Neohesperidin Dihydrochalcone versus CCl4-Induced Hepatic Injury

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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

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scavenging and Nrf2 activation

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Chuanyang Su,† Xiaomin Xia,† Qiong Shi,† Xiufang Song,† Juanli Fu,† Congxue Xiao,†

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Hongjun Chen,† Bin Lu,† Zhiyin Sun,† Shanmei Wu,† Siyu Yang,† Xuegang Li,† Xiaoli Ye,§

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Erqun Song,† Yang Song*,†

8 †

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Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest

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University), Ministry of Education, College of Pharmaceutical Sciences, Southwest

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University, Chongqing, People’s Republic of China, 400715

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§

College of Life Sciences, Southwest University, Chongqing, People’s Republic of China, 400715

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14

15

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* Corresponding author:

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Yang Song: College of Pharmaceutical Sciences, Southwest University, Beibei,

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Chongqing, 400715, P R China. Tel: +86-23-68251503. Fax: +86-23-68251225. E-mail

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address: [email protected] 1

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ACS Paragon Plus Environment


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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

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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

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(NQO1) through the activation of the nuclear factor (erythroid-derived 2)-like 2

28

(Nrf2)/antioxidant response element (ARE) signaling. Further assays demonstrated

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NHDC induces the accumulation of Nrf2 in the nucleus and augmented Nrf2-ARE binding

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activity. Moreover, NHDC inhibits the ubiquitination of Nrf2 suggested the modification of

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Kelch-like ECH-associated protein 1 (Keap1) and the disruption of Keap1/Nrf2 complex.

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c-Jun N-terminal kinase (JNK) and p38 but not extracellular signal-regulated protein

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kinase (ERK) phosphorylations were up-regulated by NHDC treatment. Taken together,

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NHDC showed its protective antioxidant effect against CCl4-induced oxidative damage

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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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INTRODUCTION

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The liver plays an important role in the detoxification of xenobiotics and drugs prior

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to excretion. However, these toxic substances can lead to hepatic injury. Carbon

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tetrachloride (CCl4) has often been used as a common model of acute liver damage. The

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toxic mechanism of CCl4 was identified to involve the excessive production of free

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radicals. CCl4 is metabolized into CCl3• and CCl3OO• radicals with the participated of

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cytochrome P4502E1, which initiates the process of lipid peroxidation and ultimately

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resulting necrosis.1 In the liver, excess free radicals inflict may result in liver failure or

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liver fibrosis.

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Previous studies have illustrated that natural compounds with antioxidant activity are

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effective in protecting liver from oxidative damage, in this regard, the use of active

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antioxidants for the prevention and treatment of liver diseases has drawn much

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attention. Our previous study indicated the protective effects of neohesperidin

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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

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restoring of antioxidant enzymes and the amelioration of oxidative biomarkers

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suggested an antioxidative activity. However, the exact molecular mechanism of NHDC

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against CCl4-insult is quite unveiled.

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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-

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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

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circumstances, Nrf2 activators trigger the dissociation of Nrf2 from Keap1 and facilitate

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the translocation of Nrf2 into the nucleus. Nucleic Nrf2 binds to the antioxidant-

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responsive

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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

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findings revealed the cytoprotective role of Nrf2 against CCl4-intoxication in vitro and in

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vivo models.

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MATERIALS AND METHODS

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Materials. NHDC (CAS: 20702-77-6, structure shown in Fig 1), ABTS (2, 2′-

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azinobis-3-ethylbenzothiazolin-6-sulfonic acid), DPPH (1, 1′-diphenyl-2-picryhydrazyl),

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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

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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),

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extracellular

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Proteintech group, Inc. (Wuhan, China), and antibodies against β-actin, rabbit IgG,

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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

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agarose beads were obtained from Beyotime Institute of Biotechnology (Nanjing, China).

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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

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diphenyltetrazoliumbromide (MTT) was purchased from Dingguo Biotechnology Co., Ltd.

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(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

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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

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hydroxyl radical generated from Fenton’s reaction Fe2+-H2O2-luminol chemiluminescence

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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)

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were provided by Chongqing Academy of Chinese Materia Medica. All animal studies

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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

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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

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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

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body weight/day, dissolved in sodium carboxymethyl cellulose, prepared immediately

121

before use) for 7 continuous days. CCl4 group, received sodium carboxymethyl cellulose

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once daily for 7 continuous days. Three hours after final intragastric administration, mice

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were injected with CCl4 (10 ml/kg body weight, 2% v/v in olive oil). NHDC + CCl4 group,

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treated with NHDC (200 mg/kg body weight/day) for 7 continuous days. Three hours

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after the final treatment, mice were injected with CCl4 (10 ml/kg body weight, 2% v/v

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dissolved in olive oil). Animals were sacrificed 24 h after CCl4 administration. The livers

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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

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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

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purchased from Third Military Medical University (Chongqing, China). HepG2 cells were

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cultured in DMEM containing 10% (v/v) FBS (HyClone, UT), penicillin (100 U/ml) and

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streptomycin (100 U/ml) in the humidified incubator at 37°C under 5% CO2. HepG2 cells

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were transferred onto 96-well culture plates at a density of 5×103 cells per well. After

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incubated overnight, cells were pre-treated with various inhibitors for 30 min and

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replaced with fresh serum-free medium containing NHDC or solvent vehicle for another 1

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h. Finally, cells were maintained in 0.5% v/v CCl4 for 3 h, and then cells were treated

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with MTT (5 mg/ml final concentration) at 37°C for 4 h. The supernatant was removed

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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-

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paraffinized and rehydrated. 3% H2O2 in methanol was used to block endogenous

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peroxidase activity for 15 min. Antigen retrieval was measured by 1 mM EDTA buffer

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(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

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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

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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

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AAA TC-3’, 3’-TGA CTC CCA CTG AGT CGT TTT AG-5’. Briefly, 5 µg of nuclear protein

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was incubated with the biotin-labeled ARE probe (10 ng/µL) in the binding buffer at

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25°C for 30 min. Mixtures were fractionated on 6.5% polyacrylamide gels, and then

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transferred onto a Magmaprobe nylon membrane (Dingguo, Beijing, China). The

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membrane was incubated with a blocking buffer for 30 min at room temperature, and

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immersed in blocking buffer that containing Streptavidin-HRP conjugate for 20 min. The

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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.

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Transfection of Small Interfering RNA (siRNA). Predesigned siRNA against

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human Nrf2 was purchased from GenePharma Co., Ltd (Shanghai, China). Transfection

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of siRNA was performed as previously described.8 Briefly, HepG2 cells were transferred

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onto the 6-well culture plates, and transfected with 100 nM siRNA against Nrf2 by

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Lipofectamine 2000. After 6 h of incubation, added to the fresh medium and the cells

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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,

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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

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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

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Western blotting.

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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

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RESULTS AND DISCUSSION

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NHDC Direct Scavenges Free Radicals. As shown in Table 1, NHDC has shown

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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

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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

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ABTS radical completely (99.1±0.2%). O2•- is an important oxygen-center radical that

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can occur during in vivo metabolism, and the basal level of O2•- may be upregulated

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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.

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.

13



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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-

15



Page 16 of 31

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


Page 17 of 31


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.

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-

17



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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22


Page 23 of 31


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 (%)

23



Page 24 of 31

Figure 1. Chemical structure of NHDC

24


Page 25 of 31


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



Page 26 of 31

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.

26


Page 27 of 31


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.

27



Page 28 of 31

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.

28


Page 29 of 31


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



Page 30 of 31

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.

30


Page 31 of 31


Graphic for table of contents

31


Neohesperidin Dihydrochalcone versus CCl4-Induced Hepatic Injury

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