Retraction: Asiatic Acid from Potentilla chinensis Attenuate Ethanol-Induced Hepatic Injury via Suppression of Oxidative Stress and Kupffer Cell Activation

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 cytochrome P450 (CYP)2E1 activity. Moreover, the over-expression of cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (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.

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 tumor growth factor (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.

MATERIALS AND METHODS

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).

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). Fraction 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). Fraction 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 Asiatic 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.

Fig. 1. The Chemical Structure of AAPC

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:

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 Zhang¹⁵⁾ as follows: 5.0 g/kg/d from 1 to 4 weeks, 7.0 g/kg/d from 5 to 8 weeks, 9.0 g/kg/d 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 body weight (b.w.), intravenously (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.

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.

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)

Cytochrome P450 (CYP)2E1 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 ethylenediamine tetraacetic acid (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⁾

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.

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 10062 M⁻¹ cm⁻¹. The protein content was determined by using bovine serum albumin as the standard.

Liver TNF-α and IL-1β Assays

Liver samples were disintegrated in 4 volumes of icecold radio immunoprecipitation assay (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 20000×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 enzyme-linked immunosorbent assay (ELISA) kits (Minneapolis, MN, U.S.A.). Liver lysates were adjusted to equal the protein concentrations after being quantified with the Coomassie blue (BioRad, Hercules, U.S.A.). The results were expressed as pg/mg protein.

Estimation of Antioxidant Enzyme and Lipid Peroxidation

Liver tissue was homogenized on ice with Tris–HCl (5 mmol/L containing 2 mmol/L EDTA, pH 7.4). 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.

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.

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 adaptor-inducing interferon-b (TRIF), cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS). NE-PER reagents (Pierce Biotechnology, Rockford, IL, U.S.A.) 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 nuclear factor (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, U.S.A.), COX-2 (Cayman Chem, Ann Arbor, MI, U.S.A.), iNOS (Santa Cruz Biotechnology, Inc.), and NF-κB/p65 (Lab Vision, Fremont, CA, U.S.A.). The signals were normalized to that of β-actin (Sigma Chemical Co.) or lamin B1 (Abcam, Cambridge, MA, U.S.A.), 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, U.S.A.).

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.

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 Turkey’s test for post hoc multiple comparisons. The data are presented as the means±S.E. A p-value <0.05 was considered to be statistically significant.

RESULTS

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).

Fig. 2. The Effects of AAPC on Plasma Alcohol and Acetaldehyde Concentrations

The results are presented as the means±S.E. * p<0.05 when compared with the ethanol group (model group). ND: no alcohol was detected.

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.

Fig. 3. The Effects of AAPC on Hepatic Alcohol Metabolizing Enzyme Activities

The results are presented as the means±S.E. * p<0.05 when compared with the ethanol group.

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.

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±S.E. * p<0.05 when compared with the ethanol group.

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.

Fig. 5. The Effects of AAPC on Serum ALT and AST Activities

The results are presented as the means±S.E. * p<0.05 when compared with the ethanol group.

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).

Fig. 6. The Effect of AAPC on Liver Myeloperoxidase Activity

The results are presented as the means±S.E. * p<0.05 when compared with the ethanol group.

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).

Fig. 7. The Effects of AAPC on Liver TNF-α and IL-1β Levels

The results are presented as the means±S.E. * p<0.05 when compared with the ethanol group.

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).

Fig. 8. The Effects of AAPC on Antioxidant Enzymes and Lipid Peroxidation

The results are presented as the means±S.E. * p<0.05 when compared with the ethanol group.

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.

Fig. 9. The Effect of AAPC on Plasma Endotoxin Level

The results are presented as the means±S.E. * p<0.05 when compared with the ethanol group.

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.

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±S.E. * p<0.05 when compared with the ethanol group.

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).

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. 11 I). There were no pathological changes in AAPC-only control rats (Fig. 11 II). 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. 11 III). AAPC supplementation ameliorated the deleterious effects of chronic ethanol exposure on the liver, as seen in the diminished fatty infiltration, lipid change and necrosis (Figs. 11 IV–VI).

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.

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.

Acknowledgment

The authors gratefully acknowledge the financial support provided by the National Natural Science Foundation of China (Nos. 81260674, 81260505), the Guangxi Natural Science Foundation (Nos. 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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