has been demonstrated in TNFR1-deficient mice, which were

protected against alcohol-induced liver injury relative to wild type mice 38)

. Furthermore, the

neutralization of TNF-α by a specific antibody has been shown to attenuate hepatic necrosis

and inflammation caused by chronic alcohol exposure 39)

. In addition, myeloperoxidase

(MPO) is present in the cytoplasm of myeloid-derived cells, such as neutrophils, and has been

considered to be a reliable marker of tissue inflammation 40)

. In this study, AAPC treatment

significantly attenuated ethanol-induced TNF-α, IL-1β and MPO levels, suggesting that

AAPC protection against ethanol-induced liver damage might also be associated with the

inhibition of inflammatory mediator release.

One central component in the complex network of processes leading to the development

of alcoholic liver disease is the activation of Kupffer cells by endotoxin, which is released by

bacteria living in the intestine 41)

. Ethanol consumption can lead to increased endotoxin levels

in the blood and liver. When activated, Kupffer cells produce signaling molecules (i.e.,

cytokines) that promote inflammatory reactions as well as reactive oxygen species (ROS),

which can damage liver cells 42)

. In the present study, we found that the plasma endotoxin

level was enhanced by alcohol. AAPC treatment effectively suppressed the endotoxin level.

These data indicate that the protective effect of AAPC against alcohol-induced hepatotoxicity


is at least partly responsible for its ability to inhibit Kupffer cell activation by decreasing the

plasma endotoxin level.

Furthermore, there is growing evidence to suggest that endotoxin interacts with a

receptor complex consisting of proteins CD14 and TLR4. This interaction initiates a variety

of signaling cascades in the cell. One of these cascades, which involves interleukin 1

receptor-associated kinase (IRAK) and the associated proteins MyD88 and tumor necrosis

factor receptor-associated factor (TRAF), acts on a regulatory molecule called nuclear factor

kappa B (NF-κB), which is inactive in the cell if it is associated with inhibitory molecule

IκBα. In response to the signals initiated by endotoxin binding, IκBα is released from NF-κB,

leading to NF-κB activation 43,44)

. This activation in turn results in superoxide generation

through the NADPH oxidase complex and the production of inflammation mediators, such as

TNF-α, IL-1β, iNOS and COX-2, etc. 45)

. In the present study, the levels of TLR4, CD14 and

MyD88 were increased in alcohol-treated rats, and AAPC attenuated those increases.

Moreover, the nuclear translocation of NF-κB was markedly decreased by AAPC, reflecting

the decreased inflammatory responses, as demonstrated by significant decreases in TNF-α,

IL-1β, iNOS and COX-2 levels. Taken together, our results suggest that AAPC suppresses

the over-expression of TLR4, CD14 and MyD88, resulting in the suppression of NF-κB

nuclear translocation and subsequent pro-inflammatory mediators.

In conclusion, AAPC had a significant protective effect on chronic ethanol-induced liver

injury. The hepatoprotective action of AAPC is most likely mediated by its ability to

attenuate oxidative stress and inhibit the activation of Kupffer cells.

Conflict of interest

The authors do not have any conflicts of interest to disclose.

Acknowledgments

The authors gratefully acknowledge the financial support provided by the National

Natural Science Foundation of China (No. 81260674; 81260505), the Guangxi Natural

Science Foundation (No. 2013GXNSFAA019146; 2013GXNSFAA019150), and the

Foundation for the Guangxi Key Laboratory for Prevention & Treatment of Regional

High-Incidence Diseases (KFJJ2010-71; KFJJ2011-37).


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Figure legends

Fig.1. The chemical structure of AAPC.

Fig.2. The effects of AAPC on plasma alcohol and acetaldehyde concentrations. The results are

presented as the means ± SE. *P< 0.05 when compared with the ethanol group (model group). ND: no

alcohol was detected.

Fig.3. The effects of AAPC on hepatic alcohol metabolizing enzyme activities. The results are

presented as the means ± SE. *P< 0.05 when compared with the ethanol group.

Fig. 4. The effect of AAPC on CYP2E1 enzyme activity. The aniline hydroxylase was determined by

measuring p-aminophenol formation activities (A), and the microsomal protein levels were determined by

using the Bradford method with bovine serum albumin as the standard (B and C). I: normal group; II: 8

mg/kg AAPC control group; III: ethanol control group; IV-VI: ethanol plus 2, 4 or 8 mg/kg AAPC treated

groups. The results are presented as the means ± SE. *P< 0.05 when compared with the ethanol group.

Fig.5. The effects of AAPC on serum ALT and AST activities. The results are presented as the means ±

SE. *P< 0.05 when compared with the ethanol group.

Fig.6. The effect of AAPC on liver myeloperoxidase activity. The results are presented as the means ±



Fig.7. The effects of AAPC on liver TNF-α and IL-1β levels. The results are presented as the means ±


Fig.8. The effects of AAPC on antioxidant enzymes and lipid peroxidation. The results are presented

as the means ± SE. *P< 0.05 when compared with the ethanol group.

Fig.9. The effect of AAPC on plasma endotoxin level. The results are presented as the means ± SE. *P<

0.05 when compared with the ethanol group.

Fig.10. The effects of AAPC on the level of NF-κB (A), and the expression of TLR4, CD14, MyD88,

COX-2, and iNOS protein (B). I: normal group; II: 8 mg/kg AAPC control group; III: ethanol control

group; IV-VI: ethanol plus 2, 4 or 8 mg/kg AAPC treated groups. The results are presented as the means ±


Fig.11. Histomorphological examination (H&E staining, 100×). I: normal group; II: 8 mg/kg AAPC

control group; III: ethanol control group; IV-VI: ethanol plus 2, 4 or 8 mg/kg AAPC treated groups.


Fig. 1


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