全 文 ： 2012 年 5 月 第 10 卷 第 3 期 Chin J Nat Med May 2012 Vol. 10 No. 3 177

Chinese Journal of Natural Medicines 2012, 10(3): 0177−0184
doi: 10.3724/SP.J.1009.2012.00177
Chinese
Journal of
Natural
Medicines






Protective effect of Ornithogalum saundersiae Ait
(Liliaceae) against acetaminophen-induced acute
liver injury via CYP2E1 and HIF-1α
WAN Ying Δ, WU Yan-Ling Δ, LIAN Li-Hua, NAN Ji-Xing *
Key Laboratory for Natural Resources of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy,
Yanbian University, Yanji 133002, China
Available online 20 May 2012
[ABSTRACT] AIM: To investigate the hepatoprotective effect of total saponin from Ornithogalum saundersiae Ait (Liliaceae) (OC)
against acetaminophen (APAP)-induced acute liver injury in vivo and in vitro. METHODS: Mice were pretreated with OC (300, 200 or
100 mg·kg–1, body weight) or N-acetyl-L-cysteine (NAC) (300 mg·kg–1, body weight) for 3 times at 24 h intervals. APAP was admin-
istered 2 h after OC last dose. Chang liver cells were incubated with the medium containing OC (50, 100, 200 mg·mL–1) or NAC (10
mmol·L–1) with the presence or absence of APAP (10 mmol·L–1) for 24 h. RESULTS: OC showed remarkable hepatoprotective effect
12 h after APAP administration by decreased aspartate aminotransferase and alanine aminotransferase levels, reduced the products of
lipid peroxidation, improved the activity of catalase, superoxide dismutase and glutathione peroxidase, inhibited the caspase-3 cleavage
and hypoxia inducible factor-1α accumulation in vivo. In vitro, OC significantly decreased the activities of metabolism enzyme cyto-
chrome P450 2E1 (CYP2E1) and cyclooxygenase-2 (COX-2) induced by APAP. CONCLUSION: OC possesses the ability to protect
hepatocyte from APAP-induced liver damage, suggesting that the hepatoprotective mechanism of OC might be related to antioxidation
via blocking the CYP2E1, and mediating reactive oxygen species scavenging and accumulation of hypoxia-inducible factor (HIF)-1α.
[KEY WORDS] Ornithogalum saundersiae Ait; Acetaminophen; Apoptosis; Cytochrome P450 2E1; Hypoxia-inducible factor-1α
[CLC Number] R965 [Document code] A [Article ID] 1672-3651(2012)03-0177-08

1 Introduction
Ornithogalum saundersiae Ait (Liliaceae) (OC) is one of
Liliaceae family species and a valuable traditional Chinese
herb, which is mainly distributed in the south of Africa, and
is commonly used as an anti-inflammatory and anticancer
agent to treat liver disease, hepatoma, cholecystitis, etc. Re-
cently it was found that OC showed a strong anti-cancer ac-
tivity. Now, Fu Fang Wan Nian Qing Jiao Nang (Chinese
name, and approval number from the State Food & Drug
Administration in China is B20020717) is a commercial
Chinese medicine, and the main functional ingredient is OC
extract. It was reported that the solid bulb of OC contains lots

[Received on] 08-Dec.-2011
[Research funding] This project was supported by the National
Natural Science Foundation of China (Nos. 81160538 and
30960511).
[*Corresponding author] NAN Ji-Xing: Prof., E-mail:
jxnanybu@gmail.com, Tel: 86-433-243-5061, Fax: 86-433-243-5072
Δ Co-first author
These authors have no any conflict of interest to declare.
of alkaloid, saponins and polysaccharides, which can regulate
the immune system [1]. Among Ornithogalum saundersiae
saponins OSW-1 (3β, 16β, 17α-tri-hydroxycholest-5-
en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-
(1→3)-(2-O-acetyl-α-L-arabinopyranoside)) is one of the
most efficiently active components isolated from OC. It has
been reported that OSW-1 was cytotoxic against several
types of malignant cells at nanomolar concentrations, which
is approximately 10 to 100 times more potent than such
clinically applied anticancer agents as camptothecin, adria-
mycin and paclitaxel [2]. Total polysaccharides, total saponin,
and total alkaloids of OC could significantly inhibit cell vi-
ability on different cancer cell lines. Toxicity studies for
safety evaluation also verified that OC has no obvious toxic-
ity or side-effects [3]. Previously, we reported that OC could
prevent fulminant hepatic failure in mice by suppressing
oxidative stress and regulating the expression of HIF-1α [4].
APAP is commonly used as an analgesic and antipyretic
agent and has shown to be safe when taken at normal thera-
peutic doses. However, an overdose can lead to severe liver
failure. The hepatotoxicity of APAP is caused by
WAN Ying, et al. /Chinese Journal of Natural Medicines 2012, 10(3): 177−184
178 Chin J Nat Med May 2012 Vol. 10 No. 3 2012 年 5 月 第 10 卷 第 3 期

N-acetyl-para-benzoquinoneimine (NAPQI), a toxic meta-
bolic product of APAP. NAPQI is rapidly conjugated with
glutathione, a sulfhydryl donor. Excessive NAPQI formula-
tion or reduction in glutathione stores, which will interact
with vital cellular proteins and the lipid bilayer of hepatocyte
membranes, results in hepatocellular death and centrilobular
hepatic necrosis [5-6]. When conjugated with cellular macro-
molecules, it can lead to the production of reactive oxygen
species (ROS). Increased ROS formation may lead to the
development of oxidative stress. The antioxidant enzymes
include glutathione peroxidase (GSH-Px), superoxide dismu-
tase (SOD) and catalase (CAT), which convert active oxygen
molecules into non-toxic compounds and consequently pro-
tect the liver against oxidative damage [7]. N-acetylcysteine
(NAC) is a thiol-containing compound that has been used for
over 30 years as the antidote for APAP toxicity in human [8].
NAC acts as a glutathione (GSH) precursor, promoting GSH
synthesis and increasing hepatic GSH stores, eventually de-
toxifing NAPQI. So we selected NAC as a positive control to
evaluate the effect of OC.
Previous studies indicated that inflammation and hy-
poxia are closely linked in cellular responses [9-11]. Metabolic
process of APAP is associated with a series of sever inflam-
matory response and activates the inflammatory factor.
HIF-1a is strictly regulated by the cellular oxygen tension.
HIF-1a plays an important role in inflammation and activa-
tion of the immune response. Under normoxic conditions,
HIF-1α protein is post-translationally hydroxylated on spe-
cific proline residues to enable binding to pVHL
(von-Hippel–Lindau protein), which targets HIF-1α for ubiq-
uitinylation and proteasomal degradation. Under hypoxic
conditions, proline hydroxylation is blocked, and this leads to
an increase in HIF-1α protein and expression of HIF-1α tar-
get genes [12].
In spite of its distinct pharmacological effects, few stud-
ies have been carried out on total saponin of OC against
APAP-induced liver injury. Thus, the hepatoprotective effect
of OC against APAP-induced acute liver injury in vivo and in
vitro was investigated to explore the potential protective
mechanism.
2 Materials and Methods
2.1 Plant materials
Ornithogalum saundersiae Ait (Liliaceae) afforded by
the Affiliated Hospital of Yanbian University was purchased
from Changbai Country, Jilin Province and was authenticated
by Prof. LV Hui-Zi of the College of Pharmacy, Yanbian
University. A voucher specimen (YBUCP200509) was de-
posited in the Herbarium of College of Pharmacy, Yanbian
University, China. Total saponins from OC were prepared by
the following procedure. Briefly, the dried whole plant (500 g)
was powdered firstly and extracted with 95% ethyl alcohol (2
000 mL) at room temperature for three times, then combined
under reduced pressure to obtain extract (20 g). The ethanolic
extract was defatted with petroleum ether, diluted with water,
separated by an AB-8 resin column (Nankai University
Chemical Industry), and eluted with 95% ethanol. Finally, the
yield of total saponins fraction was 0.79% (W/W) [4].
2.2 Materials
APAP and NAC were purchased from Sigma-Aldrich Inc.
(St. Louis, MO, USA). The alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) Reagent Strips were
purchased from Arkray Incorporated (Kyoto, Japan). Cas-
pase-3, HIF-1α, COX-2 and α-tubulin monoclonal antibody
were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). CYP2E1 was obtained from Abcam Company
(Cambridge, Britain). Malonaldehyde (MDA), glutathione
(GSH), superoxide dismutase (SOD), catalase (CAT) and
glutathione peroxidase (GSH-Px) were purchased from Nan-
jing Jiancheng Biology Engineering Institute (Nanjing, Ji-
angsu, China). DAB (3, 3-diaminobenzidin) Plus Substrate
System was purchased from Lab Vision Corporation (Fre-
mont, CA, USA). Dulbeccos modified Eagles medium
(DMEM) (Solarbio Science & Technology Co., Ltd., Beijing,
China). Fetal bovine serum (FBS) and 0.5% trypsin-EDTA
were purchased from Gibco Co., Ltd. (New York, USA).
2.3 Experimental animals
Male C57BL/6 mice (21–25 g) were obtained from the
Animal Division of Jilin University (Jilin, China). Mice were
kept in an environmentally controlled room with temperature
(24 ± 1) °C and relative humidity (55 ± 1) %. The mice were
acclimatized 1 week prior to the experiment. Food and water
were freely available. Animal experiments were performed
under the latest edition of “Guide to the Care and Use of
Laboratory Animals” (National Research Council, 1996).
2.4 Experimental design in vivo
The mice were randomly assigned into six experimental
groups, each containing 8 mice. OC (300, 200 or 100 mg·kg–1,
body weight) or NAC (300 mg·kg–1, body weight) were
orally administered every day for 3 consecutive days. Two
hours after the last OC or NAC administration, mice were
injected intraperitoneally with APAP (300 mg·kg–1) except
normal group. All mice were fasted 16–18 h prior to treat-
ment with APAP dissolved in saline warmed to 40 °C. The
animals were sacrificed 12 h after APAP injection. The blood
samples were collected from the carotid artery and liver tis-
sues were promptly removed. Serum was separated by cen-
trifugation at 3 000 r·min–1, 4 °C, then stored at –20 °C to
analyze ALT and AST levels. The liver tissue samples were
divided into two parts. One was immersed in 10% neutral
buffered formaldehyde for immunohistochemical examina-
tions and the other was stored at −80 °C for further analysis.
2.5 Serum enzymes and biochemical index
Serum ALT and AST activities 12 h after APAP admini-
stration were measured by an Autodry chemistry analyzer
(Spotchem SP4430, Arkray, Kyoto, Japan). MDA level, GSH,
SOD, CAT, GSH-Px activities were determined according to
manufacturer’s instructions.
WAN Ying, et al. /Chinese Journal of Natural Medicines 2012, 10(3): 177−184
2012 年 5 月 第 10 卷 第 3 期 Chin J Nat Med May 2012 Vol. 10 No. 3 179

2.6 Histopathology and immunohistochemistry
Liver tissue was fixed in 10% buffered formalin saline,
and then dehydrated in graded ethanol and embedded in par-
affin max. The tissue was cut into 4 µm sections. Immuno-
histochemical analysis was deparaffinaged and hydrated, and
then blocked with endogenous peroxydase. Sections were
incubated with goat anti-caspase-3 or goat anti-HIF-1α
monoclonal antibody respectively, and followed by Max
VisionTM kit and DAB kit. Finally, slides were counterstained
with hematoxylin, mounted, observed by light microscopy
and examined in a blind fashion. Positive expression area was
performed with Image-Pro Plus software (Media Cybernetics,
Inc., Georgia, USA).
2.7 Cell culture and treatment in vitro
Chang liver cells were cultured in DMEM supplemented
with 10% fetal bovine serum (FBS), in a humidified normal
atmosphere containing 5% CO2 at 37 °C. The cells were cul-
tured in a 6-well plate in 3 mL complete medium and incu-
bated for 24 h. The cultures were then washed twice in cold
phosphate buffered saline (PBS) and cultured in either APAP
(10 mmol·L–1) or APAP + OC (50, 100, 200 mg·mL–1) or
APAP + NAC (10 mmol·L–1) for 24 h. At the end of the in-
cubation, cells were prepared for Western blotting.
2.8 Cell viability assay
Cell viability was assessed with 3-(4, 5-dimethylthia-
zol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.
Chang liver cells were incubated in 96-well plates (2 × 104
cells per well) and treated with OC at various concentrations.
MTT solution (0.5 mg·mL–1 in DMEM containing 10% FBS)
was added for the last 3 h of incubation. The reduction of MTT
to formazan was read at 540 nm using a microplate reader.
2.9 Western blotting analyses
Harvested cells were washed with cold PBS for twice
and suspended in a protein lysis buffer containing protease
inhibitor (Beyotime, Jiangsu, China). The protein content was
determined using a BCA protein assay kit (Beyotime, Jiangsu,
China). Fifty micrograms of cellular protein was subjected to
SDS-PAGE. The separated proteins were electrophoretically
transferred onto the PVDF membrane, blocked with PBST
(0.05% tween 20 in PBS) containing 5% skim milk for 1 h at
the room temperature. The membrane was incubated over-
night at 4 °C with CYP2E1, COX-2 and α-tubulin antibodies;
then incubated with HRP conjugated secondary antibodies
for 1 h at the room temperature. Protein bands were visual-
ized with enhanced chemiluminescence using WEST-ZOLTM
(plus) (iNtRON Biotechnology Co., Seongnam, Korea) and
exposed with X-ray film. Densities of the immunoreactive
bands were analyzed with Quantity One software (Bio-Rad,
USA)
3 Statistical Analysis
Statistical analysis was performed by statistical software
PRISM 5.0 (Graphpad Software, Inc., San Diego, CA). Data
were analyzed using one-way analysis of variance (ANOVA)
followed by Turkey’s test. Results were present as mean ±
standard error of mean (S.E.M.). Statistical significance was
considered at P < 0.05, P < 0.01 and P < 0.001.
4 Results
4.1 Effects of OC on ALT and AST levels
Firstly, we examined the effect of OC on serum of ALT
and AST levels. The dose of 300 mg·kg–1 APAP successfully
induced liver injury and significantly elevated the activities
of ALT and AST 12 h after administration in mice, while in
the mice pretreated with different doses of OC or NAC, the
levels of ALT and AST lowered to the normal level (Fig. 1A,
1B).
4.2 Effects of OC on lipid peroxidant products and anti-
oxidase levels
MDA is a secondary metabolite of lipid peroxidation.
The hepatotoxicity of APAP leads to the development of
oxidative stress and increases the products of lipid peroxida-
tion. The MDA content was increased in APAP group (12.52
± 1.43 nmol·mg–1 protein) compared with the normal group
(4.47 ± 0.13 nmol·mg–1 protein). Mice were treated with OC
(300, 200, 100 mg·kg–1); liver MDA production were de-
creased significantly and the contents were 7.39 ± 1.35, 8.82
± 1.63, 8.89 ± 2.9 nmol·mg–1 protein respectively, and NAC
group (5.72 ± 0.93 nmol·mg–1 protein) was closer to the nor-
mal (Fig. 2A). GSH plays an important role in the detoxifica-
tion of APAP. Due to the toxicity, metabolic products of
APAP caused GSH depletion. GSH levels in APAP group
were decreased markedly (255.78 ± 25.50 mg·g–1 protein).
OC pretreatment (300, 200, 100 mg·kg–1) or NAC (300
mg·kg-1), GSH levels were (381.62 ± 30.49), (373.63 ±
41.96), (349.47 ± 38.02), (418.68 ± 21.47) mg·g–1 protein
respectively, and NAC group could keep the GSH levels
close to the normal (418.68 ± 21.47 mg·g–1 protein) (Fig. 2B).
The production of reactive oxygen species in liver was
eliminated by antioxidant enzyme to reduce liver damage.
APAP-induced levels of CAT, SOD and GSH-Px were de-
creased approximately by half compared with those of the
normal group. However, OC or NAC pretreatment recovered
the antioxidant enzyme levels to the normal (Table 1).
4.3 Histopathological examination after OC treatment
Liver tissues from the normal group showed a normal
lobular architecture with central veins. However, the liver
tissues from the APAP-treated group showed that a large
number of inflammatory cells infiltrated to the intralobular
and interlobular regions. The liver structure was destroyed
and there were more necrotic and fatty degenerated liver cells
than the normal. OC treatment reversed the hepatic regions to
a great extent, and the liver morphological appearance was
similar with that of NAC group (Fig. 1C).
4.4 Immunohistochemistry examination after OC treatment
In view of immunohistochemical results, it was obvious
that caspase-3 expressed mainly in cytoplasm and HIF-1α
WAN Ying, et al. /Chinese Journal of Natural Medicines 2012, 10(3): 177−184
180 Chin J Nat Med May 2012 Vol. 10 No. 3 2012 年 5 月 第 10 卷 第 3 期



Fig. 1 Effects of OC on ALT and AST levels and histopathological analysis. The serum and liver tissues were collected 12 h
after APAP treatment. (A) Effects of OC on ALT levels. (B) Effects of OC on AST levels. Data were represented as mean ± S.E.M.
(n = 8). *** P < 0.001 vs Normal group; ### P < 0.001, ## P < 0.01 vs APAP group. (C) Histopathological analysis of liver sections
with hematoxylin and eosin staining. A. Normal B. APAP C. APAP + OC300 D. APAP + OC200 E. APAP + OC100 F. APAP +
NAC. All slides are 100 × magnifications. Arrows present the severe inflammatory infiltration hepatocyte


Fig. 2 Liver MDA and GSH levels. Liver tissues were collected 12 h after APAP treatment. (A) Effects of OC on liver MDA
formation. (B) Effects of OC on liver GSH levels. Data were represented as mean ± S.E.M. (n = 8). Compared with normal group,
***P<0.001 vs normal group; ###P < 0.001, ## P < 0.01 vs APAP group

mainly in nucleus and cytoplasm, positive staining appear-
ance in buff and yellow brown areas respectively. The hepa-
totoxicity of APAP increased the expressions of caspase-3
and HIF-1α, but treated with different doses of OC, the posi-
tive areas were decreased observably just the same as NAC
(Figs. 3A, 3B).
4.5 Effect of OC on CYP2E1 and COX-2 expression
We chose MTT method to assess the toxicity of OC on
Chang liver cells. Under different concentrations of OC
treatment cell viability was similar to that of the normal. It in-
dicated that OC has no cytotoxicity in Chang liver cell (Fig. 4).
In Chang liver cell line, the expression of CYP2E1
WAN Ying, et al. /Chinese Journal of Natural Medicines 2012, 10(3): 177−184
2012 年 5 月 第 10 卷 第 3 期 Chin J Nat Med May 2012 Vol. 10 No. 3 181

Table 1 Effect of OC on liver antioxidant enzymes CAT,
SOD, GSH-Px levels
Group CAT(U/mg protein)
SOD (U/mg
protein)
GSH-Px
(U·L–1)
Normal 64.27 ± 4.22 80.51 ± 6.48 956.48 ± 34.46
APAP 38.04 ± 2.48** 46.27 ± 5.05** 687.32±25.01***
APAP+OC300 65.22 ± 3.34## 77.40 ± 5.07## 851.67 ± 27.81##
APAP+OC200 63.85 ± 5.52## 76.02 ± 6.82## 819.91 ± 34.19#
APAP+OC100 64.04 ± 6.30## 72.61 ± 5.94# 829.62 ± 16.9##
APAP+NAC 58.91 ± 3.63# 78.70 ± 3.36## 907.86 ±21.21###
Data were represented as mean ± S.E.M. (n = 8). ***P < 0.001 vs
normal group; ###P < 0.001, ##P < 0.01, #P < 0.05 vs APAP group
(One-way analysis of variance (ANOVA) followed by Turkey test).
was highly increased with APAP treatment, while the expres-
sion of CYP2E1 with OC pretreatment was reduced markedly.
However, NAC did not show a significant inhibition on the
expression of CYP2E1. The same result appeared in the ex-
pression of COX-2, which was induced in macrophages and
endothelial cells by proinflammatory cytokines, damaging
the liver function [13]. APAP-induced COX-2 expression was
increased much more than the normal in Chang liver cell,
nevertheless OC and NAC treatment recovered the COX-2
levels. This indicated that OC reversed the inflammatory
factor induced by APAP and inhibited CYP2E1 the metabo-
lite enzyme of APAP (Fig. 5).

Fig. 3 Immunohistochemistry with caspase-3 and HIF-1α in APAP-intoxicated Liver. (A) Immunohistochemistry with caspase-3
in APAP-intoxicated Liver. A. Normal B. APAP C. APAP + OC300 D. APAP + OC200 E. APAP + OC100 F. APAP + NAC. Slides
stained with caspase-3 are observed under 100 × magnifications. (B) Immunohistochemistry with HIF-1α in APAP-intoxicated
Liver. Slides are observed under 400 × magnification. Analysis data was determined by Image - pro plus tool and represented as
mean ± S.E.M. of three independent experiments

Fig. 4 Effect of OC on cell viability. Chang liver cells were
incubated with OC (3.125–400 µg) for 24 h. Cell viability was
measured using MTT assay. Results are expressed as the
percent of control cell viability. Data are presented as mean ±
S.E.M. for each dose
5 Discussions
APAP is a well-known, long trusted, and widely avail-
able over-the-counter analgesic and antipyretic drug, how-
ever, its overdose causes acute liver damage. The level of
serum ALT and AST and histopathological results of liver
tissues are the common measures taken to assess APAP toxic-
ity in the experimental setting. In our recent work, 300
mg·kg–1 APAP significantly increased liver serum ami-
notransferase activities, serious hemorrhagic hepatic necrosis
and inflammation appeared in histopathological examination
results (Fig. 1A, 1B, 1C). After APAP administration, cas-
pase-3 cleavage was observed from the immunohistochemical
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182 Chin J Nat Med May 2012 Vol. 10 No. 3 2012 年 5 月 第 10 卷 第 3 期


Fig. 5 Effect of OC on CYP2E1 and COX-2. The cell CYP2E1 and COX-2 protein levels were detected at 24 h after APAP
treatment on Chang liver cell. Each immunoreactive band was digitized and expressed as a ratio of α-tubulin levels. The ratio of
the normal group band was set to 100%. Values of densitometric analysis were mean ± S.E.M. of three independent experiments.
###P < 0.001 vs control group. ***P < 0.001 vs APAP alone group
data (Fig. 3A). Caspase-3 activation seems to play a key role
in the initiation of cellular events during the apoptotic proc-
ess, which interacts with Fas system, Bcl-2 family, NO and
finally facilitates apoptosis [14-16]. OC administration not only
suppressed the activities of ALT and AST, and decreased the
level of caspase-3 cleavage, but also ameliorated the extent of
inflammatory infiltration in liver and improved the liver ar-
chitecture. The results were coincident with the expression of
COX-2 in vitro (Fig. 5). COX-2 is one of the key proinflam-
matory cytokines and well-known for their inflammation and
immune response effects. COX-2, an inducible isoenzyme,
plays a crucial role in exaggerating inflammation [17]. These
results demonstrated that OC might prevent the liver injury
caused by APAP overdose. Moreover, regulating the in-
flammation infiltration and inhibiting COX-2 expression
might be related with the hepatoprotective mechanism of OC
against APAP.
APAP-induced hepatotoxicity has been linked to lipid
peroxidation and ROS production. Oxidative damage medi-
ated by ROS are toxic to cells, because they can react with
most cellular macromolecules inactivating enzymes or dena-
turing proteins, causing DNA damage, and thereby disrupting
cellular function and integrity. Similar effects were observed
with relatively more production of MDA after APAP admini-
stration. MDA, a marker for oxidative stress, is one of reac-
tive species and occurs naturally. ROS can degrade polyun-
saturated lipids, and form MDA [18]. GSH plays a key role in
the progress of detoxification of APAP, and GSH stores are
markedly depleted when liver necrosis begins [19]. GSH defi-
ciency leads to cellular damage followed by severe liver mi-
tochondrial degeneration. CAT, SOD and GSH-PX are all
related with antioxidant system in the internal environment
and reduction of hydrogen peroxide and other peroxide. In
our study, MDA products were significantly increased after
APAP administration (Fig. 2A), while GSH levels, CAT,
SOD and GSH-PX activities were depressed, which owed to
the hepatotoxicity of APAP (Fig. 2B, Table 1). However, OC
pretreatment effectively suppressed these alterations and
reversed the levels of oxidative stress. These results indicated
that antioxidative activity of OC might be an important effect
involved in the pathological process of OC against
APAP-induced acute liver injury.
The biotransformation of APAP mainly occurs via con-
jugation of glucuronide and sulfate by the transferase en-
zymes that are rarely responsible for toxic metabolite forma-
tion and their nontoxic products are generally ready for ex-
cretion [20-23]. Toxicity caused by APAP overdose is believed
to occur by initial hepatic metabolism through cytochrome
P450 enzymes to the highly reactive metabolite NAPQI,
which would exacerbate oxidation stress in conjunction with
mitochondrial dysfunction, and would especially lead to
massive hepatocytes necrosis, liver damage or death.
CYP2E1 is the important enzyme in the metabolic process of
APAP, which metabolizes numerous small molecules of
toxicological interest including ethanol, APAP, halothane,
carbon tetrachloride and carcinogens, such as nitrosamines.
CYP2E1 also transforms several endogenous substrates in-
cluding acetone, glycerol, and different fatty acids [24-26] and
generates large amounts of ROS, which can damage cellular
and mitochondrial components. In order to evaluate the role
WAN Ying, et al. /Chinese Journal of Natural Medicines 2012, 10(3): 177−184
2012 年 5 月 第 10 卷 第 3 期 Chin J Nat Med May 2012 Vol. 10 No. 3 183

of CYP2E1 in the mechanism of OC against APAP-induced
hepatotoxicity, we detected the levels of CYP2E1 in Chang
liver cell and found that OC could effectively inhibit its ex-
pression (Fig. 5). The result revealed that OC might decrease
the metabolism activity of CYP2E1, and further reduce the
toxic reactive metabolite of APAP.
The high energy consumption of hepatocytes renders
them vulnerable to reductions in oxygen availability. There-
fore, hypoxia is an important factor for cell damage in the
liver [27]. HIF-1α, an oxygen-sensitive transcription factor,
affects cellular response to injury. Hypoxia regulates cell
damage, inflammation, liver regeneration, stimulus of angio-
genesis and fibrogenesis, and promotes liver carcinogenesis.
Most of these events are mediated by HIF-1α under hypoxia.
In addition to hypoxia, oxidative stress might also might
promote HIF-1α induction [28]. In recent years, it has become
evident that disturbances of liver microcirculation, hypoxia,
and angiogenesis occur for a long time before the onset of
damaged liver [29]. Also, COX-2 expression is known to
catalyze the synthesis of prostaglandins which could induce
the expression of HIF-1α and the degradation of vHL protein
[30]. Immunohistochemical results revealed that the positive
expression of HIF-1α was highly increased in APAP-exposed
mice. And this increase was significantly attenuated by OC
(Fig. 3B), which indicated that hepatocytes might be stricken
by hypoxia caused by APAP. Therefore, we could understand
that hepatoprotective effect of OC might be associated with
the inhibition of HIF-1α. The results were coincident
withthose in vitro.

Fig. 6 Potential hepatoprotective mechanism of OC against
APAP toxicity. OC decreased liver injury degree through
inhibited caspase-3 expression and effectively attenuated
oxidative stress via declined MDA content and raised the
activity of antioxidase, that may be due to the inhibition of
P450 2E1 activity. At meanwhile, OC showed prominent
anti-inflammatory effect during the liver injury process via
decreased proinflammatory COX-2 and HIF-1α expressions
significantly
The results of the present study indicate that OC mark-
edly prevented acute liver injury caused by APAP and its
protective effects were comparable to NAC. Our findings
outline a mechanistic understanding of how OC protects liver
form APAP-induced toxicity. The potential hepatoprotective
effect of OC against APAP might be related with antioxida-
tion, inhibition of HIF-1α. Also, the exact mechanism of OC
hepatoprotective effect against APAP requires further re-
search (Fig. 6).
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虎眼万年青通过抑制 CYP2E1和 HIF-1α保护对乙酰氨基酚诱导
的急性肝损伤
宛 莹 Δ, 吴艳玲 Δ, 廉丽花, 南极星*
延边大学长白山生物资源与功能分子教育部重点实验室, 延吉 133002
【摘 要】 目的：通过对乙酰氨基酚体内体外实验建立急性肝损伤模型, 观察虎眼万年青总皂苷的肝保护作用。方法：体
内实验, 小鼠预先口服给予虎眼万年青总皂苷(300, 200, 100 mg·kg–1) 或 N-乙酰-L-半胱氨酸(300 mg·kg–1)3 次, 每次间隔 24 小时,
末次给药后 2 小时腹腔注射对乙酰氨基酚。体外实验, 张氏肝细胞培养在含有虎眼万年青(50, 100, 200 mg·mL–1)或 N-乙酰-L-半
胱氨酸(10 mmol·L–1)的培养基中, 同时加入对乙酰氨基酚(10 mmol·L–1)培养 24 小时。结果：虎眼万年青总皂苷在对乙酰氨基酚
诱导肝损伤 12 小时后具有较强的肝保护作用, 抑制了血清中谷草转氨酶和谷丙转氨酶的活性, 减少了脂质过氧化物的生成, 提
高了过氧化氢酶、超氧化物歧化酶和谷胱甘肽过氧化物酶的活性, 抑制了细胞凋亡蛋白酶 caspase-3 和缺氧诱导因子-1α 的表达。
体外实验结果显示虎眼万年青总皂苷显著减少了对乙酰氨基酚代谢过程中关键酶细胞色素 P450 2E1 的活性及环氧化酶 2 的表
达。结论：虎眼万年青总皂苷对对乙酰氨基酚诱导的肝损伤具有保护作用其肝保护作用机制可能与抗氧化, 抑制细胞色素 P450
2E1 的活性和减少缺氧诱导因子 1α 的生成有关。
【关键词】 虎眼万年青; 对乙酰氨基酚; 凋亡; 细胞色素 P450 2E1; 缺氧诱导因子-1α

【基金项目】 国家自然科学基金(Nos. 81160528, 30960511)