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Neohesperidin dihydrochalcone against CCl4-induced hepatic injury through different mechanisms: The implication of free radical scavenging and Nrf2 activation Chuanyang Su, Xiaomin Xia, Qiong Shi, Xiufang Song, Juanli Fu, Congxue Xiao, Hongjun Chen, Bin Lu, Zhiyin Sun, Shanmei Wu, Siyu Yang, Xuegang Li, Xiaoli Ye, Erqun Song, and Yang Song J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01750 • Publication Date (Web): 15 May 2015 Downloaded from http://pubs.acs.org on May 18, 2015
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Journal of Agricultural and Food Chemistry
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Neohesperidin dihydrochalcone against CCl4-induced hepatic injury
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through different mechanisms: The implication of free radical
3
scavenging and Nrf2 activation
4
5
Chuanyang Su,† Xiaomin Xia,† Qiong Shi,† Xiufang Song,† Juanli Fu,† Congxue Xiao,†
6
Hongjun Chen,† Bin Lu,† Zhiyin Sun,† Shanmei Wu,† Siyu Yang,† Xuegang Li,† Xiaoli Ye,§
7
Erqun Song,† Yang Song*,†
8 †
9
Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest
10
University), Ministry of Education, College of Pharmaceutical Sciences, Southwest
11
University, Chongqing, People’s Republic of China, 400715
12
§
College of Life Sciences, Southwest University, Chongqing, People’s Republic of China, 400715
13
14
15
16
* Corresponding author:
17
Yang Song: College of Pharmaceutical Sciences, Southwest University, Beibei,
18
Chongqing, 400715, P R China. Tel: +86-23-68251503. Fax: +86-23-68251225. E-mail
19
address: [email protected] 1
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ABSTRACT
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Neohesperidin dihydrochalcone (NHDC), a sweetener derived from citrus, belongs to
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the family of bycyclic flavonoids dihydrochalcones. NHDC has been reported to against
24
CCl4-induced hepatic injury, but its mechanism is still unclear. We first discovered NHDC
25
showed a strong ability to scavenge free radicals. Also, NHDC induces phase II
26
antioxidant enzymes heme oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase 1
27
(NQO1) through the activation of the nuclear factor (erythroid-derived 2)-like 2
28
(Nrf2)/antioxidant response element (ARE) signaling. Further assays demonstrated
29
NHDC induces the accumulation of Nrf2 in the nucleus and augmented Nrf2-ARE binding
30
activity. Moreover, NHDC inhibits the ubiquitination of Nrf2 suggested the modification of
31
Kelch-like ECH-associated protein 1 (Keap1) and the disruption of Keap1/Nrf2 complex.
32
c-Jun N-terminal kinase (JNK) and p38 but not extracellular signal-regulated protein
33
kinase (ERK) phosphorylations were up-regulated by NHDC treatment. Taken together,
34
NHDC showed its protective antioxidant effect against CCl4-induced oxidative damage
35
via the direct free radical scavenging and indirect Nrf2/ARE signaling pathway.
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KEYWORDS: NHDC; Keap1; HO-1; NQO1; MAPK; liver; ubiquitination
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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The liver plays an important role in the detoxification of xenobiotics and drugs prior
40
to excretion. However, these toxic substances can lead to hepatic injury. Carbon
41
tetrachloride (CCl4) has often been used as a common model of acute liver damage. The
42
toxic mechanism of CCl4 was identified to involve the excessive production of free
43
radicals. CCl4 is metabolized into CCl3• and CCl3OO• radicals with the participated of
44
cytochrome P4502E1, which initiates the process of lipid peroxidation and ultimately
45
resulting necrosis.1 In the liver, excess free radicals inflict may result in liver failure or
46
liver fibrosis.
47
Previous studies have illustrated that natural compounds with antioxidant activity are
48
effective in protecting liver from oxidative damage, in this regard, the use of active
49
antioxidants for the prevention and treatment of liver diseases has drawn much
50
attention. Our previous study indicated the protective effects of neohesperidin
51
dihydrochalcone (NHDC) on CCl4-induced acute hepatic injury in vivo and in vitro.2 CCl4-
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induced acute hepatotoxicity is the most used experiment model. The remarkable
53
restoring of antioxidant enzymes and the amelioration of oxidative biomarkers
54
suggested an antioxidative activity. However, the exact molecular mechanism of NHDC
55
against CCl4-insult is quite unveiled.
56
One of the strategies to avoid free radical-mediated damage is to directly increase
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the radical scavenging capacity, numerous of studies suggested polyphenol compounds
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showed antioxidant effects via a scavenging action on both carbon- and oxygen-
59
centered radicals. The second approach is to regulate intrinsic antioxidant defenses at 3
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the transcriptional level. Recent reviews have summarized that natural products
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upregulate a group of cytoprotective genes via nuclear factor erythroid-derived 2-like 2
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(Nrf2) signaling.3,
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cytoplasm by its negative regulator protein Kelch-like ECH associated protein 1 (Keap1)
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binds tightly to Nrf2, which directing Nrf2 to proteasome degradation. Under certain
65
circumstances, Nrf2 activators trigger the dissociation of Nrf2 from Keap1 and facilitate
66
the translocation of Nrf2 into the nucleus. Nucleic Nrf2 binds to the antioxidant-
67
responsive
68
cytoprotective genes.
69
4
Under physiological conditions, Nrf2 was tightly anchored in the
element
(ARE)
consensus
sequence
to
initiate
the
transcription
of
Thus, the current study was designed to identify the protective mechanisms of NHDC.
70
We discovered that NHDC showed a significant capacity in free radical scavenging. We
71
also illustrated that NHDC potently induces the activation of Nrf2 signaling. These
72
findings revealed the cytoprotective role of Nrf2 against CCl4-intoxication in vitro and in
73
vivo models.
74
75
MATERIALS AND METHODS
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Materials. NHDC (CAS: 20702-77-6, structure shown in Fig 1), ABTS (2, 2′-
77
azinobis-3-ethylbenzothiazolin-6-sulfonic acid), DPPH (1, 1′-diphenyl-2-picryhydrazyl),
78
TPTZ (2, 4, 6-tripyridyl-s-triazine) and luminol were supplied by Aladdin Reagent
79
Database Inc. (Shanghai, China). CCl4 (≥99.5%) was purchased from Kelong Chemical
80
Co., Ltd. (Chengdu, China). MG132, the proteasome inhibitor was obtained from Selleck
81
Chemicals (Shanghai, China). The antibodies against Nrf2, Heme oxygenase-1 (HO-1), 4
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NAD(P)H:quinone oxidoreductase 1 (NQO1), Lamin B, c-Jun N-terminal kinase (JNK),
83
extracellular
84
Proteintech group, Inc. (Wuhan, China), and antibodies against β-actin, rabbit IgG,
85
EasyBlot ECL kit and nuclear/cytosol fractionation kit were purchased from Sangon
86
Biotech Co., Ltd. (Shanghai, China). Chemiluminescent electrophoretic mobility shift
87
assay (EMSA) Kit, Biotin-labeled ARE probe, SP600125 (a JNK inhibitor), PD98059 (an
88
ERK inhibitor), SB203580 (a p38 inhibitor), Western stripping buffer and Protein A
89
agarose beads were obtained from Beyotime Institute of Biotechnology (Nanjing, China).
90
Antibodies against p-JNK, p-ERK, p-p38, and Keap1 were purchased from Biosynthesis
91
Biotechnology Co., Ltd. (Beijing, China). Ubiquitin antibody was from Santa Cruz
92
Biotechnology
93
diphenyltetrazoliumbromide (MTT) was purchased from Dingguo Biotechnology Co., Ltd.
94
(Beijing, China). Lipofectamine 2000 transfection reagent was supplied by Promega
95
(Madison, WI). Nrf2 siRNA was synthesized by GenePharma Co., Ltd (Shanghai, China).
signal-regulated
(Santa
protein
Cruz,
kinase
CA).
(ERK),
3-(4,
p38
were
purchased
5-dimethylthiazol-z-yl)-3,
from
5-
96
The antioxidant activity assays. The free radical scavenging activity of NHDC was
97
determined by ferric-reducing antioxidant power (FRAP), DPPH, ABTS, hydroxyl (HO•)
98
and superoxide (O2•-) radical scavenging assays. FRAP assay is based on the reduction
99
of a ferric 2, 4, 6-tripyridyl-striazine complex (Fe3+-TPTZ) by antioxidants to the ferrous
100
form (Fe2+-TPTZ), which forming a dark blue Fe2+-TPTZ complex with an absorbance
101
maximum at 593 nm.5 DPPH assay is based on the reduction of stable radical DPPH• to
102
yellow product with the incubation of antioxidant. The absorbance was measured at 515
103
nm.5 ABTS assay is based on the ability of antioxidant inhibits the generation of blue-to-
104
green color product, which indicated the reaction of ABTS•+ and potassium persulphate. 5
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ABTS radical-scavenging efficiency was measured by spectrophotometer at 750 nm.5
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The superoxide anion scavenging ability was determined by a chemiluminescence
107
method in the pyrogallol-luminol system. The rate of pyrogallol autoxidation was
108
measured at 320 nm.6 The hydroxyl radical quenching activity was measured by using
109
hydroxyl radical generated from Fenton’s reaction Fe2+-H2O2-luminol chemiluminescence
110
method.7 All experiments were performed at least three times independently.
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Animals, Treatment and Tissue Preparation. Male Kunming mice (22 ± 2 g)
112
were provided by Chongqing Academy of Chinese Materia Medica. All animal studies
113
were approved by the Southwest University Animal Care and Use Committee.
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Experiments were conducted according to the national institution’s guidelines for the
115
care and use of laboratory animals. Animals were maintained under an air-conditioned
116
room with a 12 h light and dark cycle with free access to food and water. All animals
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were adapted in individual plastic cages for a week. Then, animals were randomly
118
distributed in four groups with six mice in each group. Control group, administered
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appropriate vehicles throughout. NHDC group, administered with NHDC (200 mg/kg
120
body weight/day, dissolved in sodium carboxymethyl cellulose, prepared immediately
121
before use) for 7 continuous days. CCl4 group, received sodium carboxymethyl cellulose
122
once daily for 7 continuous days. Three hours after final intragastric administration, mice
123
were injected with CCl4 (10 ml/kg body weight, 2% v/v in olive oil). NHDC + CCl4 group,
124
treated with NHDC (200 mg/kg body weight/day) for 7 continuous days. Three hours
125
after the final treatment, mice were injected with CCl4 (10 ml/kg body weight, 2% v/v
126
dissolved in olive oil). Animals were sacrificed 24 h after CCl4 administration. The livers
127
were removed carefully, rinsed twice with ice-cold physiological saline, and then divided 6
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into two pieces. One halves prepared for hepatic homogenate and the other were used
129
for immunohistochemical analysis. All steps were performed at 4°C.
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Cell Culture and Cell Viability Assay. The human hepatoma HepG2 cell line was
131
purchased from Third Military Medical University (Chongqing, China). HepG2 cells were
132
cultured in DMEM containing 10% (v/v) FBS (HyClone, UT), penicillin (100 U/ml) and
133
streptomycin (100 U/ml) in the humidified incubator at 37°C under 5% CO2. HepG2 cells
134
were transferred onto 96-well culture plates at a density of 5×103 cells per well. After
135
incubated overnight, cells were pre-treated with various inhibitors for 30 min and
136
replaced with fresh serum-free medium containing NHDC or solvent vehicle for another 1
137
h. Finally, cells were maintained in 0.5% v/v CCl4 for 3 h, and then cells were treated
138
with MTT (5 mg/ml final concentration) at 37°C for 4 h. The supernatant was removed
139
and 150 µl of DMSO was added, measured spectrophotometrically at 570 nm.
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Immunohistochemical Staining. The paraffin-embedded liver sections were de-
141
paraffinized and rehydrated. 3% H2O2 in methanol was used to block endogenous
142
peroxidase activity for 15 min. Antigen retrieval was measured by 1 mM EDTA buffer
143
(pH = 9.0) in a microwave for 3 min. The non-specific protein binding was blocked by
144
goat serum for 30 min. Then, the following steps were followed on the instruction of
145
Histostain™-plus and DAB substrate kits. The slides were incubated with HO-1, NQO1 or
146
Nrf2 antibodies at 4°C overnight, and incubated with biotin-labeled goat anti-rabbit IgG
147
for 1 h. 3, 3′-diaminobenzidine (DAB) solution was used in color development and
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counterstained with hematoxylin. The images were captured using a light microscope
149
(magnification, 400 ×, Nikon Eclipse Ti-SR). Representative images were presented. 7
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Preparation of Cytosolic and Nuclear Extracts. Cytosolic and nuclear proteins
151
were extracted by a nuclear/cytosol fractionation kit according to the manufacturer’s
152
instructions. The cytosolic and nuclear extractions were stored at -20°C and -80°C,
153
respectively.
154
EMSA.
Nrf2/ARE
binding
activity
was
determined
by
EMSA
using
the
155
Chemiluminescent EMSA Kit. The double stranded oligonucleotides were designed
156
according to the sequences of biotin-labeled ARE probe 5’-ACT GAG GGT GAC TCA GCA
157
AAA TC-3’, 3’-TGA CTC CCA CTG AGT CGT TTT AG-5’. Briefly, 5 µg of nuclear protein
158
was incubated with the biotin-labeled ARE probe (10 ng/µL) in the binding buffer at
159
25°C for 30 min. Mixtures were fractionated on 6.5% polyacrylamide gels, and then
160
transferred onto a Magmaprobe nylon membrane (Dingguo, Beijing, China). The
161
membrane was incubated with a blocking buffer for 30 min at room temperature, and
162
immersed in blocking buffer that containing Streptavidin-HRP conjugate for 20 min. The
163
membrane was washed four times at room temperature with washing buffer. Western
164
blotting reagents were visualized with EasyBlot ECL kit (ECL) that showed the gel shifts.
165
Transfection of Small Interfering RNA (siRNA). Predesigned siRNA against
166
human Nrf2 was purchased from GenePharma Co., Ltd (Shanghai, China). Transfection
167
of siRNA was performed as previously described.8 Briefly, HepG2 cells were transferred
168
onto the 6-well culture plates, and transfected with 100 nM siRNA against Nrf2 by
169
Lipofectamine 2000. After 6 h of incubation, added to the fresh medium and the cells
170
were cultured for another 24 h. The cells were then pre-treated with 30 µM NHDC for 1
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h and treated with CCl4 (0.5% v/v) an additional 6 h for the Western blotting assay. 8
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Immunoprecipitation. After treatment with NHDC, cells were lysed with RIPA lysis
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buffer (50 mM Tris-HCl, pH=7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40,
174
1 mM PMSF, 0.1% SDS and proteinase inhibitor cocktail). The lysates were incubated on
175
ice for at least 30 min, and homogenized with an ultrasonicator for 10 min. The
176
homogenates were centrifuged at 13,000 rpm at 4°C for 10 min. The supernatants were
177
collected, the concentrations of proteins were detected by Bradford Protein Assay Kit.
178
Whole cell lysates were pre-cleared with Protein A Agarose beads for 10 min. The
179
solutions were incubated with 2 µg of Keap1 or Nrf2 antibody at 4°C overnight, and
180
further incubated with 30 µl of Protein A agarose beads at 4°C for 3 h. Then, solution
181
was centrifuged and the beads were washed five times with lysis buffer. The complexes
182
mixed with 15 µl loading buffer, heated at 98°C for 10 min, subsequently analyzed by
183
Western blotting.
184
Western Blotting. The whole cell lysates and the immunoprecipitation products
185
were separated by 8% or 10% SDS-PAGE and transferred onto the PVDF membrane.
186
The membranes were blocked with 5% nonfat milk for 2 h, then, incubated with
187
different primary antibodies at 4°C overnight. The membranes were incubated with the
188
secondary
189
Representative blots were chosen from 3 or 4 separate experiments that showing similar
190
results.
antibody
for
2
h
followed
by
visualization
using
the
ECL
system.
191
Redox Western Blotting. Redox Western blotting for Keap1 was performed based
192
on our previously reported method.9 Briefly, HepG2 cells were treated with NHDC (0-30
193
µM) for 6 h. Cells were lysed with lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 9
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mM NaCl, 0.25% sodium deoxycholate, 1% NP-40, EDTA, 1 mM PMSF, sodium fluoride,
195
sodium orthovanadate and leupeptin protease inhibitor. The cell lysates were centrifuged
196
at 12,000 rpm at 4°C for 10 min, and the supernatant was collected as the redox cell
197
extracts.
198
mercaptoethanol (βME). After heat denaturation, extracts were separated by 8% SDS-
199
PAGE, and then Keap1 was detected by Western blotting.
Half
of
the
extracts
were
diluted
in
the
buffer
that
containing
β-
200
Statistical Analysis. Values were expressed as the mean ± SD. Statistical analysis
201
was performed using SPSS 18.0. Differences between the means of data were compared
202
by one-way variance analysis (ANOVA) test and post hoc analysis of group differences
203
was performed by least significant difference (LSD) test and a P value of < 0.05 was
204
considered to be statistically significant.
205
206
RESULTS AND DISCUSSION
207
NHDC Direct Scavenges Free Radicals. As shown in Table 1, NHDC has shown
208
significant free radical scavenging activities in the range of 0.05-500 µM. In FRAP assay,
209
the ability of NHDC to reduce Fe3+ to Fe2+ was increased concentration-dependently. The
210
FRAP values were varied from 37.3±0.9 to 1292.5±56.1 µM at the concentration range
211
of 0.05 to 500 µM. DPPH is a nitrogen-centered free radical, which has been widely used
212
in assessment of free radical quenching capabilities of antioxidants. NHDC possess
213
concentration-dependent scavenging activities against DPPH radical. According to
214
previous
215
hydroxytoluene (BHT), α-tocopherol and trolox on the DPPH radical were found as 61,
report,
IC50
values
for
butylated
hydroxyanisole
(BHA),
butylated
10
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220, 31 and 50 µM, respectively.10 Therefore, the DPPH radical scavenging efficiency of
217
NHDC is in the same order of magnitude with BHA, α-tocopherol and trolox, and one
218
magnitude higher than BHT. In ABTS assay, NHDC exhibited a concentration-dependent
219
scavenging activity, and the highest concentration of NHDC (500 µM) almost scavenged
220
ABTS radical completely (99.1±0.2%). O2•- is an important oxygen-center radical that
221
can occur during in vivo metabolism, and the basal level of O2•- may be upregulated
222
under certain pathological conditions.11 The toxicity of O2•- was often identified from
223
neurodegenerative patients or experimental model, which have relative low SOD
224
activities.12 In the current study, pyrogallol was autoxidized to generate O2•-, which can
225
be detected by luminol. HO• is the most reactive oxygen radical, which readily reacts
226
with biomacromolecules and resulting the denaturation of proteins, lipid peroxidation
227
and DNA modification.13 Here, the generation of HO• was performed by reaction of Fe2+
228
and H2O2. The scavenging effects of NHDC on superoxide and hydroxyl radical were
229
shown in Table 1. Our result indicated 30.4±1.2% of superoxide generated by a
230
pyrogallol autoxidation system and 45.7±2.4% of hydroxyl radical generated by a
231
Fenton’s reaction were inhibited by 500 µM of NHDC, respectively. Together, our results
232
demonstrated that NHDC is a potent antioxidant with high antioxidant capacity, which is
233
consistent with previous investigations.14, 15
234
NHDC Enhances the Protein Levels of HO-1, NQO1 and Nrf2 in Mice. Our
235
previous studies have demonstrated that NHDC play an important role in the attenuation
236
of CCl4-induced liver injury in vivo and in vitro,2 but the specific mechanism is still
237
unclear. HO-1 is an endogenous, cytoprotective enzyme, which have both antioxidative
238
and anti-inflammatory effects by catalyzes the first and rate limiting step in the 11
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catabolism of the prooxidant heme to carbon monoxide, biliverdin, and free iron.16 NQO1
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is a cytosolic flavoprotein, which catalyzes two-electron reduction and detoxification of
241
quinones and other redox cycling endogenous and exogenous chemicals.17 Growing
242
evidences indicated that natural antioxidants play important roles in the activation of
243
HO-1 and NQO1.18 In the present study, the levels of HO-1 and NQO1 expression
244
showed significant increases in NHDC group (Fig 2A, lane 2 vs 1, lane 4 vs 3).
245
Interestingly, CCl4 group also showed the upregulation of these two gene expressions
246
(Fig 2A, lane 3 vs 1), which is consistent with previous study.19 However, controversial
247
result also has been reported, e.g., Lee et al showed NQO1 protein level in the CCl4
248
group was significantly reduced than that in the control group.20 Although there are
249
disputable effect of CCl4, it is widely accepted that antioxidants upregulated the
250
expressions of these protective genes.
251
The
activation
of
Nrf2
signaling
and
the
upregulation
of
downstream
252
antioxidant/detoxifying enzymes are important to inhibit oxidative stress and maintain
253
the cellular homeostasis.21 In view of these, we investigated whether NHDC actives HO-
254
1 and NQO1 expression through Nrf2 pathway. As shown in Fig 2A, Western blotting
255
assay clearly indicated that Nrf2 has markedly increased after treatment with NHDC,
256
which suggested a post-transcriptional regulation mechanism of Nrf2. Furthermore, we
257
investigated the level of HO-1, NQO1 and Nrf2 by immunohistochemical staining.
258
Consistently, HO-1, NQO1 and Nrf2 immunopositive cell numbers were markedly
259
increased in NHDC-treated animals compared with control or CCl4 group, Fig 2B.
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To further confirm that NHDC-induced HO-1 and NQO1 expression is mediated
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through Nrf2 activation, HepG2 cells were transfected with Nrf2 siRNA for 6 h followed
262
by CCl4 stimulation or CCl4+NHDC co-treatment. Transfection of Nrf2 siRNA reduced the
263
expression of Nrf2, as well as NHDC-induced HO-1 and NQO1 expression, Fig 2C.
264
Together, these findings suggested that the protective mechanism of NHDC against
265
CCl4-induced hepatic injury is associated with the activation of Nrf2 signaling.
266
NHDC Induces the Translocation of Nrf2 and ARE-binding in HepG2 Cells.
267
Nrf2 is an important transcription factor, which is sequestered by the actin binding
268
protein Keap1 in the cytosol under homeostatic or non-stressed conditions. Cytosolic
269
Nrf2 was subjected to ubiquitination and subsequent proteasomal degradation, thus,
270
Nrf2 is inactive in cytosol.22 However, once it translocates into nucleus, Nrf2
271
heterodimerizes with a small musculo-aponeurotic fibrosarcoma protein and then binds
272
to ARE. We therefore examined whether NHDC facilitates the translocation of Nrf2 from
273
cytosolic into nucleus. As shown in Fig 3A, Nrf2 in the cytosol fraction were reduced, on
274
the contrary, the amounts of nuclear Nrf2 were significantly increased after NHDC, CCl4
275
or NHDC+CCl4 treatment. Housekeeping genes Lamin B and β-actin have not been
276
identified in cytosolic and nucleus fraction, respectively, indicated the purity of these two
277
fractions. To evaluate the effect of NHDC on Nrf2-ARE binding, EMSA was performed. In
278
Fig 3B, our result suggested that the treatment of NHDC increased the ARE binding
279
activity of Nrf2 in nuclear fraction. Together, our result suggested that NHDC increased
280
the translocation of Nrf2 and ARE-binding activity.
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NHDC Inhibits the Ubiquitination of Nrf2 in HepG2 Cells. Previous studies
282
revealed that under homeostatic conditions Nrf2 is rapidly degraded through the
283
ubiquitin dependent proteasome pathway.23 To determine whether the up-regulation of
284
Nrf2 by NHDC is due to the inhibition of Nrf2 ubiquitination, we detected the
285
ubiquitination of Nrf2 through immunoprecipitation after treatment with MG132, a
286
proteasome and protease inhibitor which allow the detection of ubiquitinated proteins.
287
The treatment with MG132 also resulted in an increased association of Nrf2 with Keap1
288
in the cytoplasm.24 As shown in Fig 4A, the Nrf2 protein level was increased after
289
treatment with NHDC or MG132 alone or in their combination, interestingly the level of
290
Keap1 was reduced by such treatment. Meanwhile, the treatment of MG132 caused an
291
enhancement in ubiquitin level, whilst NHDC has not effect on ubiquitin level.
292
Interactions between ubiquitin and Keap1 or Nrf2 were analyzed by immunoprecipitation
293
with anti-Keap1 or anti-Nrf2 antibodies followed by blotting with anti-ubiquitin,
294
respectively (Fig 4B). NHDC showed no effect on the level of ubiquitinated Keap1 (left
295
panel, lane 4 vs 3), however, a significantly reduction of the ubiquitinated Nrf2 was
296
observed in HepG2 cells co-treated with MG132 and NHDC (right panel, lane 4 vs 3).
297
These results suggested that NHDC enhanced Nrf2 is, at least partially, due to an
298
inhibitory effect of the ubiquitination of Nrf2. Similarly, quercetin8 and isothiocyanate
299
sulforaphane25 inhibit Nrf2 ubiquitination without induces Keap1 ubiquitination. However,
300
quinone-induced oxidative stress enhances ubiquitination of Keap1, which increases the
301
level of Nrf2 correspondingly.26,
302
halogenated quinone, tetrachlorobenzoquinone induces an ubiquitination switch from
303
Nrf2 to Keap1.9 The upregulation of Keap1 ubiquitination may interrupt the ability of
27
Our recently study also demonstrated that
14
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Keap1 to assemble into a Cullin-dependent E3 (Cul3) ubiquitin ligase complex, which is
305
critical for the control steady-state levels of Nrf2.26 Although quinone or oxidative stress-
306
mediated Nrf2 activation is independent on the dissociation of Keap1-Cul3 complex,
307
oxidized fatty acids induces the dissociation of Keap1 from Cullin3, thereby inducing the
308
activation of Nrf2 signaling.28 These differences in the activation of Nrf2 signaling may
309
suggest a structure-activity relationship between these inducers.
310
NHDC Induces the Modification of Keap1 in HepG2 Cells. Keap1 contains 25
311
and 27 cysteine residues in mouse and human homologues, respectively.23 Due to the
312
high activity of these cysteine residues, inducers are able to binding with Keap1 at
313
different cysteine sites. For example, cysteine 151 is important for the binding of Keap1
314
to Cul3, and the modification of cysteine 151 causes a conformational change of Keap1,
315
which resulting the dissociation of Keap1 from Cul3 and the inhibition of Nrf2
316
ubiquitination.27 Numbers of studies has indicated that some electrophilic compounds
317
induce the formation of Keap1 dimer via S-alkylation29 or intramolecular disulfide bond
318
formation.9, 30 To determine whether modification of Keap1 might occur with NHDC, the
319
migrations of Keap1 were evaluated by SDS-PAGE analysis. As shown in Fig 5, Keap1
320
protein with a molecular mass of 69 kDa has been designed as “normal” Keap1.
321
However, the intensities of 69 kDa band were slightly decreased in NHDC groups,
322
whereas a band with slower migration appeared in a concentration-dependent manner.
323
This band, which has an approximate molecular mass of 150 kDa, could refer as
324
“modified”
325
butylhydroquinone
326
biotinylhexylenediamine (BMCC) generated high molecular weight Keap1 forms (> 150
Keap1.
Hong (tBHQ)
et
al and
demonstrated a
that
thiol-reactive
a
typical
Nrf2-inducer
electrophile,
tert-
N-iodoacetyl-N-
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327
kDa), which were identified as K-48-linked polyubiquitin conjugates.31 However, 1-
328
biotinamido-4-(4’-[maleimidoethyl-cyclohexane]-carboxamido)butane
329
sulforaphane32 have no effect on the ubiquitination of Keap1. Thus, IAB and
330
sulforaphane display a different pattern of Keap1 modification than tBHQ or BMCC.
331
Combined with the result of unaffected ubiquitination of Keap1 upon NHDC treatment in
332
Fig 4B, we concluded that NHDC may share the same mechanism with IAB and
333
sulforaphane. Interestingly, sulforaphane modified Keap1 most readily in the Kelch
334
domain, but IAB modified Keap1 in the central linker domain. However, whether NHDC
335
induces Keap1 modification in Kelch or central linker domain needs further investigation.
336
The Activation of JNK and p38 Signalings Involved in the Cytoprotective
337
Effect of NHDC. Mitogen-activated protein kinase (MAPK) signaling cascade can be
338
activated by various extracellular signals. Interestingly, previous studies have indicated
339
that Nrf2 inducers have the ability to modulate MAPK activities. For instance, Yu et al
340
reported tBHQ and sulforaphane upregulate the activity of ERK2 and p38 but not JNK1,
341
however, the inhibition of ERK2 or p38 activity blocked the induction of phase II
342
detoxifying enzyme, and ARE-linked reporter gene.33,
343
for 6 h up-regulated the phosphorylation of JNK and p38, but not ERK in HepG2 cells, in
344
a concentration-dependent manner, Fig 6A. Time-dependent analysis indicated the
345
phosphorylation of JNK increased time-dependently, but the phosphorylation of p38
346
reached the summit at 6 h. To further confirm the activation of JNK and p38 was
347
involved in the cytoprotective effects related to NHDC, cells were pretreated with the
348
JNK inhibitor (SP600125), ERK inhibitor (PD98059) or p38 inhibitor (SB203580) for 1 h
349
prior to addition of NHDC. We found that JNK and p38 inhibitors, but not ERK inhibitor,
34
(IAB)31
and
In our study, NHDC treatment
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attenuated the protective effects of NHDC against CCl4-induced HepG2 cells damage, Fig
351
6B. We also detected the expressions of antioxidant enzyme by Western blotting
352
analysis. As expected, the inhibition of JNK and p38, but not ERK, strongly decreased
353
the NHDC-induced expressions of HO-1 and NQO1. These results indicated that JNK and
354
p38 MAPK involved in Nrf2-mediated expressions of HO-1 and NQO-1, and the
355
cytoprotective function of NHDC possibly link with the activation of JNK and p38 MAPK
356
signaling Pathways.
357 358
AUTHOR INFORMATION
359
*Corresponding author
360 361
Phone:
+86-23-68251503.
Fax:
+86-23-68251225.
E-mail
address:
[email protected]
362
363
364 365
Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
366
367
368 369
Funding This work is supported by National Natural Science Foundation of China (21477098), Science
and Technology Talent Cultivation
Project of
Chongqing
(cstc2014kjrc-
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370
qnrc00001)
and
Fundamental
Research
371
(XDJK2014A020, XDJK2015A017).
Funds
for
Page 18 of 31
the
Central
Universities
372
373
Notes The authors declare no competing financial interest.
374
375
376
ABBREVIATIONS ABTS, 2, 2′-azinobis-3-ethylbenzothiazolin-6-sulfonic acid; ARE, antioxidant response
377 378
element; BMCC; 1-biotinamido-4-(4’-[maleimidoethyl-cyclohexane]-carboxamido)butane;
379
Cul3, Cullin-dependent E3; DPPH, 1, 1′-diphenyl-2-picryhydrazyl; EMSA, electrophoretic
380
mobility shift assay; ERK, extracellular signal-regulated protein kinase; FRAP, ferric-
381
reducing
382
biotinylhexylenediamine;
383
associated protein 1; MAPK, mitogen-activated protein kinase; NQO1, NAD(P)H: quinone
384
oxidoreductase 1; Nrf2, nuclear factor (erythroid-derived 2)-like 2; siRNA, small
385
interfering RNA; tBHQ, tert-butylhydroquinone; TPTZ, 2, 4, 6-tripyridyl-s-triazine;
antioxidant
power; JNK,
HO-1, c-Jun
heme
oxygenase
N-terminal
kinase;
1;
IAB,
Keap1,
N-iodoacetyl-NKelch-like
ECH-
386
387
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468
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Table 1. Free radical scavenging activities of NHDC. Concentration 0.05
0.5
5
50
500
37.3±0.9
83.7±4.2
635.7±17.3
1264.2±43.1
1292.5±56.1
7.0±3.6
8.2±4.1
18.2±0.2
59.9±4.6
82.0±3.6
9.8±0.1
11.3±0.1
16.1±0.4
66.7±0.1
99.1±0.2
1.0±0.1
1.9±0.2
3.6±0.2
6.3±0.3
30.4±1.2
-
-
6.2±2.7
20.0±2.6
45.7±2.4
of NHDC (µM) FRAP level (µM) DPPH radical scavenging (%) ABTS radical scavenging (%) Superoxide scavenging (%) Hydroxyl radical scavenging (%)
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Figure 1. Chemical structure of NHDC
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Figure 2. Effects of NHDC on the expression of HO-1, NQO1 and Nrf2. (A) NHDC stimulates HO-1, NQO1 and Nrf2 expressions by Western blotting assay. Animals were treated with NHDC or/and CCl4 as described in the method section. The test was repeated three times and representative blots were shown. β-actin served as a control. (B) Effects of NHDC on the expressions of HO-1, NQO1 and Nrf2 with the immunohistochemical method. Animals were treated with NHDC or/and CCl4 as described in the method section. Bar = 50 µM. (C) Nrf2 siRNA down-regulates Nrf2, HO1 and NQO1 protein expressions. HepG2 cells were transfected with a specific Nrf2 siRNA as described in the method section, and pre-treated with NHDC (30 µM) for 1 h. The cells were then subjected to CCl4 (0.5%, v/v) challenge. After 6 h of CCl4 treatment, the levels of Nrf2, HO-1 and NQO1 were measured by Western blot analysis. The results were from three independent experiments. 25
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Figure 3. Effects of NHDC on nuclear Nrf2 accumulation and Nrf2-ARE binding activity. (A) Western blot analysis of the Nrf2 protein in cytosol and nucleus, respectively. β-actin and Lamin B were tested as loading controls in both cytosolic and nuclear fractions. (B) EMSA detected for the Nrf2-ARE binding activity. Data were representative of three separate experiments showing similar results.
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Figure 4. Effects of NHDC on the ubiquitination of Keap1 and Nrf2. (A) Western blot analysis of endogenous Keap1, Nrf2 and ubiquitin. HepG2 cells were treated with MG132 (10 µM) for 1 h and then treated with or without 30 µM NHDC for 6 h. Whole cell lysates were used to investigate Nrf2, Keap1 and ubiquitin with their antibodies. (B) The levels of Keap1 and Nrf2 ubiquitination. The proteins were immunoprecipitated with Keap1 and Nrf2 antibodies, then, the precipitated proteins visualized by Western blot analysis with ubiquitin antibody. Results were representative of three or four experiments.
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Figure 5. NHDC induces the modification of Keap1. Cells were treated with the indicated concentrations of NHDC for 6 h, analyzed by Western blot with Keap1 antibody and βactin as a loading control. Data were representative of three separate experiments.
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Figure 6. The cytoprotective effect of NHDC involves JNK and p38 MAPK signaling. (A) Concentration and time response of NHDC on the activation of JNK and p38 signaling. HepG2 cells were treated with NHDC at the indicated concentrations or times, and then the proteins were detected by Western blotting. (B) The pretreatment of JNK and p38 inhibitors, but not the ERK inhibitor, blocks the cytoprotective effect of NHDC. HepG2 cells were pretreated with 10 µM JNK inhibitor (SP600125), ERK inhibitor (PD98059) or p38 inhibitor (SB203580) for 30 min prior to incubation with 30 µM NHDC for another 1 h, then, CCl4 was introduced to induce cell damage. MTT assay was performed to investigate the cell viability. Data from three independent experiments were expressed as means ± SD. (C) The pretreatment of JNK and p38 inhibitors, but not the ERK inhibitor, blocks the expressions of HO-1 and NQO1. Cells were treated with 10 µM 29
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SP600125, PD98059 or SB203580 for 1 h and then exposed with 30 µM NHDC for another 6 h. Total cell extracts were prepared and analyzed by Western blot for the detection of the levels of HO-1 and NQO1. Data were representative of four separate experiments.
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Article
Neohesperidin dihydrochalcone against CCl4-induced hepatic injury through different mechanisms: The implication of free radical scavenging and Nrf2 activation Chuanyang Su, Xiaomin Xia, Qiong Shi, Xiufang Song, Juanli Fu, Congxue Xiao, Hongjun Chen, Bin Lu, Zhiyin Sun, Shanmei Wu, Siyu Yang, Xuegang Li, Xiaoli Ye, Erqun Song, and Yang Song J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01750 • Publication Date (Web): 15 May 2015 Downloaded from http://pubs.acs.org on May 18, 2015
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Journal of Agricultural and Food Chemistry
1
Neohesperidin dihydrochalcone against CCl4-induced hepatic injury
2
through different mechanisms: The implication of free radical
3
scavenging and Nrf2 activation
4
5
Chuanyang Su,† Xiaomin Xia,† Qiong Shi,† Xiufang Song,† Juanli Fu,† Congxue Xiao,†
6
Hongjun Chen,† Bin Lu,† Zhiyin Sun,† Shanmei Wu,† Siyu Yang,† Xuegang Li,† Xiaoli Ye,§
7
Erqun Song,† Yang Song*,†
8 †
9
Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest
10
University), Ministry of Education, College of Pharmaceutical Sciences, Southwest
11
University, Chongqing, People’s Republic of China, 400715
12
§
College of Life Sciences, Southwest University, Chongqing, People’s Republic of China, 400715
13
14
15
16
* Corresponding author:
17
Yang Song: College of Pharmaceutical Sciences, Southwest University, Beibei,
18
Chongqing, 400715, P R China. Tel: +86-23-68251503. Fax: +86-23-68251225. E-mail
19
address: [email protected] 1
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ABSTRACT
22
Neohesperidin dihydrochalcone (NHDC), a sweetener derived from citrus, belongs to
23
the family of bycyclic flavonoids dihydrochalcones. NHDC has been reported to against
24
CCl4-induced hepatic injury, but its mechanism is still unclear. We first discovered NHDC
25
showed a strong ability to scavenge free radicals. Also, NHDC induces phase II
26
antioxidant enzymes heme oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase 1
27
(NQO1) through the activation of the nuclear factor (erythroid-derived 2)-like 2
28
(Nrf2)/antioxidant response element (ARE) signaling. Further assays demonstrated
29
NHDC induces the accumulation of Nrf2 in the nucleus and augmented Nrf2-ARE binding
30
activity. Moreover, NHDC inhibits the ubiquitination of Nrf2 suggested the modification of
31
Kelch-like ECH-associated protein 1 (Keap1) and the disruption of Keap1/Nrf2 complex.
32
c-Jun N-terminal kinase (JNK) and p38 but not extracellular signal-regulated protein
33
kinase (ERK) phosphorylations were up-regulated by NHDC treatment. Taken together,
34
NHDC showed its protective antioxidant effect against CCl4-induced oxidative damage
35
via the direct free radical scavenging and indirect Nrf2/ARE signaling pathway.
36
KEYWORDS: NHDC; Keap1; HO-1; NQO1; MAPK; liver; ubiquitination
37
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INTRODUCTION
39
The liver plays an important role in the detoxification of xenobiotics and drugs prior
40
to excretion. However, these toxic substances can lead to hepatic injury. Carbon
41
tetrachloride (CCl4) has often been used as a common model of acute liver damage. The
42
toxic mechanism of CCl4 was identified to involve the excessive production of free
43
radicals. CCl4 is metabolized into CCl3• and CCl3OO• radicals with the participated of
44
cytochrome P4502E1, which initiates the process of lipid peroxidation and ultimately
45
resulting necrosis.1 In the liver, excess free radicals inflict may result in liver failure or
46
liver fibrosis.
47
Previous studies have illustrated that natural compounds with antioxidant activity are
48
effective in protecting liver from oxidative damage, in this regard, the use of active
49
antioxidants for the prevention and treatment of liver diseases has drawn much
50
attention. Our previous study indicated the protective effects of neohesperidin
51
dihydrochalcone (NHDC) on CCl4-induced acute hepatic injury in vivo and in vitro.2 CCl4-
52
induced acute hepatotoxicity is the most used experiment model. The remarkable
53
restoring of antioxidant enzymes and the amelioration of oxidative biomarkers
54
suggested an antioxidative activity. However, the exact molecular mechanism of NHDC
55
against CCl4-insult is quite unveiled.
56
One of the strategies to avoid free radical-mediated damage is to directly increase
57
the radical scavenging capacity, numerous of studies suggested polyphenol compounds
58
showed antioxidant effects via a scavenging action on both carbon- and oxygen-
59
centered radicals. The second approach is to regulate intrinsic antioxidant defenses at 3
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the transcriptional level. Recent reviews have summarized that natural products
61
upregulate a group of cytoprotective genes via nuclear factor erythroid-derived 2-like 2
62
(Nrf2) signaling.3,
63
cytoplasm by its negative regulator protein Kelch-like ECH associated protein 1 (Keap1)
64
binds tightly to Nrf2, which directing Nrf2 to proteasome degradation. Under certain
65
circumstances, Nrf2 activators trigger the dissociation of Nrf2 from Keap1 and facilitate
66
the translocation of Nrf2 into the nucleus. Nucleic Nrf2 binds to the antioxidant-
67
responsive
68
cytoprotective genes.
69
4
Under physiological conditions, Nrf2 was tightly anchored in the
element
(ARE)
consensus
sequence
to
initiate
the
transcription
of
Thus, the current study was designed to identify the protective mechanisms of NHDC.
70
We discovered that NHDC showed a significant capacity in free radical scavenging. We
71
also illustrated that NHDC potently induces the activation of Nrf2 signaling. These
72
findings revealed the cytoprotective role of Nrf2 against CCl4-intoxication in vitro and in
73
vivo models.
74
75
MATERIALS AND METHODS
76
Materials. NHDC (CAS: 20702-77-6, structure shown in Fig 1), ABTS (2, 2′-
77
azinobis-3-ethylbenzothiazolin-6-sulfonic acid), DPPH (1, 1′-diphenyl-2-picryhydrazyl),
78
TPTZ (2, 4, 6-tripyridyl-s-triazine) and luminol were supplied by Aladdin Reagent
79
Database Inc. (Shanghai, China). CCl4 (≥99.5%) was purchased from Kelong Chemical
80
Co., Ltd. (Chengdu, China). MG132, the proteasome inhibitor was obtained from Selleck
81
Chemicals (Shanghai, China). The antibodies against Nrf2, Heme oxygenase-1 (HO-1), 4
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NAD(P)H:quinone oxidoreductase 1 (NQO1), Lamin B, c-Jun N-terminal kinase (JNK),
83
extracellular
84
Proteintech group, Inc. (Wuhan, China), and antibodies against β-actin, rabbit IgG,
85
EasyBlot ECL kit and nuclear/cytosol fractionation kit were purchased from Sangon
86
Biotech Co., Ltd. (Shanghai, China). Chemiluminescent electrophoretic mobility shift
87
assay (EMSA) Kit, Biotin-labeled ARE probe, SP600125 (a JNK inhibitor), PD98059 (an
88
ERK inhibitor), SB203580 (a p38 inhibitor), Western stripping buffer and Protein A
89
agarose beads were obtained from Beyotime Institute of Biotechnology (Nanjing, China).
90
Antibodies against p-JNK, p-ERK, p-p38, and Keap1 were purchased from Biosynthesis
91
Biotechnology Co., Ltd. (Beijing, China). Ubiquitin antibody was from Santa Cruz
92
Biotechnology
93
diphenyltetrazoliumbromide (MTT) was purchased from Dingguo Biotechnology Co., Ltd.
94
(Beijing, China). Lipofectamine 2000 transfection reagent was supplied by Promega
95
(Madison, WI). Nrf2 siRNA was synthesized by GenePharma Co., Ltd (Shanghai, China).
signal-regulated
(Santa
protein
Cruz,
kinase
CA).
(ERK),
3-(4,
p38
were
purchased
5-dimethylthiazol-z-yl)-3,
from
5-
96
The antioxidant activity assays. The free radical scavenging activity of NHDC was
97
determined by ferric-reducing antioxidant power (FRAP), DPPH, ABTS, hydroxyl (HO•)
98
and superoxide (O2•-) radical scavenging assays. FRAP assay is based on the reduction
99
of a ferric 2, 4, 6-tripyridyl-striazine complex (Fe3+-TPTZ) by antioxidants to the ferrous
100
form (Fe2+-TPTZ), which forming a dark blue Fe2+-TPTZ complex with an absorbance
101
maximum at 593 nm.5 DPPH assay is based on the reduction of stable radical DPPH• to
102
yellow product with the incubation of antioxidant. The absorbance was measured at 515
103
nm.5 ABTS assay is based on the ability of antioxidant inhibits the generation of blue-to-
104
green color product, which indicated the reaction of ABTS•+ and potassium persulphate. 5
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ABTS radical-scavenging efficiency was measured by spectrophotometer at 750 nm.5
106
The superoxide anion scavenging ability was determined by a chemiluminescence
107
method in the pyrogallol-luminol system. The rate of pyrogallol autoxidation was
108
measured at 320 nm.6 The hydroxyl radical quenching activity was measured by using
109
hydroxyl radical generated from Fenton’s reaction Fe2+-H2O2-luminol chemiluminescence
110
method.7 All experiments were performed at least three times independently.
111
Animals, Treatment and Tissue Preparation. Male Kunming mice (22 ± 2 g)
112
were provided by Chongqing Academy of Chinese Materia Medica. All animal studies
113
were approved by the Southwest University Animal Care and Use Committee.
114
Experiments were conducted according to the national institution’s guidelines for the
115
care and use of laboratory animals. Animals were maintained under an air-conditioned
116
room with a 12 h light and dark cycle with free access to food and water. All animals
117
were adapted in individual plastic cages for a week. Then, animals were randomly
118
distributed in four groups with six mice in each group. Control group, administered
119
appropriate vehicles throughout. NHDC group, administered with NHDC (200 mg/kg
120
body weight/day, dissolved in sodium carboxymethyl cellulose, prepared immediately
121
before use) for 7 continuous days. CCl4 group, received sodium carboxymethyl cellulose
122
once daily for 7 continuous days. Three hours after final intragastric administration, mice
123
were injected with CCl4 (10 ml/kg body weight, 2% v/v in olive oil). NHDC + CCl4 group,
124
treated with NHDC (200 mg/kg body weight/day) for 7 continuous days. Three hours
125
after the final treatment, mice were injected with CCl4 (10 ml/kg body weight, 2% v/v
126
dissolved in olive oil). Animals were sacrificed 24 h after CCl4 administration. The livers
127
were removed carefully, rinsed twice with ice-cold physiological saline, and then divided 6
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into two pieces. One halves prepared for hepatic homogenate and the other were used
129
for immunohistochemical analysis. All steps were performed at 4°C.
130
Cell Culture and Cell Viability Assay. The human hepatoma HepG2 cell line was
131
purchased from Third Military Medical University (Chongqing, China). HepG2 cells were
132
cultured in DMEM containing 10% (v/v) FBS (HyClone, UT), penicillin (100 U/ml) and
133
streptomycin (100 U/ml) in the humidified incubator at 37°C under 5% CO2. HepG2 cells
134
were transferred onto 96-well culture plates at a density of 5×103 cells per well. After
135
incubated overnight, cells were pre-treated with various inhibitors for 30 min and
136
replaced with fresh serum-free medium containing NHDC or solvent vehicle for another 1
137
h. Finally, cells were maintained in 0.5% v/v CCl4 for 3 h, and then cells were treated
138
with MTT (5 mg/ml final concentration) at 37°C for 4 h. The supernatant was removed
139
and 150 µl of DMSO was added, measured spectrophotometrically at 570 nm.
140
Immunohistochemical Staining. The paraffin-embedded liver sections were de-
141
paraffinized and rehydrated. 3% H2O2 in methanol was used to block endogenous
142
peroxidase activity for 15 min. Antigen retrieval was measured by 1 mM EDTA buffer
143
(pH = 9.0) in a microwave for 3 min. The non-specific protein binding was blocked by
144
goat serum for 30 min. Then, the following steps were followed on the instruction of
145
Histostain™-plus and DAB substrate kits. The slides were incubated with HO-1, NQO1 or
146
Nrf2 antibodies at 4°C overnight, and incubated with biotin-labeled goat anti-rabbit IgG
147
for 1 h. 3, 3′-diaminobenzidine (DAB) solution was used in color development and
148
counterstained with hematoxylin. The images were captured using a light microscope
149
(magnification, 400 ×, Nikon Eclipse Ti-SR). Representative images were presented. 7
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Preparation of Cytosolic and Nuclear Extracts. Cytosolic and nuclear proteins
151
were extracted by a nuclear/cytosol fractionation kit according to the manufacturer’s
152
instructions. The cytosolic and nuclear extractions were stored at -20°C and -80°C,
153
respectively.
154
EMSA.
Nrf2/ARE
binding
activity
was
determined
by
EMSA
using
the
155
Chemiluminescent EMSA Kit. The double stranded oligonucleotides were designed
156
according to the sequences of biotin-labeled ARE probe 5’-ACT GAG GGT GAC TCA GCA
157
AAA TC-3’, 3’-TGA CTC CCA CTG AGT CGT TTT AG-5’. Briefly, 5 µg of nuclear protein
158
was incubated with the biotin-labeled ARE probe (10 ng/µL) in the binding buffer at
159
25°C for 30 min. Mixtures were fractionated on 6.5% polyacrylamide gels, and then
160
transferred onto a Magmaprobe nylon membrane (Dingguo, Beijing, China). The
161
membrane was incubated with a blocking buffer for 30 min at room temperature, and
162
immersed in blocking buffer that containing Streptavidin-HRP conjugate for 20 min. The
163
membrane was washed four times at room temperature with washing buffer. Western
164
blotting reagents were visualized with EasyBlot ECL kit (ECL) that showed the gel shifts.
165
Transfection of Small Interfering RNA (siRNA). Predesigned siRNA against
166
human Nrf2 was purchased from GenePharma Co., Ltd (Shanghai, China). Transfection
167
of siRNA was performed as previously described.8 Briefly, HepG2 cells were transferred
168
onto the 6-well culture plates, and transfected with 100 nM siRNA against Nrf2 by
169
Lipofectamine 2000. After 6 h of incubation, added to the fresh medium and the cells
170
were cultured for another 24 h. The cells were then pre-treated with 30 µM NHDC for 1
171
h and treated with CCl4 (0.5% v/v) an additional 6 h for the Western blotting assay. 8
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Immunoprecipitation. After treatment with NHDC, cells were lysed with RIPA lysis
173
buffer (50 mM Tris-HCl, pH=7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40,
174
1 mM PMSF, 0.1% SDS and proteinase inhibitor cocktail). The lysates were incubated on
175
ice for at least 30 min, and homogenized with an ultrasonicator for 10 min. The
176
homogenates were centrifuged at 13,000 rpm at 4°C for 10 min. The supernatants were
177
collected, the concentrations of proteins were detected by Bradford Protein Assay Kit.
178
Whole cell lysates were pre-cleared with Protein A Agarose beads for 10 min. The
179
solutions were incubated with 2 µg of Keap1 or Nrf2 antibody at 4°C overnight, and
180
further incubated with 30 µl of Protein A agarose beads at 4°C for 3 h. Then, solution
181
was centrifuged and the beads were washed five times with lysis buffer. The complexes
182
mixed with 15 µl loading buffer, heated at 98°C for 10 min, subsequently analyzed by
183
Western blotting.
184
Western Blotting. The whole cell lysates and the immunoprecipitation products
185
were separated by 8% or 10% SDS-PAGE and transferred onto the PVDF membrane.
186
The membranes were blocked with 5% nonfat milk for 2 h, then, incubated with
187
different primary antibodies at 4°C overnight. The membranes were incubated with the
188
secondary
189
Representative blots were chosen from 3 or 4 separate experiments that showing similar
190
results.
antibody
for
2
h
followed
by
visualization
using
the
ECL
system.
191
Redox Western Blotting. Redox Western blotting for Keap1 was performed based
192
on our previously reported method.9 Briefly, HepG2 cells were treated with NHDC (0-30
193
µM) for 6 h. Cells were lysed with lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 9
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mM NaCl, 0.25% sodium deoxycholate, 1% NP-40, EDTA, 1 mM PMSF, sodium fluoride,
195
sodium orthovanadate and leupeptin protease inhibitor. The cell lysates were centrifuged
196
at 12,000 rpm at 4°C for 10 min, and the supernatant was collected as the redox cell
197
extracts.
198
mercaptoethanol (βME). After heat denaturation, extracts were separated by 8% SDS-
199
PAGE, and then Keap1 was detected by Western blotting.
Half
of
the
extracts
were
diluted
in
the
buffer
that
containing
β-
200
Statistical Analysis. Values were expressed as the mean ± SD. Statistical analysis
201
was performed using SPSS 18.0. Differences between the means of data were compared
202
by one-way variance analysis (ANOVA) test and post hoc analysis of group differences
203
was performed by least significant difference (LSD) test and a P value of < 0.05 was
204
considered to be statistically significant.
205
206
RESULTS AND DISCUSSION
207
NHDC Direct Scavenges Free Radicals. As shown in Table 1, NHDC has shown
208
significant free radical scavenging activities in the range of 0.05-500 µM. In FRAP assay,
209
the ability of NHDC to reduce Fe3+ to Fe2+ was increased concentration-dependently. The
210
FRAP values were varied from 37.3±0.9 to 1292.5±56.1 µM at the concentration range
211
of 0.05 to 500 µM. DPPH is a nitrogen-centered free radical, which has been widely used
212
in assessment of free radical quenching capabilities of antioxidants. NHDC possess
213
concentration-dependent scavenging activities against DPPH radical. According to
214
previous
215
hydroxytoluene (BHT), α-tocopherol and trolox on the DPPH radical were found as 61,
report,
IC50
values
for
butylated
hydroxyanisole
(BHA),
butylated
10
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220, 31 and 50 µM, respectively.10 Therefore, the DPPH radical scavenging efficiency of
217
NHDC is in the same order of magnitude with BHA, α-tocopherol and trolox, and one
218
magnitude higher than BHT. In ABTS assay, NHDC exhibited a concentration-dependent
219
scavenging activity, and the highest concentration of NHDC (500 µM) almost scavenged
220
ABTS radical completely (99.1±0.2%). O2•- is an important oxygen-center radical that
221
can occur during in vivo metabolism, and the basal level of O2•- may be upregulated
222
under certain pathological conditions.11 The toxicity of O2•- was often identified from
223
neurodegenerative patients or experimental model, which have relative low SOD
224
activities.12 In the current study, pyrogallol was autoxidized to generate O2•-, which can
225
be detected by luminol. HO• is the most reactive oxygen radical, which readily reacts
226
with biomacromolecules and resulting the denaturation of proteins, lipid peroxidation
227
and DNA modification.13 Here, the generation of HO• was performed by reaction of Fe2+
228
and H2O2. The scavenging effects of NHDC on superoxide and hydroxyl radical were
229
shown in Table 1. Our result indicated 30.4±1.2% of superoxide generated by a
230
pyrogallol autoxidation system and 45.7±2.4% of hydroxyl radical generated by a
231
Fenton’s reaction were inhibited by 500 µM of NHDC, respectively. Together, our results
232
demonstrated that NHDC is a potent antioxidant with high antioxidant capacity, which is
233
consistent with previous investigations.14, 15
234
NHDC Enhances the Protein Levels of HO-1, NQO1 and Nrf2 in Mice. Our
235
previous studies have demonstrated that NHDC play an important role in the attenuation
236
of CCl4-induced liver injury in vivo and in vitro,2 but the specific mechanism is still
237
unclear. HO-1 is an endogenous, cytoprotective enzyme, which have both antioxidative
238
and anti-inflammatory effects by catalyzes the first and rate limiting step in the 11
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239
catabolism of the prooxidant heme to carbon monoxide, biliverdin, and free iron.16 NQO1
240
is a cytosolic flavoprotein, which catalyzes two-electron reduction and detoxification of
241
quinones and other redox cycling endogenous and exogenous chemicals.17 Growing
242
evidences indicated that natural antioxidants play important roles in the activation of
243
HO-1 and NQO1.18 In the present study, the levels of HO-1 and NQO1 expression
244
showed significant increases in NHDC group (Fig 2A, lane 2 vs 1, lane 4 vs 3).
245
Interestingly, CCl4 group also showed the upregulation of these two gene expressions
246
(Fig 2A, lane 3 vs 1), which is consistent with previous study.19 However, controversial
247
result also has been reported, e.g., Lee et al showed NQO1 protein level in the CCl4
248
group was significantly reduced than that in the control group.20 Although there are
249
disputable effect of CCl4, it is widely accepted that antioxidants upregulated the
250
expressions of these protective genes.
251
The
activation
of
Nrf2
signaling
and
the
upregulation
of
downstream
252
antioxidant/detoxifying enzymes are important to inhibit oxidative stress and maintain
253
the cellular homeostasis.21 In view of these, we investigated whether NHDC actives HO-
254
1 and NQO1 expression through Nrf2 pathway. As shown in Fig 2A, Western blotting
255
assay clearly indicated that Nrf2 has markedly increased after treatment with NHDC,
256
which suggested a post-transcriptional regulation mechanism of Nrf2. Furthermore, we
257
investigated the level of HO-1, NQO1 and Nrf2 by immunohistochemical staining.
258
Consistently, HO-1, NQO1 and Nrf2 immunopositive cell numbers were markedly
259
increased in NHDC-treated animals compared with control or CCl4 group, Fig 2B.
12
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To further confirm that NHDC-induced HO-1 and NQO1 expression is mediated
261
through Nrf2 activation, HepG2 cells were transfected with Nrf2 siRNA for 6 h followed
262
by CCl4 stimulation or CCl4+NHDC co-treatment. Transfection of Nrf2 siRNA reduced the
263
expression of Nrf2, as well as NHDC-induced HO-1 and NQO1 expression, Fig 2C.
264
Together, these findings suggested that the protective mechanism of NHDC against
265
CCl4-induced hepatic injury is associated with the activation of Nrf2 signaling.
266
NHDC Induces the Translocation of Nrf2 and ARE-binding in HepG2 Cells.
267
Nrf2 is an important transcription factor, which is sequestered by the actin binding
268
protein Keap1 in the cytosol under homeostatic or non-stressed conditions. Cytosolic
269
Nrf2 was subjected to ubiquitination and subsequent proteasomal degradation, thus,
270
Nrf2 is inactive in cytosol.22 However, once it translocates into nucleus, Nrf2
271
heterodimerizes with a small musculo-aponeurotic fibrosarcoma protein and then binds
272
to ARE. We therefore examined whether NHDC facilitates the translocation of Nrf2 from
273
cytosolic into nucleus. As shown in Fig 3A, Nrf2 in the cytosol fraction were reduced, on
274
the contrary, the amounts of nuclear Nrf2 were significantly increased after NHDC, CCl4
275
or NHDC+CCl4 treatment. Housekeeping genes Lamin B and β-actin have not been
276
identified in cytosolic and nucleus fraction, respectively, indicated the purity of these two
277
fractions. To evaluate the effect of NHDC on Nrf2-ARE binding, EMSA was performed. In
278
Fig 3B, our result suggested that the treatment of NHDC increased the ARE binding
279
activity of Nrf2 in nuclear fraction. Together, our result suggested that NHDC increased
280
the translocation of Nrf2 and ARE-binding activity.
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281
NHDC Inhibits the Ubiquitination of Nrf2 in HepG2 Cells. Previous studies
282
revealed that under homeostatic conditions Nrf2 is rapidly degraded through the
283
ubiquitin dependent proteasome pathway.23 To determine whether the up-regulation of
284
Nrf2 by NHDC is due to the inhibition of Nrf2 ubiquitination, we detected the
285
ubiquitination of Nrf2 through immunoprecipitation after treatment with MG132, a
286
proteasome and protease inhibitor which allow the detection of ubiquitinated proteins.
287
The treatment with MG132 also resulted in an increased association of Nrf2 with Keap1
288
in the cytoplasm.24 As shown in Fig 4A, the Nrf2 protein level was increased after
289
treatment with NHDC or MG132 alone or in their combination, interestingly the level of
290
Keap1 was reduced by such treatment. Meanwhile, the treatment of MG132 caused an
291
enhancement in ubiquitin level, whilst NHDC has not effect on ubiquitin level.
292
Interactions between ubiquitin and Keap1 or Nrf2 were analyzed by immunoprecipitation
293
with anti-Keap1 or anti-Nrf2 antibodies followed by blotting with anti-ubiquitin,
294
respectively (Fig 4B). NHDC showed no effect on the level of ubiquitinated Keap1 (left
295
panel, lane 4 vs 3), however, a significantly reduction of the ubiquitinated Nrf2 was
296
observed in HepG2 cells co-treated with MG132 and NHDC (right panel, lane 4 vs 3).
297
These results suggested that NHDC enhanced Nrf2 is, at least partially, due to an
298
inhibitory effect of the ubiquitination of Nrf2. Similarly, quercetin8 and isothiocyanate
299
sulforaphane25 inhibit Nrf2 ubiquitination without induces Keap1 ubiquitination. However,
300
quinone-induced oxidative stress enhances ubiquitination of Keap1, which increases the
301
level of Nrf2 correspondingly.26,
302
halogenated quinone, tetrachlorobenzoquinone induces an ubiquitination switch from
303
Nrf2 to Keap1.9 The upregulation of Keap1 ubiquitination may interrupt the ability of
27
Our recently study also demonstrated that
14
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Keap1 to assemble into a Cullin-dependent E3 (Cul3) ubiquitin ligase complex, which is
305
critical for the control steady-state levels of Nrf2.26 Although quinone or oxidative stress-
306
mediated Nrf2 activation is independent on the dissociation of Keap1-Cul3 complex,
307
oxidized fatty acids induces the dissociation of Keap1 from Cullin3, thereby inducing the
308
activation of Nrf2 signaling.28 These differences in the activation of Nrf2 signaling may
309
suggest a structure-activity relationship between these inducers.
310
NHDC Induces the Modification of Keap1 in HepG2 Cells. Keap1 contains 25
311
and 27 cysteine residues in mouse and human homologues, respectively.23 Due to the
312
high activity of these cysteine residues, inducers are able to binding with Keap1 at
313
different cysteine sites. For example, cysteine 151 is important for the binding of Keap1
314
to Cul3, and the modification of cysteine 151 causes a conformational change of Keap1,
315
which resulting the dissociation of Keap1 from Cul3 and the inhibition of Nrf2
316
ubiquitination.27 Numbers of studies has indicated that some electrophilic compounds
317
induce the formation of Keap1 dimer via S-alkylation29 or intramolecular disulfide bond
318
formation.9, 30 To determine whether modification of Keap1 might occur with NHDC, the
319
migrations of Keap1 were evaluated by SDS-PAGE analysis. As shown in Fig 5, Keap1
320
protein with a molecular mass of 69 kDa has been designed as “normal” Keap1.
321
However, the intensities of 69 kDa band were slightly decreased in NHDC groups,
322
whereas a band with slower migration appeared in a concentration-dependent manner.
323
This band, which has an approximate molecular mass of 150 kDa, could refer as
324
“modified”
325
butylhydroquinone
326
biotinylhexylenediamine (BMCC) generated high molecular weight Keap1 forms (> 150
Keap1.
Hong (tBHQ)
et
al and
demonstrated a
that
thiol-reactive
a
typical
Nrf2-inducer
electrophile,
tert-
N-iodoacetyl-N-
15
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327
kDa), which were identified as K-48-linked polyubiquitin conjugates.31 However, 1-
328
biotinamido-4-(4’-[maleimidoethyl-cyclohexane]-carboxamido)butane
329
sulforaphane32 have no effect on the ubiquitination of Keap1. Thus, IAB and
330
sulforaphane display a different pattern of Keap1 modification than tBHQ or BMCC.
331
Combined with the result of unaffected ubiquitination of Keap1 upon NHDC treatment in
332
Fig 4B, we concluded that NHDC may share the same mechanism with IAB and
333
sulforaphane. Interestingly, sulforaphane modified Keap1 most readily in the Kelch
334
domain, but IAB modified Keap1 in the central linker domain. However, whether NHDC
335
induces Keap1 modification in Kelch or central linker domain needs further investigation.
336
The Activation of JNK and p38 Signalings Involved in the Cytoprotective
337
Effect of NHDC. Mitogen-activated protein kinase (MAPK) signaling cascade can be
338
activated by various extracellular signals. Interestingly, previous studies have indicated
339
that Nrf2 inducers have the ability to modulate MAPK activities. For instance, Yu et al
340
reported tBHQ and sulforaphane upregulate the activity of ERK2 and p38 but not JNK1,
341
however, the inhibition of ERK2 or p38 activity blocked the induction of phase II
342
detoxifying enzyme, and ARE-linked reporter gene.33,
343
for 6 h up-regulated the phosphorylation of JNK and p38, but not ERK in HepG2 cells, in
344
a concentration-dependent manner, Fig 6A. Time-dependent analysis indicated the
345
phosphorylation of JNK increased time-dependently, but the phosphorylation of p38
346
reached the summit at 6 h. To further confirm the activation of JNK and p38 was
347
involved in the cytoprotective effects related to NHDC, cells were pretreated with the
348
JNK inhibitor (SP600125), ERK inhibitor (PD98059) or p38 inhibitor (SB203580) for 1 h
349
prior to addition of NHDC. We found that JNK and p38 inhibitors, but not ERK inhibitor,
34
(IAB)31
and
In our study, NHDC treatment
16
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350
attenuated the protective effects of NHDC against CCl4-induced HepG2 cells damage, Fig
351
6B. We also detected the expressions of antioxidant enzyme by Western blotting
352
analysis. As expected, the inhibition of JNK and p38, but not ERK, strongly decreased
353
the NHDC-induced expressions of HO-1 and NQO1. These results indicated that JNK and
354
p38 MAPK involved in Nrf2-mediated expressions of HO-1 and NQO-1, and the
355
cytoprotective function of NHDC possibly link with the activation of JNK and p38 MAPK
356
signaling Pathways.
357 358
AUTHOR INFORMATION
359
*Corresponding author
360 361
Phone:
+86-23-68251503.
Fax:
+86-23-68251225.
address:
[email protected]
362
363
364 365
Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
366
367
368 369
Funding This work is supported by National Natural Science Foundation of China (21477098), Science
and Technology Talent Cultivation
Project of
Chongqing
(cstc2014kjrc-
17
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370
qnrc00001)
and
Fundamental
Research
371
(XDJK2014A020, XDJK2015A017).
Funds
for
Page 18 of 31
the
Central
Universities
372
373
Notes The authors declare no competing financial interest.
374
375
376
ABBREVIATIONS ABTS, 2, 2′-azinobis-3-ethylbenzothiazolin-6-sulfonic acid; ARE, antioxidant response
377 378
element; BMCC; 1-biotinamido-4-(4’-[maleimidoethyl-cyclohexane]-carboxamido)butane;
379
Cul3, Cullin-dependent E3; DPPH, 1, 1′-diphenyl-2-picryhydrazyl; EMSA, electrophoretic
380
mobility shift assay; ERK, extracellular signal-regulated protein kinase; FRAP, ferric-
381
reducing
382
biotinylhexylenediamine;
383
associated protein 1; MAPK, mitogen-activated protein kinase; NQO1, NAD(P)H: quinone
384
oxidoreductase 1; Nrf2, nuclear factor (erythroid-derived 2)-like 2; siRNA, small
385
interfering RNA; tBHQ, tert-butylhydroquinone; TPTZ, 2, 4, 6-tripyridyl-s-triazine;
antioxidant
power; JNK,
HO-1, c-Jun
heme
oxygenase
N-terminal
kinase;
1;
IAB,
Keap1,
N-iodoacetyl-NKelch-like
ECH-
386
387
REFERENCES
388
1.
389
as a toxicological model. Crit Rev Toxicol 2003, 33, 105-36.
Weber, L. W.; Boll, M.; Stampfl, A., Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride
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Table 1. Free radical scavenging activities of NHDC. Concentration 0.05
0.5
5
50
500
37.3±0.9
83.7±4.2
635.7±17.3
1264.2±43.1
1292.5±56.1
7.0±3.6
8.2±4.1
18.2±0.2
59.9±4.6
82.0±3.6
9.8±0.1
11.3±0.1
16.1±0.4
66.7±0.1
99.1±0.2
1.0±0.1
1.9±0.2
3.6±0.2
6.3±0.3
30.4±1.2
-
-
6.2±2.7
20.0±2.6
45.7±2.4
of NHDC (µM) FRAP level (µM) DPPH radical scavenging (%) ABTS radical scavenging (%) Superoxide scavenging (%) Hydroxyl radical scavenging (%)
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Figure 1. Chemical structure of NHDC
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Figure 2. Effects of NHDC on the expression of HO-1, NQO1 and Nrf2. (A) NHDC stimulates HO-1, NQO1 and Nrf2 expressions by Western blotting assay. Animals were treated with NHDC or/and CCl4 as described in the method section. The test was repeated three times and representative blots were shown. β-actin served as a control. (B) Effects of NHDC on the expressions of HO-1, NQO1 and Nrf2 with the immunohistochemical method. Animals were treated with NHDC or/and CCl4 as described in the method section. Bar = 50 µM. (C) Nrf2 siRNA down-regulates Nrf2, HO1 and NQO1 protein expressions. HepG2 cells were transfected with a specific Nrf2 siRNA as described in the method section, and pre-treated with NHDC (30 µM) for 1 h. The cells were then subjected to CCl4 (0.5%, v/v) challenge. After 6 h of CCl4 treatment, the levels of Nrf2, HO-1 and NQO1 were measured by Western blot analysis. The results were from three independent experiments. 25
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Figure 3. Effects of NHDC on nuclear Nrf2 accumulation and Nrf2-ARE binding activity. (A) Western blot analysis of the Nrf2 protein in cytosol and nucleus, respectively. β-actin and Lamin B were tested as loading controls in both cytosolic and nuclear fractions. (B) EMSA detected for the Nrf2-ARE binding activity. Data were representative of three separate experiments showing similar results.
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Figure 4. Effects of NHDC on the ubiquitination of Keap1 and Nrf2. (A) Western blot analysis of endogenous Keap1, Nrf2 and ubiquitin. HepG2 cells were treated with MG132 (10 µM) for 1 h and then treated with or without 30 µM NHDC for 6 h. Whole cell lysates were used to investigate Nrf2, Keap1 and ubiquitin with their antibodies. (B) The levels of Keap1 and Nrf2 ubiquitination. The proteins were immunoprecipitated with Keap1 and Nrf2 antibodies, then, the precipitated proteins visualized by Western blot analysis with ubiquitin antibody. Results were representative of three or four experiments.
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Figure 5. NHDC induces the modification of Keap1. Cells were treated with the indicated concentrations of NHDC for 6 h, analyzed by Western blot with Keap1 antibody and βactin as a loading control. Data were representative of three separate experiments.
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Figure 6. The cytoprotective effect of NHDC involves JNK and p38 MAPK signaling. (A) Concentration and time response of NHDC on the activation of JNK and p38 signaling. HepG2 cells were treated with NHDC at the indicated concentrations or times, and then the proteins were detected by Western blotting. (B) The pretreatment of JNK and p38 inhibitors, but not the ERK inhibitor, blocks the cytoprotective effect of NHDC. HepG2 cells were pretreated with 10 µM JNK inhibitor (SP600125), ERK inhibitor (PD98059) or p38 inhibitor (SB203580) for 30 min prior to incubation with 30 µM NHDC for another 1 h, then, CCl4 was introduced to induce cell damage. MTT assay was performed to investigate the cell viability. Data from three independent experiments were expressed as means ± SD. (C) The pretreatment of JNK and p38 inhibitors, but not the ERK inhibitor, blocks the expressions of HO-1 and NQO1. Cells were treated with 10 µM 29
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SP600125, PD98059 or SB203580 for 1 h and then exposed with 30 µM NHDC for another 6 h. Total cell extracts were prepared and analyzed by Western blot for the detection of the levels of HO-1 and NQO1. Data were representative of four separate experiments.
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Graphic for table of contents
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