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Journal of Ethnopharmacology 141 (2012) 1071– 1076

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

journa l h o me page: www.elsev ier .com/ locate / je thpharm

thnopharmacological communication

orean red ginseng and its primary ginsenosides inhibit ethanol-inducedxidative injury by suppression of the MAPK pathway in TIB-73 cells

ye-Min Parka,b,1, Shang-Jin Kima,b,c,1, A-Reum Munb, Hyeon-Kyu Goa,b, Gi-Beum Kima,b,c,ung-Zoo Kimb, Seon-Il Jangd, Sei-Jin Leee, Jin-Shang Kima,c, Hyung-Sub Kanga,b,c,∗

Department of Pharmacology and Toxicology, College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Republic of KoreaCenter for Healthcare Technology Development, Chonbuk National University, Jeonju 561-756, Republic of KoreaKorean Zoonoses Research Institute, Chonbuk National University, Jeonju 561-756, Republic of KoreaSchool of Alternative Medicine & Health Science, College of Alternative Medicine, Jeonju University, Jeonju 560-759, Republic of KoreaKorea Basic Science Institute Jeonju Center, Jeonju 561-756, Republic of Korea

r t i c l e i n f o

rticle history:eceived 17 September 2011eceived in revised form 12 March 2012ccepted 18 March 2012vailable online 26 March 2012

eywords:thanolinsenoside Rg3insenoside Rh2epatocyte

a b s t r a c t

Ethnopharmacological relevance: Panax ginseng (P. ginseng) is one of the most widely used medicinal plantsdue to its wide spectrum of medicinal effects. Among the currently available Panax ginseng products, Koreared ginseng (KRG) has been shown to exhibit a variety of antioxidative and hepatoprotective action.Aim of the study: Our aim was to investigate the effects of KRG and its primary ginsenosides (Rg3 andRh2) on EtOH-induced injury to mouse hepatocytes (TIB-73).Materials and methods: We investigated the effects of KRG and its primary ginsenoside on EtOH-inducedinjury to TIB-73 cells and evaluated MAPKs signals as a possible mechanism of action. Hepatocytic injurywas evaluated by biochemical assays as cell viability, lactate dehydrogenase (LDH), aspartate amino-transferase (AST), ROS and mitochondria membrane potential (MMP) level in TIB-73 cells. The levels ofMAPK activation were analyzed by Western blots.

xidative injuryAPK

Results: The results showed that exposure of EtOH to TIB-73 cells led to cell death and membrane damage,accompanied by a decrease in cell viability, MMP, and Mg2+ concentrations, but an increase in LDH, AST,ROS and MAPK activation. KRG and its primary ginsenosides reduced EtOH-induced generation of ROSand the activation of ERK and JNK, and increased Mg2+ concentrations.Conclusion: These results suggest that KRG and its primary ginsenosides inhibit EtOH-induced oxidative

he M

injury by suppression of t

. Introduction

Ethanol (EtOH) induces severe hepatic cell damage by inducinghe alteration of several biological functions (Lieber, 1991). Oxida-ive stress has been shown to be one of the main components intOH-induced cell injury and apoptosis, resulted from the exces-ive exposure to reactive oxygen species (ROS) (Koop, 2006). AntOH-induced increase in ROS has been shown to be involved inediating several signal transduction pathways leading to injury

nd apoptosis in hepatocytes (Lee and Shukla, 2005).

Panax ginseng C.A. Meyer reportedly has various biological

ffects including antioxidant activity (Abdel-Wahhab and Ahmed,004). Korean red ginseng (KRG, Ginseng Radix Rubra) is made by

∗ Corresponding author at: Department of Pharmacology and Toxicology, Collegef Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Republic oforea. Tel.: +82 63 270 3883; fax: +82 63 270 3780.

E-mail address: kang-hs@jbnu.ac.kr (H.-S. Kang).1 These authors equally contributed to this study.

378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2012.03.038

APK pathway in TIB-73 cells.© 2012 Elsevier Ireland Ltd. All rights reserved.

steaming and drying fresh ginseng, which suggests that chemicaltransformations of active physiological properties occur involvingginsenosides, polysaccharides, peptides, polyacetylenic alcoholsand fatty acids (Park, 1996). Ginsenosides are believed to be respon-sible for the pharmacological actions of Panax ginseng, whichinclude anti-neoplastic, anti-stress, anti-inflammatory, antioxidantand hepatoprotective activities (Lee et al., 2006; Cho et al., 2006).Several studies have identified specific ginsenosides in KRG thatare not usually found in Panax ginseng. The major component ofPanax ginseng is ginsenoside Rb1, otherwise the major componentof KRG is ginsenoside Rg3 (GRg3) (Kitagawa et al., 1983). GRg3is reported to reduce hepatotoxicity induced by lipopolysaccha-ride (Kang et al., 2007) and has antioxidant and neuroprotectiveeffects (Tian et al., 2005). GRg3 can be metabolized to ginsenosideRh2 (GRh2) by intestinal microflora (Bae et al., 2002). However,the preventive mechanism of KRG on EtOH-induced liver injury

remains unclear. Therefore, we examined the protective poten-tial of KRG and its primary ginsenosides (GRg3 and GRh2) againstEtOH-induced oxidative injury in mouse hepatocytes (TIB-73 cellline).

1 opharmacology 141 (2012) 1071– 1076

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Fig. 1. The effects of EtOH with/without KRG and its primary ginsenosides (GRg3and GRh2) on cell viability of TIB-73 cells. The data are reported as the mean ± SEM

072 H.-M. Park et al. / Journal of Ethn

. Materials and methods

.1. Preparation of KRG extracts and ginsenosides

KRG extracts were provided by Korea Ginseng CorporationDaejeon, Korea) in a standardized and reproducible process. KRGxtracts were extracted from red ginseng manufactured from freshoots of 6-year-old Panax ginseng plants whose botanical identityad been verified. Red ginseng was made by steaming fresh ginsengt 90–100 ◦C for 3 h and then drying at 50–80 ◦C. Red ginseng wasxtracted seven times with 10 volumes of distilled water at 85 ◦C for

h followed by cooling. KRG extract contained major ginsenoside-g1: 0.52 mg/g, -Rb1: 4.03 mg/g, -Rg3(s): 2.89 mg/g, -Re: 1.18 mg/g,Rc: 1.98 mg/g, -Rb2: 1.97 mg/g, -Rd: 1.51 mg/g, and other minorinsenosides.

GRg3 and GRh2 were purchased from Chromadex (Irvain, CA).o use KRG extract aseptically, it was first dissolved in phosphateuffered solution at a concentration of 100 mg/ml, and then filteredith 0.2 �m microfiltration apparatus (Millipore, Billerica, MA).

.2. Cell culture

TIB-73 cells were obtained from the American Type Cul-ure Collection (ATCC, Manassas, USA) and grown in Dulbecco’s

odified Eagle’s medium supplemented with 10% fetal bovineerum (Sigma–Aldrich, St. Louis, USA), 5 mM l-glutamine, 50 U/mlenicillin and 50 �g/ml streptomycin in a humidified 5% CO2–95%ir environment at 37 ◦C. 2,7′-Dichlorodihydrofluorescin diac-tate (DCFH-DA) was purchased from Molecular Probes (Eugene,SA). The 4′,6-diamidino-2-phenylindole (DAPI) and 5,5′,6,6′-

etrachloro-1,1′,3,3′ tetraethylbenzimidazolyl-carbocyanineodide (JC-1) were purchased from Enzo Life Sciences (Plymouth

eeting, USA).

.3. Cell viability assay

After treatment with/without EtOH, KRG, GRg3 or GRh2or 24 h, cell viability of TIB-73 cells was detected by a 3-(4,5-imethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)ssay, as described previously (Jeon et al., 2010).

.4. Determination of lactate dehydrogenase (LDH) and aspartateransaminase (AST) release

After the treatment mentioned above, LDH level in cultureedium were measured by using cytotoxicity detection kit (Takara

io Inc., Shiga, Japan). The culture medium was then analyzed forST levels using an Idexx Vettest 8008 Chemistry Analyzer (IDEXXab Inc., Westbrook, USA).

.5. Nuclear staining with DAPI

After the treatment mentioned above, TIB-73 cells were fixedith 70% EtOH and counter-stained with DAPI mounting medium

or 15 min at room temperature. Apoptotic nuclei were then visu-lized under a fluorescence microscope (IX-81; Olympus Corp.,okyo, Japan).

.6. Mitochondrial membrane potential (MMP) assessment byC-1 staining

After the treatment mentioned above, TIB-73 cells werencubated with 10 �g/ml JC-1 for 10 min at 37 ◦C. The fluores-ence was measured using a spectrophotometer with 550 nm

(n = 8) for each group. **p < 0.01, ***p < 0.005: Bonferroni’s post hoc test vs. control;#p < 0.5, ###p < 0.001: Bonferroni’s post hoc test vs. EtOH.

excitation/600 nm emission for red fluorescence, and 485 nm exci-tation/535 nm emission for green fluorescence.

2.7. Measurement of intracellular ROS generation

After the treatment mentioned above, TIB-73 cells were treated

with 10 �M DCFH-DA for 30 min. DCFH fluorescence was deter-mined using a spectrophotometer at excitation and emissionwavelengths of 488 nm and 515 nm, respectively.

H.-M. Park et al. / Journal of Ethnopharmacology 141 (2012) 1071– 1076 1073

Fig. 2. The effects of EtOH with/without KRG and its primary ginsenosides (GRg3 and Goptical microscopy (A). Fluorescent photographic assessment of apoptosis in TIB-73 cells

Fig. 3. The effects of EtOH with/without KRG and its primary ginsenosides (GRg3and GRh2) on LDH, AST and MMP levels in TIB-73 cells. The levels of LDH (A) andAST (B) released into the medium and MMP (C) levels in TIB-73 cells were detected.The data are reported as a mean ± SEM (n = 8) for each group. **p < 0.01, ***p < 0.005:Bonferroni’s post hoc test vs. control; #p < 0.5, ###p < 0.001: Bonferroni’s post hoc testvs. EtOH.

Rh2) on morphological changes in TIB-73 cells. Cell morphology was assessed by stained with DAPI (×200) (B).

2.8. Magnesium measurements

After the treatment mentioned above, magnesium concentra-tion was measured by atomic absorption spectrophotometry, asdescribed previously (Jeon et al., 2009). Briefly, harvested TIB-73cells were homogenized immediately and microcentrifuged for10 min at 10,000 rpm. The supernatant was used to measure with anatomic absorption spectrophotometer (Analab 9200, Seoul, Korea)at a wavelength of 285.2 nm.

2.9. Western blot analysis of the mitogen-activated proteinkinases (MAPK)

After the treatment mentioned above, the levels of MAPK acti-vation were analyzed by Western blots, as described previously(Jeon et al., 2010). All primary antibodies were purchased fromSigma. Representative Western blots were quantified with a Bio-Rad ChemiDoc XRS (Bio-Rad, Hercules, USA).

2.10. Statistical analysis

Results are expressed as means ± the standard error of the mean(SEM). The data were analyzed by Student’s t-test or analysis ofvariance (ANOVA) with the Bonferroni post hoc test, as appropriate,using Prism 5.03 (GraphPad Software Inc., San Diego, USA). A p-value <0.05 was considered significant.

3. Results

3.1. Effect of EtOH with/without KRG or its primary ginsenosideson the viability of TIB-73 cells

As shown in Fig. 1, 1% EtOH induced a dramatic decrease inthe viability of TIB-73 cells to 71 ± 4% of the control (p < 0.001).Co-treatment with EtOH and KRG attenuated the EtOH-induceddecrease in cell viability (132 ± 6% at 100 �g/ml as a percentageof EtOH). Co-treatment with EtOH and GRg3 attenuated the EtOH-induced decrease in cell viability (112 ± 5% at 10 �M as a percentageof EtOH). Co-treatment with EtOH and GRh2 (1–30 �M) attenuatedthe EtOH-induced decrease in cell viability (129 ± 6% at 10 �M as apercentage of EtOH).

In transmitted light images (Fig. 2A), EtOH led to a significantreduction in the number of cells, whereas co-treatment with EtOHand KRG, GRg3 or GRh2 recovered the EtOH-induced decrease in

cell numbers. Staining of cells with DAPI (Fig. 2B) revealed an EtOH-related increase in the number of apoptotic cells and co-treatmentwith EtOH and KRG, GRg3 or GRh2 inhibited the EtOH-inducedapoptotic changes.

1074 H.-M. Park et al. / Journal of Ethnopharmacology 141 (2012) 1071– 1076

Fig. 4. The effects of EtOH with/without KRG and its primary ginsenosides (GRg3and GRh2) on the generation of intracellular ROS and magnesium in TIB-73 cells.The involvement of ROS in EtOH-induced cell death was determined by measuringthe levels of ROS in TIB-73 cells. The cells were incubated with 10 �M DCFH-DA. Thefluorescence intensity was evaluated using fluorescent microscopy (×200) (A) anda spectrophotometer (B). The magnesium level was measured by atomic absorptions*B

3oc

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Fig. 5. The effects of EtOH with/without KRG and its primary ginsenosides (GRg3and GRh2) on MAPK expression in TIB-73 cells. The amount of MAPK was measuredby Western blot analysis. This image shows typical changes in the MAPK levels afterexposure to EtOH with/without KRG and its primary ginsenosides (GRg3 and GRh2)

pectrophotometry (C). The data are reported as a mean ± SEM (n = 8) for each group.**p < 0.005: Bonferroni’s post hoc test vs. control; #p < 0.5, ##p < 0.1, ###p < 0.001:onferroni’s post hoc test vs. EtOH.

.2. Effect of EtOH with/without KRG or its primary ginsenosidesn LDH, AST and the mitochondria membrane potential of TIB-73

ells

EtOH induced an increase in LDH released from TIB-73 cellsFig. 3A); 111 ± 1% of the control (p < 0.001). Co-treatment with

(A). The blots were quantified by scanning densitometry. The data are reported asa mean ± SEM (n = 4) for each group (B). *p < 0.05, ***p < 0.005: Bonferroni’s post hoctest vs. control; ##p < 0.1, ###p < 0.001: Bonferroni’s post hoc test vs. EtOH.

EtOH and KRG or its primary ginsenosides attenuated the EtOH-induced increase in LDH release (KRG; 91 ± 1% of the LDHrelease observed with EtOH treatment alone, GRg3; 85 ± 2%,GRh2; 86 ± 1%). As shown in Fig. 3B, EtOH induced an increasein AST release by TIB-73 cells (0.37 ± 0.02 U/mg protein), whilecontrol conditions resulted in 0.25 ± 0.01 U/mg protein release.Co-treatment with EtOH and KRG or its primary ginsenosides atten-uated the EtOH-induced increase in AST (KRG; 0.28 ± 0.01 U/mgprotein, GRg3; 0.28 ± 0.01 U/mg protein, GRh2; 0.20 ± 0.03 U/mgprotein). As shown in Fig. 3C, 1% EtOH induced a decrease inthe ratio of JC-1 (87 ± 3% of the control). Co-treatment with EtOHand KRG or its primary ginsenosides attenuated the EtOH-induceddecrease in the ratio of JC-1 (KRG; 117 ± 4% of the ratio observedwith EtOH treatment alone, GRg3; 120 ± 3%, GRh2; 119 ± 3%).

3.3. Effect of EtOH with/without KRG or its primary ginsenosideson the generation of ROS and magnesium concentrations of

TIB-73 cells

Fig. 4A and B shows that EtOH increased the level of ROS pro-duction (123 ± 5% of the control). Co-treatment with EtOH and KRG

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r its main ginsenosides attenuated the EtOH-induced increase inOS (KRG; 80 ± 2% of the levels observed under EtOH treatmentlone, GRg3; 78 ± 3%, GRh2; 79 ± 3%).

As shown in Fig. 4C, EtOH significantly decreased the mag-esium concentration ([Mg2+]); 87 ± 2% of the control. ThetOH-induced [Mg2+] decrease was inhibited significantly by co-reatment with EtOH and KRG or its primary ginsenosides (KRG;2 ± 2% of the control, GRg3; 93 ± 1%, GRh2; 101 ± 2%).

.4. Effect of EtOH with/without KRG or its primary ginsenosidesn the MAPK expression of TIB-73 cells

As shown in Fig. 5, EtOH activated extracellular signal regulatedinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAPKn hepatocytes. Using densitometry, the percent variations in thetOH treated group compared to the control group (100%) wereetermined to be 177 ± 19% (p-p38MAPK), 287 ± 22% (p-ERK1/2),nd 133 ± 3% (p-JNK). Co-treatment with KRG or its primary gin-enosides inhibited the EtOH-induced increase in phosphorylationevels of ERK1/2 (as a percentage of EtOH: KRG, 67 ± 8%; GRg3,7 ± 8%; GRh2, 69 ± 8%), and JNK (as a percentage of EtOH: KRG,7 ± 1%; GRg3, 83 ± 2%; GRh2, 84 ± 2%).

. Discussion and conclusion

EtOH induces hepatocytic injury in human and animal models.tOH produces excessive ROS via alcohol dehydrogenase, catalasend especially by CYP 2E1 (Shimada et al., 2006). Our results showhat EtOH caused an increase in ROS and membrane damage inIB-73 cells, leading to increase the levels of AST and LDH, which isonsistent with other observation in human hepatocytes (Liu et al.,004). We reported that in the presence of KRG, EtOH-induced LDHelease was decreased in perfused rat livers (Park et al., 2011). Inhis study, we demonstrated for the first time that the treatment ofRG, GRg3 and GRh2 effectively inhibited EtOH-induced oxidativeamages in TIB-73 cells.

ROS are generated in many stressful situations and induce celleath by either apoptosis or necrosis (Klaunig and Kamendulis,004) and can induce MAPK activation by which hepatocytesediate reaction to oxidative stress (Wang et al., 1998). TheAPK constitute a family of serine/threonine kinases that includes

RK1/2, JNK and p38 MAPK. MAPKs play central roles in the inte-ration of signal transduction processes leading to various cellesponses including proliferation, differentiation and apoptosisZhang and Liu, 2002). It has been suggested that chronic activationf p42/44 MAPK will lead to cellular growth arrest (Woods et al.,997). In this study, we showed that EtOH caused the increase inctivation of ERK1/2, JNK and p38 MAPK in TIB-73 cells. KRG, GRg3nd GRh2 reduced the EtOH-induced activation of the ERK1/2 andNK. Although there was no significant difference, KRG, GRg3 andRh2 reduced activation of the p38 protein. These results indicated

hat KRG, GRg3 and GRh2 could have protective effect against EtOH-nduced hepatocyte injury by the attenuation of the activation ofRK1/2 and JNK. Moreover, it was proposed that JNK and p38 MAPKctivation may promote death of hepatocyte (Kurata, 2000). In arimary culture of rat hepatocytes, EtOH caused the increase in thectivation of ERK1/2 and JNK (Lee et al., 2002). Also, nuclear p38APK phosphorylation was increased by EtOH (Lee and Shukla,

007).In this work, EtOH induced a decrease in [Mg2+], significantly

nhibited by co-treatment with KRG, GRg3 and GRh2. Clinical and

xperimental evidence indicates a decrease in tissue and plasmaMg2+] under pathological conditions, including alcoholism (Flink,986). Also, lower [Mg2+] enhanced free radical-induced intracel-

ular oxidation and cytotoxicity (Dickens et al., 1992). In association

acology 141 (2012) 1071– 1076 1075

with MAPK and [Mg2+], the previous study reported that changesin extracellular and/or intracellular [Mg2+] can modulate the acti-vation of ERK1/2 and affect cell cycle progression and regulation(Touyz and Yao, 2003). Moreover, ERK 1/2 and p38 MAPK play akey role in modulating PKC-induced Mg2+ accumulation in hepa-tocytes (Torres et al., 2006). Therefore, the inhibitive effect of KRG,GRg3 and GRh2 on the EtOH-induced decrease in [Mg2+] could cor-relate with the attenuation of cytotoxicity induced by increased thelevel of ROS and ERK 1/2 activation.

In summary, EtOH induced cell death in mouse hepatocytes byan increase in membrane damage and intracellular ROS and bydecreasing Mg2+. KRG, GRg3 and GRh2 reduced the EtOH-inducedincrease in the level of ROS, the activation of ERK and JNK, and thedecrease in [Mg2+]. Therefore, KRG and its primary ginsenosides(GRg3 and GRh2) exert a protective effect on the EtOH-inducedhepatocytic injury.

Acknowledgements

This research was supported by the research funds of ChonbukNational University in 2010, Korean Ministry of Science (2010-0021909 and 2011-0013872) and Technology through the Centerfor Healthcare Technology Development.

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