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Acanthoic acid, a diterpene in Acanthopanax koreanum, protects acetaminophen-induced hepatic toxicity in mice.

ABSTRACT

The protective effect of a diterpenoid acanthoic acid (AA) isolated from Acanthopanax koreanum Nakai was investigated in acetaminophen (APAP)-induced hepatic toxicity. Drug-induced hepatotoxicity induced by an intraperitoneal (i.p.) injection of 300 mg/kg (sub-lethal dose) of APAP. Pretreatment with AA (50 and 100 mg/kg) orally 2 h before the APAP administration attenuated the APAP-induced acute increase in serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activites, replenished the depleted hepatic glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) activities, decreased malondialdehyde (MDA) level and considerably reduced the histopathological alterations in a manner similar to silymarin (Sily). Immunohistochemical analyses also demonstrated that AA could reduce the appearance of necrosis regions as well as caspase-3 and hypoxia inducible factor-1[alpha] (HIF-1[alpha]) expression in liver tissue. Our results indicated that AA protected liver tissue from the oxidative stress elicites by APAP-induced liver damage and suggestes that the hepatic protection mechanism of AA would relate to antioxidation and hypoxia factor on APAP-induced hepatotoxicity.

ARTICLE INFO

Keywords:

Acanthopanax koreanum

Acanthoic acid

Acetaminophen

Hepatotoxicity

Antioxidation

Hypoxia inducible factor-1[alpha]

Introduction

APAP in excessive doses has a potential to cause a fatal hepatotoxicity due to hepatic centrilobular necrosis and has become an important problem (Larson et al. 2005). APAP is metabolized by a cytochrome P450 system to N-acetyl-p-benzo-quinoneimine (NAPQI), a highly reactive metabolite that depletes the intracellular pool of GSH (Potter and Hinson 1986). Normally, the free radical level can neutralize, metabolize, or subtract the toxic effects by free radical scavengers. Once the balance is destroyed, these radicals attack macromolecule, then result in histiocytes damage and induce oxidative stress. In many forms of liver injury, including ischemia and fulminant hepatic failure, TNF-[alpha] signaling appears to play an important role (Malhi et al. 2006). Hypoxia-inducible factor (HIF) participates hypoxia genetic transcription regulation, while HIF-1[alpha] expression and activity are tightly regulated by oxygen concentration (Semenza 2002). In addition to hypoxia, oxidative stress may promote HIF-1[alpha] induction (Wu et al. 2008).

Acanthoic acid (AA), (-)-primara-9(11),15-dien-19-oic acid, is a pimaradiene diterpene (Fig. 1) isolated from the root bark of Acanthopanax koreanum Nakai. It was found that AA suppressed the production of interleukin-1 (IL-1), TNF-[alpha], and interleukin-8 (IL-8) (Kang et al. 1996; Park et al. 2004). In our previous research, we found that AA protected against D-GalN/LPS-induced fulminant liver failure at least in part by a mechanism associated with the down-regulation of TNF-[alpha] secretion (Nan et al. 2008). In this research we have also investigated the protection of AA on APAP-induced hepatotoxicity.

[FIGURE 1 OMITTED]

Materials and methods

Plant material

A. koreanum were provided by Susin Ogapi Co., Cheonan, Korea and identified by Dr. Y. H. Kim, College of Pharmacy, chungnam National University. A voucher specimen (KRIBB96076) was deposited in the Herbarium of the Korea Research Institute of Bioscience and Biotechnology, Korea.

Isolation of AA

AA was isolated from the roots of A. koreanum as described previously (Kim et al. 1988). Briefly, the roots of A. koreanum were extracted with methanol under reflux for 15 h. After evaporating solvent, the residue suspended in distilled water was extracted with methylene chloride. The methylene chloride soluble fraction was repeatedly chromatographed on a silica gel column chromatography eluted with hexane-ethylacetate gradient system (20:1 to 1:1). Finally, AA (0.82% recovery: > 95% purity) (Fig. 1) was obtained by a preparative HPLC using a J'sphere ODS-H80 column (YMC Co., LTD, Kyoto, Japan) eluted with methanol.

Materials

APAP ([greater than or equal to] 98.0% HPLC) were purchased from Sigma-Aldrich Inc. Silymarin was purchased from Aldrich Chemical Co., Inc. (Milwaukee, USA). Detection kits for glutathione and malondialdehyde were purchased from Oxis International, Inc. (Portland, OR). Nanjing Jiancheng Bioengineering institute supplied SOD, CAT and GSH-Px Kit. Mouse TNF ELISA Kit was purchased from BD Biosciences (San Diego, CA). Caspase-3 p20 (sc-1226) and HIF-1[alpha] (sc-53546) monoclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The ALT and AST Reagent Strips were purchased from Arkray Incorporated (Kyoto, Japan).

Animals

All studies were conducted in 6-7 week-old male C57BL/6 mice weighing 21-24 g. Mice were maintained under a controlled ambient temperature between 21 and 23 [degrees]C with 12 h light and dark cycles and relative humidity of 50%. Mice were acclimated for 7 days and had free access to normal chow and water ad libitum prior to initiation of any procedures. Animal experiments were carried out under the "Guiding Principles in the Use of Animals in Toxicology" adopted by the Society of Toxicology (USA) in July 1989 and revised in March 1999. The Animal Care Committee of our institution approved the present study.

Experimental design

The mice were randomly assigned into six experimental groups. Each group contained 8 mice, including normal group, AA-100 group, APAP group, APAP+AA-50 group, APAP+AA-100 group and APAP+Sily-100 group. Normal group was administrated with sterile saline only. AA-100 group was administrated only with AA (100 mg/kg), except for normal chow. APAP+AA-50 group, APAP+AA-100 group and APAP+ Sily-100 groups were administrate mice with AA (50 and 100 mg/kg) or silymarin (100 mg/kg) for three consecutive days, which dose consult reference (Park et al. 2004). Two hours after the last AA or Silymarin administration, mice were injected with APAP (300 mg/kg). The animals were sacrificed 12 h after APAP administration and blood was collected from the carotid artery 2 h after APAP injection to measure TNF-[alpha] levels and 12 h after APAP injection to measure serum ALT and AST activities. Blood samples were allowed to coagulate at 4 [degrees]C for 30 min. Serum was then separated by centrifugation at 4 [degrees]C, RCF 1990 x g. The same lobe of liver in each animal were excised and immersed in neutral buffered formaldehyde for histopathological and immunohistochemical examinations and the other kept at -80 [degrees]C for analyses of MDA level, GSH, SOD, CAT and GSH-Px activies.

In survival experiments, mice were divided and administrated as above mentioned, administrated with AA or silymarin 2 h before APAP, while intoxicated with 500 mg/kg (lethal dose) of APAP, and observed the mortality in 24 h after administration of APAP. Each group contained 16 mice.

Serum enzyme and TNF-[alpha] assay

Serum ALT and AST activities 12 h after APAP injection were measured by an Autodry chemistry analyzer (Spotchem SP4430, Arkray, Kyoto, Japan). TNF-[alpha] level was determined 2 h after APAP injecton with a TNF-[alpha] ELISA kit (BD Bioscience, San Diego, CA) according to manufacturer's instructions.

Antioxidation system determination

The frozen liver slices obtained after 12 h APAP administration were washed in ice-cold EDTA solution (0.02 mol/l), blotted, dissected to remove connective tissues, weighed, and homogenized with 10% saline and then determined MDA level, GSH, total SOD, CAT, GSH-Px activities according to manufacturer's instructions. Liver protein was determined by Coomassie brilliant blue. Results were expressed in MDA nmol/mg prot, GSH mg/g prot, SOD U/mg prot, CAT U/mg prot and GSH-Px U/L.

Immunohistochemistry

In immunohistochemistry staining, briefly, paraffin section were deparaffinaged and hydrated, and then followed with antigen reparation. After blocking with endogenous peroxydase by 3% hydrogen dioxide, sections were incubated with goat anti-caspase-3 (1:50) or goat anti-HIF-1[alpha]monoclonal antibody (1:100) respectively, and then followed by Max Vision TM kit, DAB kit. Finally, re-stained with hematoxylin, mounting, and assessed by light microscopy.

Statistical analysis

All values were expressed as means [+ or -]SD. The results were evaluated by one-way ANOVA and Tukey's multiple comparison tests. Statistically significant differences between groups were defined as P values less than 0.05. Calculations were performed with the GraphPad Prism program (Graphpad Software, Inc. San Diego, USA).

Results

When mice were intoxicated with 500 mg/kg (lethal dose) of APAP, 17% of animals survived after 24 h. However, as mice were administrated with AA (50 and 100 mg/kg), or silymarin (100 mg/ kg) 2 h before APAP (500 mg/kg) injection, the survival rate was increased to 52%, 74%, and 57% at 24 h, respectively (Fig. 2). The administration of APAP induced markedly increase in serum ALT and AST activities. APAP+AA-50, APAP+AA-100 and APAP+Sily-100 groups reduced serum ALT activities to 138 [+ or -] 54, 102 [+ or -] 46 and 225 [+ or -] 57 IU/L; reduced serum AST activities to 119 [+ or -] 50, 94 [+ or -] 35 and 195 [+ or -] 49 IU/L respectively. APAP-induced increase in the TNF-[alpha] level. In APAP+AA-50, APAP+AA-100 and APAP+ Sily-100 groups, TNF-[alpha] levels were reduced significantly (80.0 [+ or -] 14.6, 65.9 [+ or -] 23.2, and 93.9 [+ or -] 19.4 pg/ml, P < 0.001, respectively) compared to APAP group (Fig. 3). As shown in Table 1, in the liver of APAP-induced group, tissue lipid peroxidation levels as evidenced by MDA determination increased significantly as compared to normal group, however, the activities of GSH, SOD, CAT and GSH-Px in liver decreased. In the APAP+AA-50, APAP+AA-100 and APAP+ Sily-100 groups, GSH, SOD, CAT and GSH-Px activities were remarkably increased and MDA level was decreased in comparison with the APAP group, except for SOD activity of APAP+AA-50 group. AA-100 group provided similar all parameters levels with the normal group. Immunohistochemistry results demonstrated that caspase-3 expression in APAP group was observed around central vein (Fig. 4A), which focused on endochylema and positive staining and presented in pale brown or brown. In contrast to the APAP group, caspase-3 expression decreased and even recovered to normal level APAP+AA-50, APAP+AA-100 and APAP+Sily-100 groups (Fig. 4D-F). Less caspase-3 positive staining expressed in normal and AA-100 groups (Fig. 4B, C). HIF-1[alpha] expression in APAP group focused on nucleus and endochylema, especially in endochylema, and positive staining presented in tan (Fig. 5A). However, there was no HIF-1[alpha] expression in normal and AA-100 groups (Fig. 5B, C). In APAP+AA-50, APAP+AA-100 and APAP+Sily-100 groups, the immunohistochemical labeling of HIF-1[alpha] decreased (Fig. 5D-F) and this decrease presented dose-effect relationship obviously.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]
Table 1
Protective effect of acanthoic acid (AA) against hepatotoxicity induced
by acetaminophen (APAP) in mice

 Treatments        MDA (nmol/mgprot)            GSH (mg/gprot)

Normal           1.95 [+ or -] 0.70        498.63 [+ or -] 46.16
AA-100           1.93 [+ or -] 0.71 ***    500.47 [+ or -] 99.63 ***
APAP             8.90 [+ or -] 0.82 #      213.61 [+ or -] 30.45 #
APAP+AA-50       7.24 [+ or -] 0.56 ***    417.25 [+ or -] 20.49 ***
APAP+AA-100      5.05 [+ or -] 0.66 ***    431.19 [+ or -] 32.42 ***
APAP+Sily-100    5.84 [+ or -] 0.52 ***    401.36 [+ or -] 44.2l ***

 Treatments          SOD (U/mgprot)             CAT (U/mgprot)

Normal           76.28 [+ or -] 8.88        54.39 [+ or -] 6.37
AA-100           74.93 [+ or -] 9.25 ***    54.86 [+ or -] 3.13 ***
APAP             61.47 [+ or -] 5.18 #      38.14 [+ or -] 5.24 #
APAP+AA-50       69.22 [+ or -] 3.53        45.71 [+ or -] 2.97 *
APAP+AA-100      73.46 [+ or -] 2.40 **     52.92 [+ or -] 4.22 ***
APAP+Sily-100    73.13 [+ or -] 2.04 **     52.58 [+ or -] 4.42 ***

 Treatments            GSH-Px (U/L)

Normal           908.46 [+ or -] 83.97
AA-100           899.84 [+ or -] 76.38 ***
APAP             639.23 [+ or -] 121.56 #
APAP+AA-50       783.39 [+ or -] 99.55 *
APAP+AA-100      847.13 [+ or -] 86.38 ***
APAP+Sily-100    850.35 [+ or -] 77.29 ***

* P < 0.5, ** P < 0.01, *** P < 0.001, significantly different
from APAP group.
# P < 0.001, significantly different from normal group.


[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Discussion

The aim of this study was to investigate the protective effect and the protective mechanism of AA on the APAP-induced hepatotoxicity. Intraperitoneal injection of APAP caused increases in serum levels of ALT, AST, TNF-[alpha], a depletion of hepatic GSH. However, AA increased the survival rates of mice intoxicated with a lethal dose of APAP and improved all the biological parameters including ALT, AST, TNF-[alpha], MDA, GSH SOD, CAT, GSH-Px, suggesting that AA was able to protect mice from the APAP-induced hepatotoxicity.

NAPQI is the reactive metabolite product caused by APAP-induced hepatic toxicity, leads to GSH depletion and covalently binds to cysteine residues on proteins, which results in lipid peroxidation reaction. Reactive oxygen species are normally detoxified by antioxidation system, consisted with non enzyme antioxidation system and antioxidase. SOD, a special antioxidase, converts superoxide anion into hydrogen peroxide, which is detoxified by CAT and GSH-Px. And GSH-Px, making GSH as its substrate, removes redundant free radicals and peroxidate in coordination with SOD. In our study, AA remarkably reduced serum ALT and AST levels, restored the GSH level from APAP-induced depletion, inhibited the APAP-induced hepatic MDA production, decreased TNF-[alpha] level, and increased SOD, CAT and GSH-Px activities. However, AA abrogated the TNF-[alpha] release that would be coincided with the reduction of APAP-induced oxidative injury.

Caspase-3 is one of the caspases that are responsible for apoptosis. Our result demonstrated that caspase-3 protein expression in the hepatocytes would play an apoptosis effect in APAP-induced liver injury and AA could inhibit the expression of caspase-3. Our data indicated AA could alleviate oxidation stimulation, which related with oxidative stress might cause induction of HIF-1[alpha] Thus we should notice the same tendency in other antioxidases alternation, oxidative stress might be a potential role in APAP-mediated induction of HIF-1[alpha] The finding that AA and silymarin blocked HIF-1[alpha] induction would show that HIF-1[alpha] induction occurred because of the toxic metabolite NAPQI. This metabolite detoxified by GSH leading to its depletion. AA may increase GSH synthesis and recover antioxidase (Gao et al. 2002), therefore AA would inhibit the expression of HIF-1[alpha] by preventing the metabolite from depleting GSH and the happening of the oxidation.

In summary, our results indicates that AA protected liver tissue from the oxidative stress elicited by APAP-induced liver damage and suggestes that the hepatic protection mechanism of AA may be due to antioxidation, which refers to hypoxia factor. Further study will be required to confirm that HIF-1[alpha] level is affected by the degree of antioxidation.

Acknowledgements

This work was supported in part by a research grant (PF06204-00 to JJ Lee) from Plant Diversity Research Center of 21st Frontier Research Program funded by the Korean Ministry of Science and Technology and by a research grant (No. 30660225 to JX Nan) from National Natural Science Foundation of China.

References

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Kang, K.S., Kim, Y.H., Lee, C.S., Lee, J.J., Choi, I., Pyun, K.H., 1996. Suppression of interleukin-1 and tumor necrosis factor-alpha production by acanthoic acid, (-)-pimara-9(11), 15-dien-19-oic acid, and its antifibrotic effects in vivo. Cell. Immunol. 170, 212-221.

Kim, Y.H., Chung, B.S., Sankawa, U., 1988. Pimaradiene diterpenes from Acantho-panax koreanum. J. Nat. Prod. 51, 1080-1083.

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Nan, J.X., Jin, X.J., Lian, L.H., Cai, X.F., Jiang, Y.Z., Jin, H.R., Lee, J.J., 2008. A diterpenoid acanthoic acid from Acanthopanax koreanum protects against D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure in mice. Biol. Pharm. Bull. 31, 738-742.

Park, E.J., Zhao, Y.Z., Young, H.K., Lee, J.J., Sohn, D.H., 2004. Acanthoic acid from Acanthopanax koreanum protects against liver injury induced by tert-butyl hydroperoxide or carbon tetrachloride in vitro and in vivo. Planta Med. 70, 321-327.

Potter, D.W., Hinson, J.A., 1986. Reactions of N-acetyl-p-benzoquinone imine with reduced glutathione, acetaminophen and NADPH. Mol. Pharmacol. 30, 33-41.

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Wu, Y.L., Dong, M.P., Han, X.H., Nan, J.X., 2008. Protective effects of salidroside against acetaminophen induced toxicity in mice. Biol. Pharm. Bull. 31, 1523-1529.

* Corresponding author. Tel.: +86433 266 0603; fax: +86433 273 2456. E-mail addresses: jjlee@kribb.re.kr (J. Joon Lee), jxnanybu@gmail.com (J.-X. Nan).

(1) Co-corresponding author. Tel.: +8242 8604360; fax: +8242 8604595.

Yan-Ling Wu (a), Ying-Zi Jiang (a), Xue-Jun Jin (b), Li-Hua Lian (a), Juan-Yu Piao (a), Ying Wan (a), Hong-Ri Jin (b), Jung Joon Lee (b), (1), Ji-Xing Nan (a), *

(a) Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji 133002 Jilin Province, China

(b) Molecular Cancer Research Center, Korea Research Institute of Bioscience and Biotechnology; 52 Uheundong, Yuseonggu, Daejeon, 305-333, Korea

doi:10.1016/j.phymed.2009.07.011
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Title Annotation:Short Communication
Author:Wu, Yan-Ling; Jiang, Ying-Zi; Jin, Xue-Jun; Lian, Li-Hua; Piao, Juan-Yu; Wan, Ying; Jin, Hong-Ri; Le
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:1USA
Date:May 1, 2010
Words:2833
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