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Original Contribution
Caffeic acid prevents acetaminophen-induced liver injury by activating
the Keap1-Nrf2 antioxidative defense system
Chun Pang
a,c,1
, Zhiyong Zheng
a,1
, Liang Shi
a
, Yuchen Sheng
b
, Hai Wei
c
,
Zhengtao Wang
a
, Lili Ji
a,
n
a
Shanghai Key Laboratory of Complex Prescription and MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica,
Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China
b
Center for Drug Safety Evaluation and Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
c
Center for Traditional Chinese Medicine and Systems Biology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
article info
Article history:
Received 23 August 2015
Received in revised form
14 December 2015
Accepted 19 December 2015
Available online 23 December 2015
Keywords:
Caffeic acid
Acetaminophen
Hepatotoxicity
Nrf2
Keap1
abstract
Acute liver failure induced by acetaminophen (APAP) overdose is the main cause of drug-induced liver
injury (DILI). Caffeic acid (CA) is a phenolic compound from many natural products. This study aims to
investigate the protective mechanism of CA in APAP-induced liver injury. The results of serum alanine/
aspartate aminotransferases (ALT/AST), liver myeloperoxidase (MPO) activity, liver glutathione (GSH) and
reactive oxygen species (ROS) levels demonstrated the protection of CA against APAP-induced liver in-
jury. Liver histological observation provided further evidences of CA-induced protection. CA was found to
reverse the APAP-induced decreased cell viability in human normal liver L-02 cells and HepG2 cells. CA
also reduced the increased cellular ROS level induced by APAP in hepatocytes. The results of luciferase
assay and Western-blot analysis showed that CA increased the transcriptional activation of nuclear factor
erythroid 2-related factor 2 (Nrf2) in the presence of APAP. Nrf2 siRNA reduced the protection of CA
against APAP-induced hepatotoxicity. CA also reversed the APAP-induced decreased mRNA and protein
expression of heme oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase 1(NQO1). In addition,
HO-1 inhibitor zinc protoporphyrin (ZnPP) and NQO1 inhibitor diminutol (Dim) reduced the protection
of CA against APAP-induced hepatotoxicity. CA also decreased the expression of kelch-like ECH-asso-
ciated protein-1(Keap1). Molecular docking indicated the potential interacting of CA with Nrf2 binding
site in the Keap1 protein. CA had little effect on the enzymatic activity of cytochrome P450 (CYP) 3A4 and
CYP2E1 in vitro. In conclusion, we demonstrated that CA prevented APAP-induced hepatotoxicity by
decreasing Keap1 expression, inhibiting binding of Keap1 to Nrf2, and thus activating Nrf2 and leading to
increased expression of antioxidative signals including HO-1 and NQO1.
&2015 Elsevier Inc. All rights reserved.
1. Introduction
Caffeic acid (CA) is a natural polyphenolic compound derived
from coffee, some fruits and traditional Chinese medicines [1–7].
CA and its analogs have shown a variety of pharmacological ac-
tivities including anti-inflammation, anti-cancer and anti-virus [8].
In addition, this compound is easily absorbed through the gut
barrier [9], so enhancing its mode of action. Previous reports have
already shown that CA prevented liver reperfusion injury and
griseofulvin-, nickel-, or doxorubicin-induced hepatotoxicity; fur-
thermore its antioxidative capacity contributes to protecting liver
against injury [10–13]. Previously, we demonstrated that CA, as a
main compound in medicinal herb Flos Lonicerae, prevented
acetaminophen (APAP)-induced hepatotoxicity in human normal
liver L-02 cells in vitro [7]. CA has also been reported to inhibit
APAP-induced liver injury in both mice and rats in vivo [14].
However, the precise mechanism involved in its protection
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/freeradbiomed
Free Radical Biology and Medicine
http://dx.doi.org/10.1016/j.freeradbiomed.2015.12.024
0891-5849/&2015 Elsevier Inc. All rights reserved.
Abbreviations: APAP, acetaminophen; DILI, drug-induced liver injury; CA, Caffeic
acid; ALT/AST, alanine/aspartate aminotransferases; MPO, myeloperoxidase; GSH,
glutathione; ROS, reactive oxygen species; Nrf2, nuclear factor erythroid 2-related
factor 2; HO-1, heme oxygenase 1; NQO1, NAD(P)H: quinone oxidoreductase 1;
ZnPP, zinc protoporphyrin; Dim, diminutol; Keap1, kelch-like ECH-associated
protein-1; CYP, cytochrome P450; NAPQI, N-acetyl-p-benzoquinone imine; NAC,
N-acetylcysteine; H
2
DCFDA, 2ʹ-7ʹ-Dichlorodihydrofluorescein diacetate; FBS, fetal
bovine serum; BSO,
L
-Buthionine-(S, R)-sulfoximine; H&E, haematoxylin and eosin;
MTT, 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide; TRE, tran-
scription response element; DDTC, sodium diethyldithiocarbamate trihydrate; GCL,
glutamate-cysteine ligase; ARE, antioxidant-related elements; GR, glutathione
reductase
n
Corresponding author.
E-mail address: lichenyue1307@126.com (L. Ji).
1
These two authors contributed equally to this paper.
Free Radical Biology and Medicine 91 (2016) 236–246
remains unclear.
Drug-induced liver injury (DILI) is a major clinical problem.
Acute liver failure induced by APAP overdose is common, and is
reported to be the main reason for DILI in the United States and
the United Kingdom [15,16]. Despite great efforts made over the
last 40 years, the mechanism of APAP-induced liver injury is still
not completely elucidated. However, it is widely accepted that
APAP can be metabolized by liver cytochrome P450 (CYP) into a
reactive metabolite named N-acetyl-p-benzoquinone imine (NAP-
QI), which depleted cellular glutathione (GSH) and disturbed cel-
lular redox balance, and thus led to oxidative stress-induced liver
injury [17,18]. The commonly used antidote for APAP detoxifica-
tion is N-acetylcysteine (NAC), which is a precursor for cellular
GSH synthesis and also is a well-known antioxidant [19]. Although
NAC attenuates APAP-induced hepatotoxicity, some patients may
still develop liver injury despite administration of recommended
dosage [20]. Therefore, there is a need to find a more effective and
safe drug for APAP detoxification.
The transcription factor nuclear factor erythroid 2-related fac-
tor 2 (Nrf2) regulates the constitutive and inducible expression of
a variety of genes involved in drug metabolism, detoxification, and
antioxidative defenses [21]. Kelch-like ECH-associated protein 1
(Keap1), an adapter subunit of Cullin 3-based E3 ubiquitin ligase,
regulates the degradation of Nrf2 [22]. The Keap1-Nrf2 system is
thought to play a critical role in liver oxidative injury, and has been
considered as a prospective target for liver disease [21,22]. Here,
we investigated CA-induced protection against APAP-induced liver
oxidative injury, and the involvement of the Keap1-Nrf2 signaling
pathway.
2. Materials and methods
2.1. Chemical compounds and reagents
CA was purchased from Beijing Aoke Biological technology Co.,
Ltd (Beijing, China), and the purity is over 98.5%. Kits for analysis of
alanine/aspartate aminotransferase (ALT/AST) and myeloperox-
idase (MPO) were purchased from Nanjing Jiancheng Bioengi-
neering Institute (Nanjing, China). Cignal
™
Reporter Assay kit for
Nrf2&Nrf1, RNeasy
s
Plus Mini kit and Attractene were bought from
Qiagen (Hilden, German). Duan-Glo
s
Luciferase Assay System was
purchased from Promega (Madison, WI). Vivid
s
CYP2E1, Vivid
s
CYP1A2, and Vivid
s
CYP3A4 kits, Opti-MEM
s
, lipofectamine
RNAiMAX, 2ʹ-7ʹ-Dichlorodihydrofluorescein diacetate (H
2
DCFDA),
RPMI1640, and fetal bovine serum (FBS) were purchased from Life
Technology (Carlsbad, CA). NE-PER
s
nuclear and cytoplasmic ex-
traction reagents, and Pierce
s
BCA Protein Assay Kit were pur-
chased from ThermoFisher Scientific (Waltham, MA). Whole cell
protein extraction kit and enhanced chemiluminescence kit were
all obtained from Millipore (Darmstadt, Germany). Antibodies for
immunoblotting including anti-Actin, -Lamin B, and -Keap1 were
all purchased from Cell Signaling Technology (Danvers, MA) (all
1:1000 dilutions). Antibodies for immunoblotting including anti-
Nrf2, -HO-1, -GCLC, -GCLM, and -NQO1 were all bought from Santa
Cruz (Santa Cruz, CA) (all 1:200 dilutions). Peroxidase-conjugated
goat anti-rabbit immunoglobulin G (IgG) (H þL) and anti-mouse
IgG (HþL) were purchased from Jackson ImmunoResearch (West
Grove, PA). PrimeScript
s
RT Master Mix and SYBR
s
Premix Ex Taq
™
were bought from Takara (Shiga, Japan). Control siRNA and Nrf2
siRNA were both purchased from Santa Cruz (Santa Cruz, CA).
APAP, NAPQI,
L
-Buthionine-(S, R)-sulfoximine (BSO), ZnPP, Dim and
other reagents unless indicated were purchased from Sigma Che-
mical Co. (St. Louis, MO).
2.2. Animals and treatments
Specific pathogen-free male ICR mice (16–20 g body weight)
were purchased from Shanghai Laboratory Animal Center of Chi-
nese Academy of Science (Shanghai, China). The animals were
supplied with standard laboratory diet and water ad libitum at a
temperature 2271°C with a 12 h light–dark cycle (6:00–18:00)
and 6575% humidity. All animals were received humane care in
compliance with the institutional animal care guidelines approved
by the Experimental Animal Ethical Committee, Shanghai Uni-
versity of Traditional Chinese Medicine.
Forty mice were randomly divided into 4 groups: (1) vehicle
control, (2) APAP (400 mg/kg), (3) APAP (400 mg/kg) þCA (10 mg/
kg), and (4) APAP (400 mg/kg) þCA (30 mg/kg). Mice were pre-
administered orally with CA (10, 30 mg/kg per day) for 7 con-
secutive days. On the last day, mice were orally given a single dose
of APAP (400 mg/kg) after administration of CA for 1 h. Animals
were then killed 4 h after APAP intoxication, and plasma and liver
tissue were collected.
For analyzing the effects of CA alone, 24 ICR mice were ran-
domly divided into 3 groups: (1) vehicle control, (2) CA (10 mg/
kg), and (3) CA (30 mg/kg). Mice were administered orally with CA
(10, 30 mg/kg per day) for 7 consecutive days. Animals were then
killed 5 h after the last administration of CA, and plasma and liver
tissue were collected.
2.3. Analysis of serum ALT/AST activities
The blood samples obtained were kept at room temperature for
2 h. Serum was then collected after centrifugation at 840 gfor
15 min. Serum ALT and AST were measured with kits according to
the manufacturer's instructions.
2.4. Analysis of liver GSH amount
Liver GSH amount was determined according to our previous
reported method [23].
2.5. Analysis of liver MPO activity
Liver MPO enzymatic activity was determined according to the
manufacturer's instruction. Protein concentration was detected by
BCA kit and MPO activity was expressed as units/g protein.
2.6. Liver histological observation
Slices of mice livers were fixed in 10% phosphate buffered
saline (PBS)–formalin for at least 24 h and then embedded in
paraffin for histological assessment of tissue damage. Samples
were subsequently sectioned (5
μ
m), stained with haematoxylin
and eosin (H&E), and then observed under a light microscope
(Olympus, Japan) to evaluate liver damage.
2.7. Cell culture
The L-02 cell line was derived from an adult human normal
liver [24] (Cell Bank, Type Culture Collection of Chinese Academy
of Sciences, Shanghai). Hepatoma-derived HepG2 cell line was
obtained from the American Type Culture Collection (Manassas,
VA). L-02 or HepG2 cells were cultured in RPMI1640 or MEM
supplemented with 10% [v/v] fetal bovine serum, 2 mM glutamine,
100 U/ml penicillin and 100 mg/ml streptomycin.
2.8. Cell viability assay
Cells were seeded into 96-well plates. After attachment, cells
C. Pang et al. / Free Radical Biology and Medicine 91 (2016) 236–246 237
were pre-incubated with or without various inhibitors for 15 min,
and then incubated with or without CA for another 15 min, and
then incubated with or without APAP or its metabolic product
NAPQI for another 48 h. After treatment, cells were incubated with
500
μ
g/ml 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium
bromide (MTT) for 4 h. The formed formazan in surviving cells was
dissolved in 10% SDS–5% iso-butanol–0.01 M HCl as described
previously [25], and the optical density was measured at 570 nm
with 630 nm as a reference. Cell viability was calculated as the
percentage of control.
2.9. Measurement of liver and cellular ROS
Cellular ROS were measured using the probe H
2
DCFDA as de-
scribed in our previous published paper [25]. As for detecting liver
ROS level, cold liver homogenate were centrifuged (10,000 g,
15 min, 4 °C). The supernatants were incubated with 10
μ
M
H
2
DCFDA in the dark for 1 h, and then were transferred to a Black
wall with clear bottom 96-well plate. Fluorescence was im-
mediately read at excitation 485720 nm, emission 525 720 nm
using a spectrophotometer (BioTek Synergy H4, Winooski, VT).
Protein concentrations in supernatants were assayed by BCA kits,
and all the results were calculated as units of fluorescence per
microgram of protein and presented as percentage of control (% of
control).
2.10. Nrf2&Nrf1 luciferase reporter assay
Nrf2&Nrf1 luciferase reporter assay was performed as de-
scribed in Cignal
™
Reporter Assay kit. Briefly, L-02 cells (2.5 10
4
per well) were transfected with Nrf2/1 transcription response
element (TRE) containing construct diluted in Opti-MEM
s
using
Attractene reagent. After 24 h of transfection, cells were pre-
treated with various concentrations of CA for 15 min, and then
incubated with APAP (7.5 mM) for additional indicated time. Lu-
ciferase activities were measured using the Duan-Glo
s
Luciferase
Assay System, and the constitutively expressed Renilla luciferase
was used as an internal control.
2.11. Protein extraction
Cells were seeded into dishes. After attachment, cells were pre-
incubated with or without CA (10, 50
μ
M) for 15 min, and then
incubated with or without APAP for additional indicated time.
After treatment, cellular proteins were isolated using whole cell
protein extraction kit according to the manufacturer's instruction.
Cytosolic and nuclear proteins were isolated as described in
NE-PER
s
nuclear and cytoplasmic extraction kit. Protein con-
centration was detected by BCA Kits, and all the samples in the
same experiment were normalized to the equal protein
concentration.
2.12. Western-blot analysis
Protein samples were separated by SDS-PAGE gel electrophor-
esis and electrophoretically transferred to PVDF membrane, and
then the membranes were probed with appropriate combination
of primary and horseradish peroxidase-conjugated secondary an-
tibodies. Proteins in the membranes were visualized by enhanced
chemiluminescence kits. The protein bands were quantified by the
average ratios of integral optic density following normalization to
the expression of internal control
β
-actin or Lamin B, and the re-
sults were further normalized to control.
2.13. siRNA transfection
L-02 cells were cultured in 60 mm dishes (for Western-blot
analysis) or 96-well plates (for cell viability assay). Control siRNA
(no silencing) and Nrf2 siRNA were transfected into cells by using
lipofectamine RNAiMAX. For cell viability assay, cells were pre-
treated with CA (10, 50
μ
M) for 15 min, and then incubated with
APAP (7.5 mM) for another 48 h, and surviving cells were de-
termined as described above.
2.14. RNA isolation and cDNA synthesis
L-02 cells were seeded into dishes. After attachment, cells were
pre-incubated with or without CA for 15 min, and then incubated
with APAP for additional 36 h. After treatment, cellular total RNA
was isolated by using RNeasy
s
Plus Mini kits. The RNA content was
determined by measuring the optical density at 260 nm, and cDNA
was synthesized according to the instruction described in
PrimeScript
s
RT Master Mix kit.
2.15. Real-time PCR analysis
Real-time PCR was performed by using SYBR
s
Premix Ex Taq
™
kit. Relative expression of target genes was normalized to GAPDH,
analyzed by 2
ΔΔ
Ct
method and given as ratio compared with the
control. The primer sequences used in this study are shown in the
Supplementary table.
2.16. Molecular docking analysis
To investigate the possible binding mode of CA to Keap1 as the
potential inhibitor, the molecular docking analysis was performed
as described in our previous study [26]. The confirmation of CA is
generated using Comformational Search (MMFF94X force field) in
MOE2013.
2.17. CYP450 metabolic enzyme activity assay
The effects of CA on CYP2E1, CYP1A2 and CYP3A4 enzymatic
activity were analyzed by using Vivid
s
CYP2E1, CYP1A2, CYP3A4
Kits. The CYP450 positive inhibitors used for CYP2E1 is sodium
diethyldithiocarbamate trihydrate (DDTC), for CYP1A2 is furafyl-
line, for CYP3A4 is ketoconazole, respectively. The analysis of re-
sults was performed with a Kinetic Assay Mode using the fol-
lowing equation: % inhibition (of positive inhibitor)¼(
−
−
−
1
XB
AB
)
100%; X,Aand Brespectively represent the rate of test compound
CA, solvent control and positive inhibitor control.
2.18. Statistical analysis
Data were expressed as means7standard error of the mean.
The significance of differences between groups was evaluated by
one-way ANOVA with LSD post hoc test, and Po0.05 was con-
sidered as statistically significant differences.
3. Results
3.1. CA prevented APAP-induced liver injury in vivo
Fig. 1A showed that APAP (400 mg/kg) induced the elevation of
serum ALT and AST (Po0.01), whereas CA (30 mg/kg) reduced the
APAP-induced increased ALT and AST (Po0.01, Po0.05). However,
CA (10 mg/kg) had no significant inhibition on the APAP-induced
increased ALT and AST. Fig. 1B showed that CA (10, 30 mg/kg) both
C. Pang et al. / Free Radical Biology and Medicine 91 (2016) 236–246238
Fig. 1. CA prevented APAP-induced liver injury in ICR mice: (A) serum ALT/AST activity; (B) liver GSH amount; (C) liver MPO activity; and (D) liver ROS level. Data are
expressed as means7SEM (n¼10). **Po0.01, ***Po0.001 compared to control;
#
Po0.05,
##
Po0.01 compared to APAP.
Fig. 2. Histological observation of the protection of CA against APAP-induced liver injury.Typical images were chosen from each experimental group (original magnification
100 ), and the magnified images were shown in lower left corner in each picture (original magnification 200 ): (A) vehicle control, (B) APAP (400 mg/kg), (C) APAP
(400 mg/kg)þCA (10 mg/kg), and (D) APAP (400 mg/kg) þCA (30 mg/kg).
C. Pang et al. / Free Radical Biology and Medicine 91 (2016) 236–246 239
reversed APAP-induced decreased liver GSH amount (Po0.05). As
shown in Fig. 1C and D, APAP (400 mg/kg) increased liver MPO
activity and ROS level (Po0.01, Po0.001), whereas CA (30 mg/kg)
reduced the increased MPO activity (Po0.05) and ROS level
(Po0.01), and CA (10 mg/kg) also reduced the increased ROS level
(Po0.01). CA (10, 30 mg/kg) alone increased liver GSH amount
(Po0.001), but had no effect on serum ALT/AST, liver MPO activity
and ROS level in mice (Supplementary Fig. 1).
Furthermore, mice treated with APAP (400 mg/kg) showed
severe liver damage, indicated by intrahepatic hemorrhage, lym-
phocytes infiltration and the destruction of liver structure
(Fig. 2B). After CA (10, 30 mg/kg) treatment, such phenome were
all ameliorated (Fig. 2C and D).
3.2. CA attenuated APAP-induced cytotoxicity and oxidative stress
injury in vitro
As a normal hepatic cell line, L-02 cells are commonly used in
hepatotoxicity studies induced by various hepatotoxicants [27,28].
As shown in Fig. 3A, APAP (7.5, 10 mM) decreased cell viability as
compared to control (Po0.001), whereas CA (5, 10, 25, 50
μ
M)
reversed the deceased cell viability induced by APAP (10 mM) in
L-02 cells (Po0.05, Po0.001). In addition, CA (25, 50
μ
M) also
reversed the decreased cell viability induced by APAP (7.5 mM) in
L-02 cells (Po0.05, Po0.001). Further results in HepG2 cells de-
monstrated that CA (25, 50
μ
M) reversed the decreased cell via-
bility induced by APAP (10 mM) (Fig. 3B) (Po0.01, Po0.001).
As shown in Fig. 3C, APAP (7.5 mM) increased cellular ROS level
in both L-02 and HepG2 cells (Po0.001). Whereas, CA (50
μ
M)
reduced enhanced ROS level induced by APAP in both L-02 and
HepG2 cells (Po0.01). CA (10
μ
M) also reduced the enhanced ROS
level induced by APAP in L-02 cells (Po0.01).
3.3. CA induced the transcriptional activation of Nrf2
When L-02 cells were treated with APAP (7.5 mM) for 4 h, the
expression of nuclear Nrf2 was increased, but this increase was
reduced after CA (50
μ
M) treatment (Fig. 4A). While, there was no
obvious alternation of the expression of cytosolic Nrf2. After cal-
culating the gray density of protein bands, the results clearly de-
monstrated that CA (50
μ
M) reduced the increased nuclear Nrf2
expression induced by APAP when cells were treated with APAP
for 4 h (Po0.05) (Supplementary Fig. 2A). However, when L-02
cells were treated with APAP (7.5 mM) for 18 h or 36 h, the in-
creased expression of nuclear Nrf2 was diminished. Pretreatment
with CA (50
μ
M) increased the expression of nuclear Nrf2 when
cells were incubated with APAP for 36 h. Furthermore, data from
calculating the gray density of protein bands evidenced that CA
(50
μ
M) enhanced nuclear Nrf2 expression when cells were in-
cubated with APAP for 36 h (Po0.05) (Supplementary Fig. 2A). CA
(50
μ
M) also enhanced the expression of nuclear Nrf2 in HepG2
cells when cells were incubated with APAP for 36 h (Fig. 4B). The
results of densitometric analysis of Nrf2 protein bands further
implied that nuclear Nrf2 expression was enhanced by CA in
HepG2 cells (Po0.05) (Supplementary Fig. 2B).
The luciferase reporter assay demonstrated that APAP (7.5 mM)
enhanced the transcriptional activation of Nrf2 after incubated
with cells for 4, 8, and 12 h (Po0.05, Po0.001), but CA (50
μ
M)
reduced the APAP-induced increased Nrf2 transcriptional activa-
tion when cells were incubated with APAP for 4, 8 h (Po0.05)
(Fig. 4C). By extending the incubation period, APAP-induced Nrf2
activation was reduced (Fig. 4C). However, CA (50
μ
M) increased
Nrf2 transcriptional activation when cells were incubated with
APAP for 36 h (Po0.01) (Fig. 4C).
In order to confirm the important role of Nrf2 in regulating the
protection of CA against APAP-induced hepatotoxicity, Nrf2 siRNA
was used to knock down the expression of Nrf2 in L-02 cells
(Fig. 4D). As compared with control siRNA, Nrf2 siRNA decreased
the CA-induced elevated cell viability after APAP intoxication
(Po0.05) (Fig. 4E).
3.4. HO-1 and NQO1 were involved in the protection of CA against
APAP-induced hepatotoxicity
NQO1, HO-1, and glutamate-cysteine ligase (GCL) are important
cellular antioxidative enzymes, and NQO1, HO-1, and catalytic or
modify subunits of GCL (GCLC or GCLM) are all Nrf2-regulated
downstream genes [22]. As shown in Fig. 5A, APAP (7.5 mM) en-
hanced mRNA expression of GCLc (Po0.05), but CA (10, 50
μ
M)
did not further increase the mRNA expression of GCLc (P40.05).
The results in Fig. 5A also demonstrated that APAP and APAP plus
CA (10, 50
μ
M) had no significant effect on the mRNA expression
of GCLm (P40.05). Moreover, APAP (7.5 mM) decreased mRNA
expression of both NQO1 and HO-1 (Po0.001) (Fig. 5B). After CA
(50
μ
M) treatment, the decreased mRNA expression of both NQO1
and HO-1 induced by APAP was reversed (Po0.05, Po0.01)
Fig. 3. CA attenuated APAP-induced cytotoxicity and oxidative stress injury in
hepatocytes: (A) CA prevented the hepatotoxicity induced by APAP (7.5 mM and
10 mM) in L-02 cells. (B) CA prevented the hepatotoxicity induced by APAP
(10 mM) in HepG2 cells. (C) CA reduced the APAP-induced elevation of cellular ROS
level in both L-02 and HepG2 cells. Data are expressed as means 7SEM (n¼3).
***Po0.001 compared to control;
#
Po0.05,
##
Po0.01,
###
Po0.001 compared to
APAP.
C. Pang et al. / Free Radical Biology and Medicine 91 (2016) 236–246240
(Fig. 5B). Further, the protein expression of NQO1, HO-1, GCLC, and
GCLM was observed. As shown in Fig. 5C and D, there was no
obvious difference between the protein expression of GCLC and
GCLM with or without APAP. CA also had no effect on GCLC and
GCLM protein expression. APAP (7.5 mM) decreased HO-1 protein
expression (Po0.001), whereas CA (25, 50
μ
M) reversed the de-
creased HO-1 expression induced by APAP (Po0.05) (Fig. 5C and
D). APAP had no significant effect on NQO1 protein expression
(Fig. 5C and D). However, CA (25, 50
μ
M) significantly increased
NQO1 protein expression when pre-incubated with cells before
APAP intoxication (Po0.05) (Fig. 5C and D). Next, the chemical
inhibitors for NQO1, HO-1, and GCL were used to further evaluate
the roles of those antioxidative enzymes in regulating the pro-
tection of CA against APAP-induced hepatotoxicity. As shown in
Fig. 5E, GCL inhibitor BSO (both 20 and 50
μ
M) had no obvious
effect on the protection of CA against APAP-induced cytotoxicity.
Results in Fig. 5E showed that CA (50
μ
M) prevented APAP-in-
duced cytotoxicity (Po0.001), but such protection was abrogated
by both Dim (Po0.01, Po0.001) and ZnPP (Po0.05, Po0.01)
(both 20 and 50
μ
M), that are HO-1 and NQO1 inhibitors,
respectively.
3.5. The effects of CA on Keap1 expression and molecular docking
analysis
The effect of CA on the expression of Keap1, an inhibitor of Nrf2
activation, was observed. As shown in Fig. 6A and B, no apparent
effect on keap1 expression was shown after APAP (7.5 mM)
treatment in L-02 and HepG2 cells. After cells were pretreated
with CA (50
μ
M), the Keap1 protein expression was significantly
decreased in the above hepatocytes (Po0.05). Molecular docking
analysis was used to analyze the potential binding of CA to Keap1
kelch domain, as it is important to determine whether small
molecules act as direct inhibitors of the protein–protein interac-
tion between Keap1 and Nrf2. The chemical structure of CA was
shown in Fig. 6C. The docking mode of CA in the binding site of
human Keap1 was illustrated in Fig. 6D (front view) and Fig. 6E
(top view). The three-dimension (Fig. 6F) and two-dimension
(Fig. 6G) interaction-map showed that CA had one H-benzene
interaction between the benzene rings of chromone and Arg415,
which facilitated CA anchored into the binding site. In addition,
the hydrogen atom of the hydroxyl at 3-position in CA formed
water-mediated hydrogen bonds with Ser508, and the carbonyl
group of CA could form H-bond with Gly603 and Ser363, which
Fig. 4. CA induced the transcriptional activation of Nrf2: (A) L-02 cells were pre-incubated with CA (10, 50 μM) for 15 min, and then incubated with APAP (7.5 mM) for the
indicated time. The translocation of Nrf2 from cytoplasm into nucleus was detected by immunoblotting using specific antibody. β-actin and lamin B were used as loading
control for cytoplasm or nucleus, respectively. (B) HepG2 cells were pre-incubated with CA (10, 50 μM) for 15 min, and then incubated with APAP (7.5 mM) for 36 h. The
translocation of Nrf2 from cytoplasm into nucleus was detected by immunoblotting using specific antibody. β-actin and lamin B were used as loading control for cytoplasm
or nucleus, respectively. (C) L-02 cells were transfected with Nrf2/1 transcription response element (TRE) containing construct, and pre-incubated with CA (10, 50 μM) for
15 min, and then incubated with APAP (7.5 mM) for the indicated time. Luciferase activities were measured by using a luciferase assay system. Nrf2/1 luciferase activity was
expressed as a fold induction of control cells. Data are expressed as means7SEM (n¼3). *P o0.05, ***Po0.0 01 compared to control;
#
Po0.05,
##
Po0.01 compared to APAP.
(D) L-02 cells were transiently transfected with Nrf2 siRNA and the protein expression of Nrf2 was detected by immunoblotting using specific antibody, and β-actin and
Lamin B were used as loading control. Results represent three independent experiments. (E) L-02 cells were transiently transfected with Nrf2 siRNA, and then cells were pre-
incubated with or without CA for 15 min, and then cells were further incubated with or without APAP for 48 h. Finally, cell viability was detected by MTT assay, and the
results were obtained by deducting APAP alone. Data are expressed as means 7SEM (n¼6). *Po0.1 versus control siRNA.
C. Pang et al. / Free Radical Biology and Medicine 91 (2016) 236–246 241
will help to enhance the binding affinity.
3.6. The effects of CA on the metabolic activity of CYP2E1, CYP1A2,
and CYP3A4 in vitro and NAPQI-induced hepatotoxicity
CYP2E1, CYP1A2, and CYP3A4 are regarded as the main meta-
bolic enzymes for converting APAP into the toxic and reactive
metabolite NAPQI [17,18]. Next, we observed whether CA will af-
fect the metabolic activity of CYP2E1, CY1A2, and CYP3A4 in vitro.
CA appeared to have limited inhibition on CYP2E1 over a range of
concentrations (Fig. 7A). For example, only less than 20% inhibition
of the positive inhibitor was noted at CA (100
μ
M) (Po0.05). Re-
sults of Fig. 7B showed that only the highest concentration of CA
(100
μ
M) had weak inhibition on CYP3A4 metabolic activity in
vitro (less than 20% inhibition of the positive inhibitor) (Po0.05).
Fig. 7C showed that CA had no effect on CYP1A2 metabolic activity
in vitro. Meanwhile, we also observed whether CA could prevent
NAPQI-induced hepatotoxicity in vitro. The results of Fig. 7D
showed that NAPQI (200
μ
M) decreased cell viability in both L-02
(Po0.05) and HepG2 (Po0.001) cells, whereas CA (50
μ
M) re-
versed the NAPQI-induced decreased cell viability in both above
cells (Po0.05).
Fig. 5. HO-1 and NQO1 were involved in the protection of CA against APAP-induced hepatotoxicity:(A) The mRNA expression of GCLc and GCLm was detected by Real-time
PCR. Data are expressed as means7SEM (n¼4). *Po0.05 versus control. (B) The mRNA expression of HO-1 and NQO1 was detected by Real-time PCR. Data are expressed as
means7SEM (n¼4). ***P o0.001 versus control,
#
Po0.05,
##
Po0.01 versus APAP. (C) The protein expression GCLC, GCLM, HO-1, and NQO1 was detected by im-
munoblotting using specific antibody, and β-actin was used as loading control. Results represent at least three repeated experiments. (D) The quantitative densitometric
analysis of GCLC, GCLM, HO-1, and NQO1 proteins. Data are expressed as means7SEM (n¼4). ***Po0.0 01 versus control,
#
Po0.05 versus APAP. (E) L-02 cells were pre-
incubated with BSO, ZnPP or Dim for 15 min, and then incubated with or without CA (50 μM) for another 15 min, and then incubated with APAP (7.5 mM) for another 48 h.
After treatment, the surviving cells were determined by MTT assay. The results are expressed in percentage of control and presented as the means7SEM (n¼6). ***Po0.001
versus control;
###
Po0.001 versus APAP;
$
Po0.05,
$$
Po0.01,
$$$
Po0.001 versus APAP plus CA.
C. Pang et al. / Free Radical Biology and Medicine 91 (2016) 236–246242
4. Discussion
Polyphenols are the most abundant antioxidants distributed in
plant-derived food products, and phenolic acids (mainly CA) is one
of the main classes of polyphenols [29]. The measurement of
serum ALT and AST activities demonstrated that CA (30 mg/kg)
provided the protection against APAP-induced liver injury in mice
(Fig. 1A), and further liver histological evaluation confirmed this
protection (Fig. 2). Although CA (10 mg/kg) did not reduce APAP-
induced increased ALT/AST activities (Fig. 1A), it appears that CA
(10 mg/kg)-treated livers are healthier than CA (30 mg/kg)-treated
livers in histological evaluation (Fig. 2). Meanwhile, CA (10 mg/kg)
also reversed the decreased liver GSH amount (Fig. 1B) and in-
creased ROS level (Fig. 1D) induced by APAP in mice. These results
indicate the amelioration of CA (10 mg/kg) against APAP-induced
liver injury. The above in vivo results evidenced the protection of
Fig. 6. The effects of CA on Keap1 expression and molecular docking analysis: (A) the protein expression Keap1 was detected by immunoblotting using specific antibody, and
β-actin was used as loading control. Results represent four repeated experiments. (B) The quantitative densitometric analysis of Keap1 protein. Data are expressed as
means7SEM (n¼4).
#
Po0.05 versus APAP. (C) The chemical structure of CA. (D) Front view of the docking mode of CA (Green) in the binding site of Keap1 (shown in ribbon
representation and colored by structure). (E) Top view of the docking mode of CA (Green) in the binding site of Keap1 (shown in ribbon representation and colored by
structure). (F) Representative amino acid residues surrounding CA (Red) in the binding pocket of Keap1. (G) Two-dimensional interaction map of CA and the human Keap1.
The arrows indicate potential interactions between amino acid residues and CA. (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
C. Pang et al. / Free Radical Biology and Medicine 91 (2016) 236–246 243
CA against APAP-induced liver injury, which are consistent with
previous reported study [14]. CA (10, 30 mg/kg) alone had no
obvious effect on serum ALT/AST activities when mice were orally
given with CA for 7 consecutive days (Supplementary Fig. 1A). In
addition, cell viability of L-02 and HepG2 cells demonstrated that
different concentrations of CA prevented APAP-induced cytotoxi-
city (Fig. 3A and B). Furthermore, CA reduced the APAP-induced
increased liver MPO activity (Fig. 1C), but CA alone had no ap-
parent effect on this enzyme in mice (Supplementary Fig. 1B).
Enhanced liver MPO activity generally indicates the occurrence of
liver inflammation [30]. Thus, the above results suggest the po-
tential inhibition of CA on APAP-induced hepatic inflammation.
These above results demonstrated that CA prevented APAP-in-
duced hepatotoxicity in vivo and in vitro.
Cellular GSH is critical for the detoxification of APAP when
conjugating with its metabolic product NAPQI. Thus, the accu-
mulation of NAPQI will deplete cellular GSH, so generating oxi-
dative stress-induced liver injury [17,18,31]. Our results displayed
the depletion of liver GSH in mice after APAP treatment; however
this was reversed by CA (10, 30 mg/kg) (Fig. 1B). Meanwhile, CA
(10, 30 mg/kg) also enhanced liver GSH level when CA alone was
orally given to mice (Supplementary Fig. 1C), which may con-
tribute to its antioxidant capacity. CA (10, 30 mg/kg) reduced the
APAP-induced formation of liver ROS in mice in vivo (Fig. 1D), and
it also abrogated the formation of cellular ROS in L-02 and HepG2
cells induced by APAP (Fig. 3C). However, CA (10, 30 mg/kg) alone
had no obvious effect on the formation of liver ROS in mice
(Supplementary Fig. 1D). Again, these results implied the protec-
tion of CA against APAP-induced liver oxidative stress injury.
Nrf2 is an important antioxidative transcription factor, which
has been reported to play a key role in protecting against APAP-
induced liver injury [32]. Nrf2 knockout mice were more suscep-
tible to APAP-induced liver injury [33]. Moreover, a variety of re-
ports demonstrated that some nature products prevented APAP-
induced hepatotoxicity by inducing the transcriptional activation
of Nrf2, some examples being quercetin, salvianolic acid B, gin-
senoside Rg3, sauchinone, and oleanolic acid [26,34–37]. Firstly,
our findings showed that CA pretreatment enhanced the expres-
sion of Nrf2 in nucleus when cells were incubated with APAP for
36 h (Fig. 4A and B, Supplementary Fig. 2). Secondly, luciferase
reporter assay also suggested that CA-induced Nrf2 transcriptional
activation occurred when hepatocytes were incubated with APAP
for 36 h (Fig. 4C). Thirdly, Nrf2 siRNA significantly reduced the
protection of CA against APAP-induced hepatotoxicity (Fig. 4D and
E). Thus, it could be proposed that Nrf2 plays a critical role in
regulating the protection of CA against APAP-induced hepato-
toxicity. Our results also showed that APAP weakly induced Nrf2
activation when hepatocytes were incubated with APAP for 4, 8,
and 12 h (Fig. 4C). This is consistent with previous reported study
where the APAP metabolic product NAPQI can directly activate
Nrf2 [38]. However, CA reduced APAP-induced the increased Nrf2
activation after hepatocytes were incubated with APAP for 4 and
8h (Fig. 4C), which may be due to the reduced production of
NAPQI as our further results showed that CA weakly inhibited
CYP2E1 and CYP3A4 enzymatic activity (Fig. 7A and B). There may
be other signals regulating such phenome, which requires future
investigation.
The transcription factor Nrf2 regulates the expression of a
series of antioxidative genes, including NQO1, HO-1, GCLC and
GCLM, when binding to antioxidant-related elements (ARE)
[22,39]. It appears that CA reversed the APAP-induced decrease of
mRNA and protein expression of NQO1 and HO-1 (Fig. 5B–D). In
addition, ZnPP and Dim reduced the protection of CA against
APAP-induced hepatotoxicity (Fig. 5E). These observations suggest
that CA activates Nrf2, which then enhances the expression of
NQO1 and HO-1, and thus contributes to CA-induced protection
against APAP-induced hepatotoxicity. APAP and APAP plus CA had
no obvious effects on either mRNA or protein expression of GCLM
Fig. 7. The effects of CA on the metabolic activity of CYP2E1, CYP1A2, and CYP3A4 in vitro and NAPQI-induced hepatotoxicity:(A) Inhibition of CA on CYP2E1 enzymatic
activity. (B) Inhibition of CA on CYP3A4 enzymatic activity. (C) Inhibition of CA on CYP1A2 enzymatic activity. Data are expressed as means7SEM (n¼4). *Po0.05 versus
solvent control. (D) L-02 and HepG2 cells were pre-incubated with CA (25, 50 μM) for 15 min, and then incubated with NAPQI (200 μM) for another 48 h. Af ter treatment, the
surviving cells were determined by MTT assay. The results are expressed in percentage of control and presented as the means7SEM (n¼6). *Po0.05,***Po0.001 versus
control;
#
Po0.05 versus NAPQI.
C. Pang et al. / Free Radical Biology and Medicine 91 (2016) 236–246244
(Fig. 5A, C and D). Although APAP weakly enhanced the mRNA ex-
pression of GCLc (Fig. 5A), it had no significant effect on the protein
expression of GCLC (Fig. 5C and D). Similarly, CA did not further
enhance the APAP-induced increased mRNA expression of GCLc
(Fig. 5A). BSO, a specific inhibitor for GCL which is composed of GCLC
and GCLM [40], also had no effect on CA-induced protection against
APAP-induced hepatotoxicity (Fig. 5E). Thus, we conclude that GCL is
not engaged in CA-induced protection against APAP-induced hepa-
totoxicity. GCL is critical for the de novo biosynthesis of cellular GSH
[41], which is important for the detoxification of APAP. But why GCL
is not involved in the protection of CA against APAP-induced liver
injury? We think that CA may directly conjugate with NAPQI, which
will contribute to the maintenance of cellular GSH level. Otherwise,
some other important enzymes for regulating cellular GSH amount
such as glutathione reductase (GR) may be involved.
Keap1, an inhibitor of Nrf2, acts as an adapter for Cul3/Rbx1-
mediated degradation of Nrf2 [22]. Our results showed that CA
reduced the expression of Keap1 protein in the presence of APAP
(Fig. 6A and B), and this may contribute to the activation of Nrf2
induced by CA. Furthermore, the outcome of molecular docking
(Fig. 6D–G) suggests that CA may interact with Keap1 and occupy
the Nrf2 binding site in the protein, and thus lead to the dis-
sociation of Keap1 from Nrf2, and so finally induce the transcrip-
tional activation of Nrf2.
It is reported that CYP2E1, CYP3A4 and CYP1A2 are the main
metabolic enzymes for converting APAP into NAPQI, which sub-
sequently depleted cellular GSH and so caused oxidative stress-
induced injury [42,43]. In this study, we further observed if CA-
induced protection against APAP-induced hepatotoxicity is due to
inhibition on CYP2E1, CYP3A4, and CYP1A2. The results showed
that CA displayed weak inhibition on the enzymatic activity of
CYP2E1 and CYP3A4 (both less than 20% inhibition of the positive
inhibitor) in vitro (Fig. 7A and B). However, CA had no inhibition on
the enzymatic activity of CYP1A2 in vitro (Fig. 7C). These above
results indicate that CA-induced inhibition on CYP2E1 and CYP3A4
may weakly contribute to the protection of CA against APAP-in-
duced hepatotoxicity, but it is not a major determinant. Further
results showed that CA also reversed NAPQI-induced hepatotoxi-
city in hepatocytes (Fig. 7D). The above results indicate that CA can
directly inhibit the hepatotoxicity induced by APAP's toxic meta-
bolite NAPQI, and CA-induced the weak inhibition on CYP2E1 and
CYP3A4 is not required for protection.
In summary, the present study demonstrates that CA induces
Nrf2 activation by decreasing the expression of its inhibitor pro-
tein Keap1 and blocking the binding of Nrf2 with Keap1, and then
leads to the enhanced expression of downstream antioxidative
enzymes including NQO1 and HO-1, and thus prevents APAP-in-
duced liver oxidative injury.
Acknowledgments
This work was financially supported by National Natural Sci-
ence Foundation of China (81322053 and 81573679), “Shu Guang”
project supported by Shanghai Municipal Education Commission
and Shanghai Education Development Foundation (13SG43), and
State major science and technology special projects during the
12th five year plan (2015ZX09501004-002-002).
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
theonlineversionathttp://dx.doi.org/10.1016/j.freeradbiomed.2015.
12.024.
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