Accepted Manuscript

Improved protective effects of American Ginseng Berry againstAcetaminophen-induced liver toxicity through TNF-α-mediatedcaspase-3/-8/-9 signaling pathways

Xing-Yue Xu , Zi Wang , Shen Ren , Jing Leng , Jun-nan Hu ,Zhi Liu , Chen Chen , Wei Li

PII: S0944-7113(18)30514-2DOI: https://doi.org/10.1016/j.phymed.2018.09.234Reference: PHYMED 52699

To appear in: Phytomedicine

Received date: 10 February 2018Revised date: 23 August 2018Accepted date: 28 September 2018

Please cite this article as: Xing-Yue Xu , Zi Wang , Shen Ren , Jing Leng , Jun-nan Hu , Zhi Liu ,Chen Chen , Wei Li , Improved protective effects of American Ginseng Berry against Acetaminophen-induced liver toxicity through TNF-α-mediated caspase-3/-8/-9 signaling pathways, Phytomedicine(2018), doi: https://doi.org/10.1016/j.phymed.2018.09.234

This is a PDF file of an unedited manuscript that has been accepted for publication. As a serviceto our customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, andall legal disclaimers that apply to the journal pertain.

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Improved protective effects of American Ginseng Berry against

Acetaminophen-induced liver toxicity through TNF-α-mediated

caspase-3/-8/-9 signaling pathways

Xing-Yue Xua, Zi Wang

a, Shen Ren

a, Jing Leng

a, Jun-nan Hu

a, Zhi Liu

a, Chen Chen

b, and Wei Li

a, c*

a College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130118, China.

b School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia;

c National & Local Joint Engineering Research Center for Ginseng Breeding and Development, Changchun

130118, China

*Correspondence

Professor Wei Li, College of Chinese Medicinal Materials, Jilin Agricultural University,

Changchun 130118, China. E-mail: liwei7727@126.com, Tel. /Fax: +86-431-84533304.

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Abstract

Background: Similar to the leaves of P. Quinquefolius, American ginseng berry (AGB) is

another important part of P. Quinquefolius with alternative therapeutic potential. The liver

protection capabilities of the former have been demonstrated previously, however, the later

has not yet been evaluated.

Purpose: Based on our previous observation, the present work was designed to evaluate the

hepatic protective effects for novel mechanisms of AGB in acetaminophen (APAP)-induced

liver injury in vivo.

Study Design/Methods: All mice were divided into four groups as follows: normal group,

APAP group and APAP+AGB (150 mg/kg and 300 mg/kg) groups. AGB were orally

administered for one week before exposure to APAP (250 mg/kg). Severe liver injury was

observed and hepatotoxicity was evaluated after 24 h through evaluating the biochemical

markers, protein expressions levels and liver histopathology.

Results: Our study results clearly demonstrated that AGB pretreatment ameliorated

APAP-induced hepatic injury as evidenced by decreasing plasma alanine aminotransferase

(ALT), aspartate transaminase (AST), tumor necrosis factor α (TNF-α) and interleukin-1β

(IL-1β) compared to the APAP group. Western blotting analysis showed that pretreatment

with AGB decreased the expressions levels of TNF-α and nuclear transcription factor-κB

(NF-κB p65) in liver tissues. Meanwhile, the protein expression levels of caspases,

cytochrome c, and Bax were elevated by AGB treatment for seven days, while the protein

expression level of Bcl-2 was inhibited comparison with that in APAP group. Furthermore,

supplement of AGB resulted in increase of superoxide dismutase (SOD) and glutathione

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(GSH), while decrease of malondialdehyde (MDA) content and the expression levels of

4-hydroxynonenal (4-HNE) and cytochrome P450 E1 (CYP2E1). The results of

histopathological staining demonstrated that AGB pretreatment inhibited APAP-induced

hepatocyte infiltration, congestion, and necrosis.

Conclusion: The present study demonstrated that AGB pretreatment protected liver cells

against APAP-induced hepatotoxicity through inhibition of oxidative stress, inflammation

responses via TNF-α-mediated caspase-3/-8/-9 signaling pathways.

Key words: American ginseng berry; APAP-induced liver injury; oxidative stress;

anti-inflammation; anti-apoptosis

Abbreviations:

APAP Acetaminophen SOD Superoxide dismutase

AGB American ginseng berry Caspase Cysteine aspartic acid specific protease

ALI Acute liver injury NF-κB Nuclear transcription factor-κB

AST Aspartate transaminase TNF-α Tumor necrosis factor-α

ALT Alanine aminotransferase IL-1β Interleukin-1β

GSH Glutathione CYP2E1 Cytochrome P450 E1

MDA Malondialdehyde 4-HNE 4-hydroxynonenal

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1. Introduction

Acetaminophen (APAP) is commonly used as an anodyne and febrifuge, which is safe and

no side effects within therapeutic dosages. Nevertheless, the overdose APAP can induce

severe liver injuries from hepatic necrosis, fibrosis and even cirrhosis (Truong et al., 2016).

APAP-induced liver toxicity is triggered by massive accumulation of reactive metabolic

N-acetyl-p-benzoquinone imine (NAPQI), which is catalyzed by metabolism of hepatic

cytochromes P450, mainly CYP2E1 (Shanmugam et al., 2016). NAPQI is detoxified by

reacting with reduced glutathione (GSH) in the cells. Excessive NAPQI bindings to cellular

mitochondrial proteins also inhibit the mitochondrial oxidative phosphorylation, thus

generating reactive oxygen species (ROS) which leads to mitochondrial dysfunction, damage

and eventually the pathways of apoptosis are activated (Zhao et al., 2012). In addition, during

the metabolism processes, antioxidant enzymes, such as superoxide dismutase (SOD), play a

vital role in modulating the APAP-induced hepatotoxicity (Michael Brown et al., 2012).

Accumulated evidences have revealed that APAP overdose caused the transcriptional

activation of pro-inflammatory cytokines such as nuclear transcription factor-κB (NF-κB),

tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) (Song et al., 2014). Bax, Bcl-2

and cytochrome-c were mitochondria-related apoptotic factors and playing important roles in

the cell apoptosis (Zhao et al., 2014). Caspases as members of protease family exert necessary

effects in cell apoptosis. Initiator caspases (containing 8, 9) were tightly coupled to

pro-apoptotic signals (Hsiao et al., 2014). Once activated, these caspases would activate the

downstream effector caspases-3 following by cleaving cell scaffolds, nucleoproteins and

finally induce apoptosis (Peng et al., 2016). Taken together, a continuing search for a

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promising hepatoprotective medicine is warranted.

American ginseng berry (AGB) is the ripe fruit of Panax quinquefolius. Many reports have

proved that AGB exerts similar or same physiological activities as P. ginseng or P.

quinquefolius (Lin et al., 2008; Xie et al., 2004). So far, researchers have reported the

chemical constituents and biological activities of roots of American ginseng, while the studies

on its berry are scarce. In order to fully understand pharmaceutical value of American ginseng,

studies on the extracted saponin of American ginseng berry have been carried out (Sritularak

et al., 2009). A recent report indicated that AGB exerted protective effect on myocardial

ischemia by decreasing myocardial oxygen consumption and increasing coronary blood flow

in dogs (Lu et al., 2012). AGB also exerted anti-hyperglycemic effects in diabetic ob/ob mice

(Xie et al., 2002) and protective effect in chemotherapy of 5-FU on human colorectal cancer

cells (Li et al., 2009). We have showed that extracted saponins from the leaves of P.

quinquefolius (PQS) suppressed APAP-induced liver toxicity in mice (Xu et al., 2017).

Pharmacological studies have showed that AGB exerted anti-apoptotic (Wang et al., 2006b),

antioxidant (Lin et al., 2008) and anti-hyperglycemic (Xie et al., 2004) effects. Taking into

account the significant liver protection activity of American ginseng leaves, it is worth

exploring the hepatoprotective effects and mechanisms of AGB on APAP-induced liver injury.

In our study, molecular mechanisms of AGB action on APAP-induced liver injury have

been clarified, in which TNF-α-mediated caspase-3/-8/-9 signaling pathway was identified

clearly. To our knowledge, it is the first report of potential molecular mechanisms of AGB

action in protecting liver injuries against APAP-induced hepatotoxicity in mice.

2. Materials and methods

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2.1. Materials and reagents

AGB extracts from the American ginseng berry was prepared and quantified by our own as

previous description (Liu et al., 2008; Wang et al., 2006a). In brief, AGB were eluted three

times with 70% ethanol, and then collecting from AB-8 resin column chromatogram. As

described in our previous work (Xu et al., 2017), the quantitative analysis of AGB was

implemented on a Hypersil ODS column using high-performance liquid chromatography

(Waters HPLC, Milford, MA, USA) at 203 nm. The contents of the crude saponins in the

ABG were determined as follows: 0.336% Rg1, 9.107% Re, 0.504% Rb1, 8.805% Rb2,

29.523% Rb3, 3.171% Rc, and 6.022% Rd. the HPLC chromatogram and chemical structures

of seven saponins in AGB including protopanaxadiol ginsenosides (ginsenosides Rb1, Rb2,

Rb3, Rc, and Rd) and protopanaxatriol ginsenosides (ginsenosides Re and Rg1) as shown in

Fig. 1.

APAP was provided by Sigma-Aldrich (St. Louis, MO). Diagnostic kits applied to the

evaluation of ALT, AST, GSH, MDA, SOD and hematoxylin and eosin (H&E) dye kits were

obtained from Nanjing Jiancheng Institute of Biotech (Nanjing, China). Two-site sandwich

enzyme-linked immunosorbent assays (ELISA) for serum TNF-α and IL-1β were provided by

R&D systems (Minneapolis, MN, USA). Hoechst 33258 dye kit was bought from Beyotime

Biotech Co., Ltd. (Shanghai, China). The CPY2E1, 4-HNE, NF-κB (p65), phospho-p65,

TNF-α, Bax, Bcl-2, cytochrome-c, caspases-3/-8/-9, cleaved-caspases-3/-8/-9 and GAPDH

antibodies were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA).

DyLight488-labeled second antibody was obtained from BOSTER Biological Technology Co.,

Ltd. (Wuhan, China). For the analysis of cell apoptosis, TUNEL cell apoptosis diagnostic kit

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was obtained from Roche Applied Science (No. 11684817910). All the other chemical agents

during the study were of the highest grade commercially available.

2.2. Animals and experimental design

Adult male ICR mice (8 weeks old, 25~27g), SPF grade, were purchased from YISI

Experimental Animal Co., Ltd. with Certificate of Quality No. of SCXK (JI) 2017-0003

(Changchun, Jilin province, China). All mice were maintained 23~27°C and humidity of 50%

~70% with a 12-h light: 12-h dark cycle. All mice were continuously fed with standard chow

diet and libitum. All experimental protocols were strictly abided by the Committee of Animal

Care and Use. All animal protocols were in accordance with the Ethical Committee for

Laboratory Animals of Jilin Agricultural University (Permit No.: ECLA-JLAU-17626).

All animals were randomly divided into four groups (8, per group) as follows: normal

group, APAP-treated group, and APAP/AGB (150 mg/kg)-treated group and APAP/AGB (300

mg/kg)-treated group. AGB powder was dissolved in water. Mice were administered AGB

(150 and 300 mg/kg, respectively) by oral administration once daily for 7 days, the normal

and APAP-treated groups were managed with 0.9% normal saline only. On the 6th day, all

animals were intraperitoneally injected with 250 mg/kg APAP dissolved in warm saline

(65°C). The mice were administered saline in normal group. Subsequently, at 24 h post-APAP,

all mice were euthanized. Blood was collected and centrifuged at 3000 g, 4℃ for 15 min to

gain serum then stored at −80°C for biochemical analysis. Meanwhile, the fresh liver tissue

samples were harvested. A portion of the liver tissues immediately fixed in 10% buffered

formalin solution and embedded in paraffin for pathologic analysis. The remaining tissues

were maintained at −80°C for biochemical markers estimation.

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2.3. Biochemical marker assay

The contents of AST and ALT in serum were quantified using commercial available kits

(Nanjing Jiancheng Bioengineering Institute, Nanjing China) in accordance with the

manufactures’ protocols. The contents of TNF-α and IL-1β in serum were determined using

ELISA assay kits (R&D, Minneapolis, MN, USA). The absorbance was measured at 450 nm

in an ELISA reader (Bio-Rad, California, USA).

The hepatic levels of GSH, MDA and SOD were analyzed by commercial reagent kits

(Nanjing Jiancheng, Bioengineering Institute, Nanjing China) according to the manufacturer’s

protocols.

2.4. H&E and Hoechst 33258 Staining

For evaluation of hepatotoxicity, formalin-fixed liver tissues, which were embedded in

paraffin, and sliced into a thickness of 5-μm. Slices were stained with H&E according to a

commercial protocol, and subsequently performing an observation for histopathological

assessment used light microscope (Leica, DN750, Solms, Germany). These histopathological

changes would be evaluated including hepatocyte necrosis, congestion and inflammatory cell

infiltration.

As we known, Hoechst 33258 staining is commonly used for apoptosis detection which

was implemented as previously performed (Li et al., 2015). In brief, 5-μm paraffin slices were

stained by Hoechst 33258 (10 μg/ml). Stained nuclei were visualized use a fluorescent

microscope (Olympus BX-60, Tokyo, Japan). Image-Pro Plus 6.0 software (Media

Cybernetics, Maryland, USA) was applied to evaluate the degree of liver apoptosis through

quantifying the fragmented and condensed staining.

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2.5. TUNEL Staining

TUNEL analysis, in which apoptotic cells in the liver tissues would be precisely detected

by an situ cell death detection kit (Roche Applied Science, Germany), was implemented as

previously described (Li et al., 2016). In brief, the liver tissue slices were covered with 20

μg/mL of proteinase K for 10 min and then incubated in methanol including 3% H202 for 20

min. The slices were subsequently incubated with equilibrium buffer solution and terminal

deoxynucleotidyl transferase (TdT). Finally, the sections were incubated with anti-fluorescein

antibody. The activity of peroxidase (POD) in sections were revealed by applying

diaminobenzidine (DAB), every section was counterstained with hematoxylin. Subsequently

apoptotic cells assessment used light microscope (Leica, DN750, Solms, Germany).

2.6. Immunofluorescence Staining

Immunohistochemical analysis was executed on liver tissue sections as prior report

(Limsrichamrern et al., 2016). Specifically, the 5-μm paraffin slices were fixed to de-paraffin

and rehydrate with a series of xylene and different concentration alcohol solutions. After

antigen retrieval in citrate buffer solution (0.01 M, pH 6.0) for 8 min, the sections were

swashed with PBS and incubated with 1% BSA for 10 min. The blocking serum was casted

off, then incubated primary antibodies including CYP2E1 (1:200) and 4-HNE (1:100)

overnight at 4℃. The liver tissues were blotted with secondary antibody for 30 min at 37℃.

Then liver tissues slides were exposed to the DyLight 488-labeled (BOSTER, Wuhan, China).

Nuclear staining was implemented with DAPI. The sections were visualized using a Leica

microscope (Leica TCS SP8, Solms, Germany). The degree of liver expression was analyzed

by Image-Pro Plus 6.0 software analyzed (Media Cybernetics, Maryland, USA)

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2.7. Western Blotting Analysis

Western blotting analysis was implemented as previously depicted (Noh et al., 2015).

Briefly, liver tissues were homogenized with cold RIPA buffer. After centrifuging 3 times for

10 min, the protein concentrations were analyzed using the BCA protein Trizol Reagent

(Beyotime Biotechnology, China). Equivalent protein extracts from samples were subjected to

12% SDS-PAGE and then electrophoretically transferred onto a PVDF membrane.

Phosphorylated proteins were blocked with 5% bovine serum albumin (BSA) in Tris-buffered

saline (TBS) with 0.1% Tween-20, and other proteins with 5% skimmed milk in TBS with

0.1% Tween-20 as least 1 h, and then incubated overnight at 4℃ with primary antibodies

including NF-κB p65 (1:1000), p-p65 (1:1000), TNF-α (1:1000), Bcl-2 (1:1000), Bax

(1:1000), cytochrome-c (1:1000) and caspases family (caspase-3/-8/-9 1:1000). Followed by,

the secondary antibody (1:1000) incubated for 1 h. Signals were captured with Emitter

Coupled Logic (ECL) substrate (Pierce Chemical Co., Rockford, IL, USA). Protein band

intensities were assessed with Quantity One software (Bio-Rad Laboratories, Hercules, CA,

USA).

2.8. Statistical Analysis

All of the experimental data are expressed as the means ± SD. SPSS version 17.0 for

Windows (SPSS Inc., Chicago, IL, USA) was used for the least significance difference (LDS)

multiple-comparison tests. Statistically significant differences between four groups were

evaluated by using one-way analysis of variance (ANOVA) followed by Tukey-Kramer tests.

p <0.05 or <0.01 were considered dramatically. Statistical graphs were presented by GraphPad

Prism 6.0.4. (GraphPad Software, La Jolla, CA, USA).

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3. Results

3.1. AGB pretreatment reduces AST and ALT levels after APAP treatment

To evaluate the function of AGB on liver-protection from APAP-induced hepatotoxicity, we

measured the serum levels of ALT and AST, the markers of acute liver injury in all mice. As

shown in Fig 2A and B, compared to the normal group, a single injection of 250 mg/kg APAP

significantly increased the levels of ALT and AST (p < 0.05 or p < 0.01), reflecting a severe

liver injury. However, pretreatment with AGB once daily for seven days (150 and 300 mg/kg)

reduced APAP-induced ALT and AST activities. These results indicated that AGB may exert a

protective effect in APAP-induced liver injury.

3.2. AGB pretreatment inhibits oxidative stress after APAP treatment

It has been reported that oxidative stress damage was caused by the APAP-induced

molecular mechanisms linking to acute liver failure (Nagi et al., 2010). As shown in Fig 2C,

D and E, the results indicated that GSH content and SOD activity were dramatically reduced

accompanied by an increase in lipid peroxidation product MDA by APAP treatment (p < 0.05

or p < 0.01). Interestingly, oral administration with 150 mg/kg and 300 mg/kg AGB for seven

days dramatically decreased the level of MDA and restored the controlled oxidant status as

verified by reinforce of SOD activity and suppression of GSH depletion (p < 0.05 or p < 0.01).

These results demonstrated that AGB had a remarkable antioxidant capacity against

APAP-induced live injury via increasing antioxidant enzyme activity.

In order to further demonstrate whether oxidative stress was relevant with development of

APAP-induced hepatotoxicity in mice, lipid peroxidation was verified using 4-HNE staining.

Strong fluorescence intensities of 4-HNE were shown in the central veins of the liver tissues,

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after administration APAP alone for 24 h. Nevertheless, Pretreatment AGB for one week

dramatically reduced 4-HNE positive areas, especially in the group of dosage 300 mg/kg. The

sites of lipid peroxidation were closely correlated to the necrotic areas in the liver. According

to the lipid peroxidation product MDA analysis, results showed that pretreatment with AGB

significantly reduced the increase in lipid peroxidation, as shown in Fig 3A.

APAP toxicity is triggered through its metabolic activation, which is catalyzed by

cytochrome P450 enzymes, mainly CYP2E1 (Du et al., 2015). In this study, the protein

expression level of CYP2E1 in liver tissues was determined after 24 h APAP treatment. The

expression of CYP2E1 was elevated after APAP treatment. However, pretreatment with AGB

remarkably reduced APAP-induced the expression of CYP2E1 (Fig 3B). These results

demonstrated that administration of AGB may exert a protective effect on oxidative stress

caused by APAP.

3.3. AGB pretreatment inhibits Inflammation response after APAP treatment

Prior studies reported that oxidative stress increased pro-inflammatory cytokine during the

progression of APAP-induced hepatotoxicity (Barman et al., 2016). As described in Fig 4A

and B, TNF-α and IL-1β contents were markedly increased compared with normal group after

24 h APAP injection alone (p < 0.05 or p < 0.01). In contrast, significantly increased TNF-α

and IL-1β levels following exposure to APAP were diminished by AGB pretreatment for 7

days (p < 0.05 or p < 0.01).

To gain an insight into the anti-inflammatory effects of AGB with APAP-induced liver

damage, we assessed the protein expression levels of NF-κB p65, p-p65 and TNF-α in all

groups by western blotting analysis. As depicted in Fig 4C, the results clearly showed that

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exposure to APAP caused significant overproduction of pro-inflammatory cytokines

(phosphorylation of p65 and TNF-α) in the liver tissues. However, AGB pretreatment result

reduced the expressions of these pro-inflammatory proteins in the liver.

3.4. AGB pretreatment inhibits liver Histological alteration after APAP treatment

In liver tissues from 4 groups (Fig. 5A), we found that APAP treatment aroused more liver

hemorrhage than that in the normal control mice. Besides, AGB pretreatment ameliorated the

hemorrhage injury by APAP. H&E staining revealed that the livers with APAP treatment

presented more necrotic regions, congestion and inflammatory cell infiltration than that in

normal control group. Pretreatment with AGB reversed these APAP-induced hepatic lesions.

Light microscopic methods for histopathological analysis were performed on liver tissues

from all groups. As shown in Fig 5B, liver tissues in APAP treated mice showed necrosis,

congestion, and inflammatory infiltration. After treatment with low dose of AGB (150 mg/kg),

there was dramatic decrease in cytoplasm damage. Interestingly, the group treated with high

dosage AGB (300 mg/kg) after exposure to APAP markedly attenuated the hepatocyte

necrosis, inflammatory infiltration to the similar level in the normal group.

3.5. AGB pretreatment inhibits cell apoptosis after APAP treatment

To investigate whether AGB pretreatment decreased hepatocyte apoptosis induced by

APAP, Hoechst 33258 staining assay was performed to evaluate apoptosis of cells. As shown

in Fig 5C, significant nuclear fragmentation and condensation of liver cell nucleus were

observed to indicate apoptosis of liver cells in the APAP group. With AGB pretreatment, most

cell nucleus exhibited the regular homogeneous fluorescent density and normal cell structures.

To further evaluate whether there is any coexistence of apoptosis and by APAP, the liver cell

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apoptosis was verified and quantified by TUNEL staining. As described in Fig 5D, compared

to normal group, TUNEL staining showed abundant cell apoptosis in APAP group. However,

AGB pretreatment (300 mg/kg) markedly reduced the number of TUNEL positive cells.

To clarify the signaling systems in this hepatocyte apoptosis, expressions of pro-apoptotic

factor Bax, cytochrome-c, the members of caspase family (including 3, 8, 9), and

anti-apoptotic factor Bcl-2 were determined in all experimental groups. As showed in Fig 6,

The results also demonstrated that pretreatment with AGB 150 mg/kg and 300 mg/kg

significantly reduced the protein expression of Bax, cytochrome-c and cleaved caspase-3/-8/-9,

and increased the protein expression of Bcl-2 compared with that in APAP alone group.

4. Discussion

APAP was commonly used as an anodyne and febrifuge agent in clinical application when

administered at therapeutic doses. However, accidental or intentional APAP overdose could

trigger acute hepatic injury and even acute liver cirrhosis (Holubek et al., 2006). To date,

N-acetylcysteine (NAC) has considered to be the first clinical antidote against APAP

hepatotoxicity. Although NAC was very effective partially for preventing APAP-induced liver

toxicity to avoid morbidity and mortality, some patients still arouse severe toxicity with

nausea, anaphylactic reaction and headaches (Saito et al., 2010). Therefore, it was necessary

to develop therapeutic options and approaches for APAP-induced liver toxicity.

It has considered that many natural extracts and bioactive compounds exert potential

protective effects against APAP-caused liver toxicity (Yuan et al., 2010; Zhang et al., 2017).

American ginseng (P. quinquefolius), a famous medicinal plant, was originated in virgin forest

of North America (Jiao et al., 2015). In the last century, American ginseng was introduced to

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China for cultivation and breeding. So far, numerous scientists have made a great effort to

identify the chemical compositions and pharmacological effects of American ginseng (Yu et

al., 2014). Although many published reports have focused on its roots, there were little

systematic studies or reports on the active components and the utilization value of American

ginseng berry (Wang et al., 2006b). Like the roots, American ginseng berry also contains

similar ginsenosides. These ginsenosides mainly include protopanaxadiol-type saponins (PDS,

e.g. Rb1, Rb2, Rb3, Rc, and Rd) and a few protopanaxatriol-type saponins (PTS, e.g. Rg1 and

Re), of which PDS accounts for more than 80% of all saponins (Wang et al., 2006a).

A recent report indicated that AGB exerted protective effect on myocardial ischemia by

decreasing myocardial oxygen consumption and increasing coronary blood flow in dogs (Lu

et al., 2012). Several studies have found AGB exerted anti-hyperglycemic effects in diabetic

ob/ob mice (Xie et al., 2002) and enhanced chemopreventive effect of 5-FU on human

colorectal cancer cells (Li et al., 2009). AGB produced cardiovascular effects because of its

bioactive ingredients (Wang and Shi, 2011). Accordingly, Korean red ginseng extract mainly

including ginsenosides Rg3, prevented APAP-induced hepatotoxicity via metabolic enzyme

regulation (Gum and Cho, 2013), and panaxatriol saponin ameliorated APAP-induced liver

injury by restoring thioredoxin-1 and pro-caspase-12 (Wang et al., 2014). Importantly, our

recent results showed that saponins from the leaves of Panax quinquefolius attenuated

APAP-induced liver toxicity in mice (Xu et al., 2017). There were many reports about

experimental liver injury models, including APAP-induced (Xu et al., 2017), alcohol-induced

(Liu et al., 2016), CCl-4-induced (Xie et al., 2013) and LPS/D-GaLN-induced (Yan et al.,

2016). The hepatoprotective effects of AGB and the underlying mechanisms against

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APAP-induced liver toxicity have not been elucidated yet.

This study clearly showed that AGB attenuated APAP-induced toxicity via attenuating liver

cells damage, ameliorating oxidative stress, inhibiting the expressions of apoptosis protein

and pro-inflammatory factors. The serum levels of ALT and AST were commonly referred as

liver enzymes that enter the bloodstream after hepatocyte structural integrity damage, and

were generally regarded as useful quantitative markers to reflect hepatic diseases (Yuan et al.,

2010). Our research demonstrated that the serum levels of AST and ALT remarkably increased

in mice exposed to APAP, but AGB pretreatment inhibited APAP-induced increase in AST

and ALT. Meanwhile, histological analysis demonstrated that AGB attenuated liver pathologic

changes including inflammatory infiltration and cellular necrosis and apoptosis. These results

indicated that the hepatocellular damage was reduced as a direct consequence of AGB oral

administration.

Oxidative stress played an important role in APAP-induced hepatotoxicity (Nagi et al.,

2010). APAP extensively affected hepatic metabolism and then promoted the formation of

reactive metabolite NAPQI, which induced the APAP’s toxicity, as it reacted rapidly with

GSH with depleting GSH. In APAP overdose situation, GSH became exhausted, and excess

NAPQI irreversibly adhered to mitochondrial proteins (Smith et al., 2016). As a result,

accumulation of NAPQI induced hepatocellular damage by aggravating oxidative stress in

conjunction with mitochondrial dysfunction (Kang et al., 2017). Our findings clearly

displayed the depletion of hepatic GSH in the APAP-intoxicated mice compared to the normal

group. However, pretreatment with AGB (150 and 300 mg/kg) reversed the depletion of liver

GSH. APAP metabolic activation and formation of NAPQI were mediated by cytochrome

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P450-mediated enzymes, especially CYP2E1. We found overexpression of CYP2E1 in liver

tissues of APAP-treated mice reported in the previous reports. (Na et al., 2017). However,

AGB inhibited the overexpression of APAP-induced CYP2E1 expression evidenced by IHC

analyses. Furthermore, oxidative stress acted an important contribution to liver injury, which

was characterized by LPO and the accumulation of ROS (Du et al., 2016). Oxidative stress

induced by APAP was normally detoxified by the enzymatic antioxidant defense system.

Antioxidant enzymes, such as SOD, were massively depleted with APAP exposure (Wang et

al., 2017). The increase in SOD was reversed after AGB administration (150 and 300 mg/kg),

demonstrating the protective effect of AGB on APAP toxicity. The hepatic level of MDA was

generally used as a reliable biomarker of free radical mediated LPO injury (Noh et al., 2015).

The results in this investigation demonstrated that the level of hepatic MDA was remarkably

increased after the APAP challenge and these alterations were effectively alleviated by AGB

pretreatment for seven days. A study conducted by Guo et al also supported our findings (Guo

et al., 2016). In supporting MDA data, 4-HNE staining also showed an overexpression in liver

slices after exposure to APAP, and the overexpression was remarkably reduced in the groups

of AGB pretreatment.

Many pro-inflammatory mediators, like TNF-α and IL-1β, are involved in the development

of inflammatory responses. They were released due to activation of Kupffer cells by the APAP

toxic metabolite NAPQI (Legert et al., 2015). Besides, NF-κB translocation to the nucleus

was needed for the induction of cytokines that were associated with APAP-induced liver

injury (Wang et al., 2016). Inflammatory cytokines TNF-α and IL-1β were activated by the

ubiquitous NF-κB pathway and their receptors engaged in activating NF-κB (Sullivan et al.,

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2014). It was reported that APAP activated the NF-κB pathway through expression of TNF-α,

IL-1β and others in a recent study (Truong et al., 2016), where TNF-α mediated the death

receptor pathway and apoptosis. In this study, the levels of TNF-α and IL-1β, and the protein

expressions of phosphorylated NF-κB and TNF-α were highly increased in the APAP-injured

mice and these increases were significantly attenuated by AGB treatment (150 and 300

mg/kg). These results may suggest that AGB exerted the protective effect against

APAP-induced liver inflammation.

Accumulating evidences demonstrated that APAP-induced liver injury was relevant to

apoptosis (Xu et al., 2017). Apoptosis acted as an important cell death mode in APAP

hepatotoxicity (Yuan et al., 2010). Mitochondria-mediated apoptosis was often accurately

regulated by the Bcl-2 family members. Bcl-2 as a prominent pro-survival protein was

binding to the mitochondrial external membrane to antagonize Bax homodimers mediated

pro-apoptosis (Joshi et al., 2011). The Bcl-2/Bax ratio was regarded as a vital index to cause

hepatocyte apoptosis (Guo et al., 2017). Our study showed that pretreatment AGB decreased

Bax and increased Bcl-2 protein expression levels as well as the Bcl-2/Bax ratio was clearly

decreased compared to that in only APAP group. Those results demonstrated that the balance

between Bcl-2 and Bax were broken. TNF-α, a significant pro-inflammatory cytokine,

induced hepatocyte apoptosis or necrosis by activating caspase-3 (Fan et al., 2014). In this

study, we demonstrated TNF-α-mediated caspase-3/-8/-9 signaling pathway. TNF-α mediated

apoptosis by recruiting death domain-containing adaptor proteins FADD and TRADD to their

receptor, which in turn leads to the activation of the initiator caspase-8. Caspase-8 cleaved and

activated cytosolic protein caspase-3 in cell apoptosis. In fact, caspase-3 functions as a central

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effector to cleave various cellular substrates and trigger cell apoptosis eventually (Nagase et

al., 2002). Caspase-9 directly linked to the pathway of mitochondrial apoptosis, and it can be

activated by death stress (Peng et al., 2016). Caspase-3 was activated by caspase-9 and

involved in the mitochondrial apoptosis pathway (Zhu et al., 2012). Alternatively, caspase-3

can be directly triggered apoptosis via activation of the initiator caspase-8/-9 (Hayakawa et al.,

2008). Western blotting analyses in this report clearly revealed that APAP increased

caspase-3/8/9 in hepatocytes, which were similar to the results by Yuan et al (Yuan et al.,

2010). However, the protein expression of caspase-3/-8/-9 was dramatically suppressed after

AGB administration, indicating that AGB exerted anti-apoptotic properties in the presence of

APAP hepatotoxicity. These data indicated that AGB remarkably reduced apoptosis of

hepatocytes in APAP-induced hepatotoxicity. Overall, AGB ameliorated activation of

TNF-α-mediated caspase pathway by reducing the expression of cleaved caspase-3/-8/-9,

cytochrome-c and the Bcl-2/Bax signaling pathway, eventually suppressing hepatocyte

apoptosis (Fig 7).

5. Conclusions

In summary, AGB pretreatment possessed a great positive effect in ameliorating

antioxidant mechanisms, inflammation responses, and TNF-α-mediated caspase-3/-8/-9

hepatocyte apoptosis on APAP-induced hepatotoxicity. AGB exhibited anti-inflammatory

ability through the inhibition of pro-inflammatory cytokines including NF-κB, TNF-α and

IL-1β in APAP-induced acute hepatotoxicity. Furthermore, the APAP toxicity might be

reduced by preventing the production of lipid peroxidation to alleviate oxidative stress level

as well as hepatic antioxidant status. Likewise, AGB attenuated activation of TNF-α-mediated

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caspase pathway and decreased the Bcl-2/Bax ratio and the expression of cytochrome-c,

which eventually prevented hepatocyte apoptosis and necrosis. The findings from the present

work clearly revealed that AGB may be serves as a hepatoprotective agent in near future.

Acknowledgements

This work was supported by the grants of National Natural Science Foundation of China (No.

81403052 & 314707418), the Scientific Research Foundation for the Returned Overseas

Chinese Scholars (Jilin Province, 2015), Jilin Science & Technology Development Plan (No.

20160209008YY), and the Program for the Young Top-notch and Innovative Talents of Jilin

Agricultural University (2016).

Conflicts of Interest

The authors declare no conflict of interest.

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Figure legends

Figure 1. HPLC chromatogram and chemical structures of seven saponins in the AGB. These

saponins includes four protopanaxadiol ginsenosides (e.g. Rb1, Rb2, Rb3, Rc, and Rd) and

two protopanaxatriol ginsenosides (e.g. Re and Rg1).

Figure 2. Pretreatment with AGB protected against APAP-induced liver injury: The levels of

ALT (A), AST (B), GSH (C), MDA (D) and SOD (E) were measured in APAP-induced

hepatotoxicity. All data were expressed as mean ± S.D., n = 8. * p < 0.05, ** p < 0.01 vs.

normal group; #

p < 0.05, ##

p < 0.01 vs. APAP group. GSH = glutathione; MDA

=malondialdehyde; SOD = superoxide dismutase.

Figure 3. Pretreatment with AGB protected against APAP-induced liver injury. 4-HNE (A);

CYP2E1 (B), the 4-HNE of relative fluorescence intensity (C), the CYP2E1 of relative

fluorescence intensity (D). The expression level of CYP2E1 and 4-HNE (Green) in tissue

section isolated from different groups was assessed by immunofluorescence. Representative

immunofluorescence images were taken at 400×4’ 6-Diamidino-2-phenylindole (DAPI) (Blue)

was used as a nuclear counterstain.

Figure 4. Pretreatment with AGB protected against APAP-induced liver injury. The levels of:

TNF-α (A) and IL-1β (B) in liver tissues were determined by ELISA kits. The protein

expression was examined by western blotting analysis in liver tissues. p-p65 and TNF-α (C),

the relative protein expression of p-p65 (D), the relative protein expression of TNF-α (E). All

data were expressed as mean ± S.D., n = 8. * p < 0.05, ** p < 0.01 vs. normal group; # p <

0.05, ##

p < 0.01 vs. APAP group.

Figure 5. Histological examination of morphological changes in liver tissues. Livers of four

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groups were observed (A), Liver tissues stained with H&E (100×, 400×) (B), Hoechst 33258

(100×, 400×) (C), and TUNEL (400×) (D), the Hoechst 33258 percentage apoptosis (%) (E),

the TUNEL percentage apoptosis (%) (F). Arrows show hepatocyte, necrosis, congestion,

inflammatory cell infiltration. All data were expressed as mean ± S.D., n = 8. * p < 0.05, ** p

< 0.01 vs. normal group; # p < 0.05,

## p < 0.01 vs. APAP group.

Figure 6. Effects of AGB on the protein expression of Cytochrome C, Bax, Bcl-2 (A),

cspase-3, caspase-8, caspase-9 (B), relative protein expression of Cytochrome C, Bax, Bcl-2,

Bcl-2/Bax (C); caspase-3 caspase-8 caspase-9 (D) band intensities were analyzed. The protein

expression was examined by western blotting analysis in liver tissues from normal, APAP,

APAP + AGB (150 mg/kg), and APAP + AGB (300 mg/kg) groups. All data were expressed

as mean ± S.D., n = 8. * p < 0.05, ** p < 0.01 vs. normal group; # p < 0.05,

## p < 0.01 vs.

APAP group.

Figure 7. Schematic diagram of signaling pathway involved in the hepatoprotective effects of

AGB on APAP-induced hepatotoxicity. AGB exerts the liver protection effects against

APAP-induced hepatotoxicity mainly through TNF-α-mediated caspase-3/-8/-9 signaling

pathways.

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Figure 1

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A G B (m g /k g ) -

+-

-

+ +

1 5 0 3 0 0 -

+-

-

+ +

1 5 0 3 0 0

- +

--

+ +

1 5 0 3 0 0

- +

- -

+ +

1 5 0 3 0 0

## #

**

# #

# #

**#

# #

**

# #

# #

1 2 3

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Re

lati

ve

pr

ote

in e

xp

re

ss

ion

(% o

f n

or

ma

l)

c a s p a s e - 3 c a s p a s e - 8 c a s p a s e - 9

* *

#

# #

* *

#

# #

* *

## #

A P A P (m g /k g )

A G B (m g /k g ) -

+-

-

+ +

1 5 0 3 0 0 -

+-

-

+ +

1 5 0 3 0 0

- +

- -

+ +

1 5 0 3 0 0

Figure 6

A

B

D

C

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Figure 7

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Graphical Abstract