Nrf2-activators Attenuate the Progression of Nonalcoholic

MOL #84269 1

Title page

Nrf2-activators Attenuate the Progression of Nonalcoholic Steatohepatitis–Related Fibrosis

in a Dietary Rat Model

Rieko Shimozono, Yoshiji Asaoka, Yoshitaka Yoshizawa, Takumi Aoki, Hidetoshi Noda,

Masateru Yamada, Mie Kaino, and Hidenori Mochizuki

Toray Industries, Inc., Pharmaceutical Research Laboratories, Kanagawa, Japan.

Molecular Pharmacology Fast Forward. Published on April 16, 2013 as doi:10.1124/mol.112.084269

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on April 16, 2013 as DOI: 10.1124/mol.112.084269

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Running Title Page

Running title

Nrf2-activators attenuate fibrosis progression in NASH model

Contact information

Rieko Shimozono. Pharmaceutical Research Laboratories, Toray Industries, Inc., 10-1,

Tebiro 6-chome, Kamakura, Kanagawa 248-8555, Japan. Telephone number: +81 467 32

9805; fax number: +81 467 32 9862; e-mail address: [email protected]

Number of items

number of text pages: 40

number of tables: 1

number of figures: 7

number of references: 40

number of words in Abstract: 246

number of words in Introduction: 449

number of words in Discussion: 1447

List of Abbreviations

ALT, alanine aminotransferase; ARE, antioxidant response element; AST, aspartate

aminotransferase; α-SMA, α smooth muscle actin; CDAA, choline-deficient L-amino


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acid-defined; COL1A1, collagen α1(I); ECM, extracellular matrix synthesis; GST, glutathione

S-transferase; H&E, hematoxylin and eosin; HSC, hepatic stellate cell; Keap1, kelch-like

ECH-associated protein 1; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic

steatohepatitis; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear

factor-erythroid-2-related factor 2; OPZ, oltipraz; qPCR, quantitative polymerase chain

reaction; ROS, reactive oxygen species; TGF-β1, transforming growth factor-β1; TIMP-1,

tissue inhibitor of metalloproteinase-1.


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Abstract

Oxidative stress is considered to be a key mechanism of hepatocellular injury and disease

progression in patients with nonalcoholic steatohepatitis (NASH). The transcription factor

Nrf2 plays a central role in stimulating expression of various antioxidant-associated genes in

the cellular defense against oxidative stress. As the cytosolic repressor Keap1 negatively

regulates Nrf2, activation of Nrf2 facilitated by its release from Keap1 may represent a

promising strategy in the treatment of NASH. To test this hypothesis, we utilized two

chemically distinct types of Nrf2-activator. One is the thiol-reactive agent oltipraz (OPZ), a

typical Nrf2-activator, and the other is a novel biaryl urea compound, termed NK-252.

NK-252 exhibits a greater Nrf2-activating potential than OPZ. Furthermore, in vitro binding

studies revealed that NK-252 interacts with the domain containing the Nrf2-binding site of

Keap1, whereas OPZ does not. This finding indicates that NK-252 is more potent than OPZ

due to its unique mechanism of action. For in vivo animal model studies, we employed rats

on a choline-deficient L-amino acid-defined (CDAA)-diet, which demonstrate pathological

findings similar to those seen in human NASH. The administration of OPZ or NK-252

significantly attenuated the progression of histological abnormalities in rats on a CDAA diet,

especially hepatic fibrosis. In conclusion, by using Nrf2-activators with independent

mechanisms of action, we show in a rat model of NASH that the activation of Nrf2 is


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responsible for the anti-fibrotic effects of these drugs. This strategy of Nrf2 activation

presents new opportunities for treatment of NASH patients with hepatic fibrosis.


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Introduction

Nonalcoholic fatty liver disease (NAFLD) is a common, chronic disease of the liver. NAFLD

is mainly associated with the presence of obesity and multiple metabolic disorders.

Nonalcoholic steatohepatitis (NASH) is a progressive form of NAFLD and defined as the

presence of hepatic steatosis and inflammation with hepatocyte injury (ballooning) with or

without fibrosis (Chalasani et al., 2012). NASH can develop into cirrhosis, hepatic failure,

and hepatocellular carcinoma. With the growing epidemic of obesity, the prevalence and

impact of NAFLD continues to increase, making NASH potentially the most common cause

of advanced liver disease in coming decades (Vernon et al., 2011).

There is currently no broadly approved pharmacological therapy for NAFLD and the

management of patients with NAFLD consists of treating the associated metabolic disorders

(Chalasani et al., 2012). Several studies have investigated the effect of insulin sensitizing

agents in patients with NASH. For example, pioglitazone, one of the most well-established

thiazolidinediones, was demonstrated to improve liver histopathology in NASH. Furthermore,

antioxidant therapy with vitamin E has also been shown to have a beneficial effect on liver

histopathology. However, their efficacies are likely to be limited, and have not yet been

confirmed especially regarding fibrosis due to mixed results (Chalasani et al., 2012, Sanyal

et al., 2010, Comar and Sterling, 2006).


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Oxidative stress is thought to be a major contributor to the pathogenesis and progression of

NASH (Koek et al., 2011). Oxidative stress has been defined as an imbalance between

oxidants and antioxidants in favor of the former, resulting in an overall increase in cellular

levels of reactive oxygen species (ROS) (Sies, 1997). In patients with histopathologically

progressive NASH, production of antioxidants is reduced and the total antioxidant capacity

is apparently insufficient to compensate for oxidative stress (Sreekumar et al., 2003).

Therefore, agents that promote cellular antioxidant defense mechanisms are likely to

improve NASH as well as or better than direct scavengers of ROS like vitamin E.

Nuclear factor-erythroid-2-related factor 2 (Nrf2), a transcription factor that activates

antioxidant response elements (AREs), plays a central role in stimulating expression of

various antioxidant-associated genes in the cellular defense against oxidative stress (Itoh et

al., 1997). Under normal conditions, Kelch-like ECH-associated protein 1 (Keap1), a

cytosolic repressor of Nrf2, retains Nrf2 in the cytoplasm (Itoh et al., 1999, Itoh et al., 2004).

Thus, activation of Nrf2, facilitated by its release from Keap1, may represent a promising

strategy in the treatment of NASH.

To test this hypothesis, we utilized two chemically distinct types of Nrf2-activator. This study

characterized the respective mechanisms by which they activate Nrf2, and furthermore,

investigated their biochemical and histopathological effects on NASH using an established


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model of diet-induced NASH: rats on a choline-deficient L-amino acid-defined (CDAA)-diet

(Nakae et al., 1992).


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Materials and Methods

Chemicals. Oltipraz (OPZ, Fig. 1, left) was obtained from LKT Laboratories, Inc. (St. Paul,

MN). NK-252 (Fig. 1, right) was obtained from in-house synthesis. All compounds were

diluted in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) for in vitro assays, or suspended

in 0.5% (w/v) methyl cellulose (Nakarai tesque, Kyoto, Japan) for in vivo assays.

Plasmid Construntion. The 3-tandem repeat of ARE in the 5’-upstream region of NAD(P)H

quinone oxidoreductase 1 (NQO1) gene (Nioi et al., 2003) was inserted into pGL4.32

luciferase reporter vector (Promega, Madison, WI) via KpnI and HindIII sites to create ARE /

pGL4.32. A gene fragment encoding the partial length (residues 321-609) of human Keap1

(named Keap1-DC) was inserted into pGEX-6p-3 glutathione S-transferase (GST)-fusion

protein expression vector (GE Healthcare, Bucks, UK) via EcoRI and XhoI sites to create

Keap1-DC / pGEX-6p-3. All resultant plasmids were amplified in E. coli DH5α.

Stable Transformation, siRNA Transfection of Cells and Luciferase Reporter Gene

Assay. The Huh-7.5 cells, a subline derived from Huh-7 cells (Blight et al., 2002), were

obtained under license from Apath, LLC (St Louis, MO). Cells were transfected with ARE /

pGL4.32 by Lipofectamine™ LTX (Invitrogen, Carlsbad, CA). The stable clonal transfectant

was isolated by selection in hygromycin B (0.1 mg/mL). Cells derived from stable clones

were transfected with control or Nrf2 small interfering RNA (siRNA) (Stealth™ Select RNAi


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for Nrf2, HSS107130, Invitrogen) by lipofectamine RNAiMAX (Invitrogen) (30 h), then

treated with OPZ, NK-252 (0.1−30 μmol/L, 16 h) or dimethyl sulfoxide alone (control). The

luciferase activity values were measured using the Steady-Glo® Luciferase Assay System

(Promega).

Hydrogen Peroxide (H2O2)-Induced Cytotoxicity Assays. The Huh-7 cells were treated

with OPZ, NK-252 (0.1−30 μmol/L, 24 h) or dimethyl sulfoxide alone (control). Cells were

then washed and treated with H2O2 (1 mmol/L, 24 h). Cell viability values were determined

using the Cell Titer 96® AQueous One Solution Reagent (Promega).

Protein Expression and Purification. pGEX-6p-3 or Keap1-DC / pGEX-6p-3 were

transformed into E. coli BL21 (DE3) cells. The induction of GST or GST-fused Keap1-DC

gene expression was done with isopropyl-1-thio-β-D-galactoside (0.2 mmol/L). Soluble

proteins were then extracted using the BugBuster® HT Protein Extraction Reagent

(Novagen, Madison, WI) and purified on GSTrap FF columns (1 mL, GE Healthcare).

Surface Plasmon Resonance (SPR) Interaction Analysis (BIAcore™). The SPR

measurements were performed on a BIAcore™ S51 instrument (Biacore AB, Uppsala,

Sweden). GST-fusioned Keap1-DC was bound onto the surface of flow cell 1 of a Series S

Sensor Chip CM5 (GE Healthcare) using the amine coupling kit (GE Healthcare). Flow cell 2

with GST was used as the reference flow cell. OPZ or NK-252 solutions (12.5−100 μmol/L)


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were injected for 60 s at a flow rate of 10 μL/min. The resonance unit curves were

processed by subtracting the response in flow cell 1 from that in flow cell 2.

Animals and in vivo Experimental Design. The animal experiments were conducted

according to Guidelines for Animal Experiments, Research & Development Division, Toray

Industries, Inc. Six-week-old male Fischer 344 rats (Charles River Laboratories Japan Inc.,

Kanagawa, Japan) were randomly divided into four compound administration groups and

four control groups. Compound administration groups of rats fed with choline-deficient

L-amino acid-defined CDAA diet (Research Diets, Inc., New Brunswick, NJ) were orally

administered as follows: 1) with OPZ from one week after feeding at a dose of 60 mg/kg

once daily for nine weeks (CDAA+OPZ group; N=8), 2) with NK-252 from one week after

feeding at a dose of 20 mg/kg once daily for nine weeks (CDAA+NK-252_low group; N=8),

3) 2) at a dose of 60 mg/kg (CDAA+NK-252_high group; N=8), or 4) with NK-252 from six

weeks after feeding at a dose of 60 mg/kg once daily for four weeks (CDAA+NK-252_DA

[Delayed Administration] group; N=7). Two Control groups of rats fed with a CDAA diet for

six or ten weeks (pre-CDAA control or CDAA control group; N=9 each), and the other two

control groups of rats fed with CRF-1 diet (Charles River Laboratories Japan Inc.) for six or

ten weeks (pre-naive or naive; N=3 each). Laparotomy and blood sampling were performed

under isoflurane anesthesia. After blood sampling, rats were euthanized by exsanguination


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under isoflurane anesthesia and the livers were immediately extirpated.

Histopathological Examination. In all experiments, 2 μm-thick slices of formalin-fixed and

paraffin-embedded liver were processed with hematoxylin and eosin (H&E) stain and Sirius

red stain using established methods. In addition, formalin-fixed paraffin sections were

applied to immunostaining for α smooth muscle actin (α-SMA). Sections were boiled for 20

min in Immunosaver (Wako, Osaka, Japan) before the overnight incubation of the primary

antibody (M0851, DAKO, Glostrup, Denmark). They were then exposed to Histofine®

Simple Stain RAT MAX PO (Nichirei Biosciences, Tokyo, Japan) for 30 min, visualized by

the peroxidase-diaminobenzidine (DAB) reaction.

Histopathological evaluation was performed using H&E and Sirius red staining. The fibrosis

score was defined with a modified Brunt’s method (Brunt, 2001) as follows: score-1) slight

fibrosis is observed sporadically in the centrilobular or periportal area, score-2) slight fibrosis

is observed frequently in the centrilobular and periportal area, score-3) bridging fibrosis is

formed between the centrilobular and periportal area, score-4) pseudolobule is formed. The

Nano-Zoomer Digital Pathology (Hamamatsu Photonics KK, Shizuoka, Japan) was used to

acquire digital high-resolution images through the 50× objectives. Image analysis was

performed using Definiens XD software (Definiens, Munich, Germany). Three images per

specimen were examined blindly and randomly and Sirius red-positive areas were quantified


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as rate of fibrosis area.

Measurement of Plasma Transaminases. Blood samples from the abdominal aorta were

collected in tubes containing lithium heparin and plasma separator (Becton Dickinson and

Co., Franklin, NJ), centrifuged at 1500 x g, for 15 min to obtain the supernatant plasma.

Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were

measured using a DRI-CHEM system (Fujifilm Corp., Tokyo, Japan).

Real-Time qPCR. Total RNA was extracted from each liver using the RNeasy® Mini Kit

(Qiagen, Valencia, CA) and translated into complementary DNA (cDNA) with High-Capacity

cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA). Each cDNA was

subjected to real-time quantitative polymerase chain reaction (qPCR) using the ABI-Prism

7500 Sequence Detection System (Applied Biosystems) and amplified by SYBR® Premix

Ex Taq™ (TaKaRa Bio, Tokyo, Japan) reaction mixture utilizing gene specific primers

(Supplemental Table 1).The relative amount of each mRNA was determined using the

standard curve method and normalizing with GAPDH mRNA expression levels.

Measurement of Carbonyl Content of Proteins. Liver samples were rinsed with

phosphate-buffered saline to remove blood and homogenized. Following the measurement

of protein concentration by Bradford assay using the protein dye reagent concentrate

(Bio-Rad Laboratories, Richmond, CA), protein carbonyl derivatives in each homogenate


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were analyzed using the OxiSelect™ Protein Carbonylation ELISA kit (Cell Biolabs, Inc.,

San Diego, CA) based on equal protein loading (10 μg/mL).

Statistical Analysis. In vivo quantitative data were analyzed by either Dunnett’s test or

Steel’s test with unequal variance, histological scores were analyzed by Steel’s test, and P <

0.05 was considered statistically significant.


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Results

NK-252 and OPZ Activate Nrf2 and Protect against H2O2-induced Cytotoxicity. As an

indicator of Nrf2 activation, we examined activation of the NQO1-ARE, which is

predominantly regulated by Nrf2 (Kwak et al., 2001, Reisman et al., 2009a, Reisman et al.,

2009b), using a luciferase reporter gene assay. The luciferase activity in Huh-7.5 cells

treated with OPZ or NK-252 showed activation of the NQO1-ARE in a dose-dependent

manner (Fig. 2, solid line). NK-252 displayed this effect with higher potency than OPZ based

on the fact that the EC2 value (concentration for a two-fold induction above background),

calculated with linear extrapolation from the values above and below the induction threshold,

was 20.8 μmol/L for OPZ and 1.36 μmol/L for NK-252. We also confirmed that the luciferase

activities induced by OPZ or NK-252 were almost completely inhibited by Nrf2 siRNA (Fig. 2,

dotted line). These results indicate that OPZ and NK-252 have potential as Nrf2-activators in

hepatic cells. Prototypical Nrf2-activators that include OPZ have been reported to protect

microglial cells from H2O2-induced cytotoxicity (Konwinski et al., 2004). In this study, we

examined and compared the protective effects of OPZ and NK-252 against H2O2-induced

cytotoxicity using Huh-7 cells to evaluate their antioxidant properties. The cells treated with

OPZ or NK-252 showed increased resistance to H2O2-induced cytotoxicity compared to

control cells (Fig. 3). Furthermore, the protective effect of NK-252 was also considerably


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stronger than that of OPZ.

NK-252 but Not OPZ Interacts Directly with the Keap1-DC Domain that Contains the

Nrf2-binding Site. Sulfhydryl reactive compounds, such as dithiolethiones (including OPZ)

have been assumed to modify the sulfhydryl groups of Keap1 cysteines directly, and alter

the conformation of Keap1 (Kwak et al., 2003, Kobayashi et al., 2009). On the other hand,

NK-252, with no thiol reactive group, was not expected to interact with cysteine residues of

Keap1. Thus, we examined the binding affinity of NK-252 for the Keap1-DC domain

harboring the Kelch/double-glycine repeat motif, which has been reported to associate with

Nrf2 (Padmanabhan et al., 2006, Tong et al., 2006), and the C-terminal region. Using

BIAcore™ technology, the binding of NK-252 to the recombinant protein, consisting of the

Kelch domain of Keap1, was detected as an increase in resonance units in a

dose-dependent manner (Fig. 4B). In contrast, binding of OPZ to Keap1-DC was not

detected (Fig. 4A).

OPZ and NK-252 Exert Histopathological Anti-fibrotic and Anti-inflammatory Property

in the Liver of NASH Model Rats. On histopathological examination (Fig. 5A, B), the

median score of fibrosis in rats fed with a CDAA diet for 10 weeks (CDAA control) was 4,

indicating that most rats exhibited pseudolobule formation. However, rats on a CDAA diet

given OPZ or NK-252 displayed decreased fibrosis scores compared with CDAA control rats,


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with median scores of 3, corresponding to bridging fibrosis (Fig. 5D). In addition, CDAA

control rats displayed approximately 20-fold augmentation of the liver fibrosis area

compared with rats fed with a normal control diet (naive) (14.7%, 0.72%, respectively). This

augmentation was also drastically reduced by administration of OPZ or NK-252 (Fig. 5E,

5.80% for OPZ, 6.20% [low] and 4.97% [high] for NK-252). The effects of NK-252 on both

fibrosis score and fibrosis area were dose-dependent. Furthermore, immunohistochemistry

for α-SMA (Fig. 5C), an indicator of activation of hepatic stellate cells (HSCs), demonstrated

that there was a marked increase in HSCs activation in livers of CDAA control rats,

especially at the advancing edge of the fibrosis. In contrast, livers of rats on a CDAA diet

given OPZ or NK-252 showed markedly reduced numbers of α-SMA positive cells.

Regarding inflammation, cellular infiltration of liver tissue was also assessed on H&E

stained liver sections. Infiltration with inflammatory cells, which was rarely observed in the

liver of naive rats, appeared occasionally (slight, +) or frequently (moderate, ++) in the

peribiliary region of CDAA control rats. On the other hand, the incidence and severity of

inflammatory lesions were decreased in rats given OPZ or NK-252, demonstrating the

anti-inflammatory properties of OPZ and NK-252 (Supplemental Table 2).

OPZ and NK-252 Have a Hepatoprotective Effect as Manifested by Levels of Plasma

Aminotransferases in NASH Model Rats. The biochemical liver function tests showed


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mild elevation of plasma aminotransferase (ALT and AST) levels, indicators of

hepatocellular damage, in CDAA control rats (Table 1). In contrast, a significant reduction in

plasma ALT levels were observed in rats on a CDAA diet with high-dose administration of

NK-252, and plasma AST levels were decreased significantly in each of the compound

administration groups when compared with CDAA control rats (Table 1), which indicates the

hepatoprotective effects of OPZ and NK-252.

OPZ and NK-252 Upregulate NQO1 Gene Expression and Exert Antioxidant Property

in the Liver of NASH Model Rats. To demonstrate Nrf2 activation by OPZ and NK-252 in

vivo, using real-time qPCR we examined the level of gene expression of NQO1, one of the

prototypic Nrf2-regulated antioxidant enzymes, in the liver of rats. There was no significant

difference between CDAA control rats and naive rats. However, administration of OPZ or

NK-252 significantly up-regulated NQO1 gene expression in the liver of rats on a CDAA diet

(Fig. 6A). Carbonyl content of proteins as a marker of protein oxidation was also measured

to validate oxidative stress in the liver of rats on a CDAA diet and antioxidant properties of

OPZ and NK-252 in vivo. CDAA control rats showed markedly higher carbonyl content in

liver than Naive rats, which indicates elevation of oxidative damage. In contrast, the

carbonyl content in liver of rats on a CDAA diet given OPZ or NK-252 were lower compared

to these of CDAA control rats (Fig. 6B). These results suggest that OPZ and NK-252 exerted


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both Nrf2-activating and antioxidant properties in those livers affected by NASH.

OPZ and NK-252 Downregulate Fibrogenic Gene Expression in the Liver of NASH

Model Rats. To investigate the molecular mechanism of Nrf2-activator’s anti-fibrotic

properties, we examined levels of gene expression of fibrogenic mediators including

transforming growth factor-β1 (TGF-β1), collagen α1(I) (COL1A1) and tissue inhibitor of

metalloproteinase-1 (TIMP-1). The expression of all of these genes was elevated in the liver

of rats on a CDAA diet compared to naive rats (4.73, 52.5, 13.4-fold, respectively). On the

other hand, rats on a CDAA diet given OPZ or NK-252 showed substantially lower

expression of all three genes, although not all changes were statistically significant (Fig.

6C-E). The effect of NK-252 on the expression of these genes and the associated

histopathological properties (Fig. 5D, E) were both dose-dependent, suggesting that these

genes are involved in the anti-fibrotic mechanism of NK-252.

NK-252 Prevents Further Progression of Established Fibrosis in the Liver of NASH

Model Rats. Thus far, we used a dosing regimen in which administration of the test

compounds was started after one week of pre-feeding with a CDAA diet. In a preliminary

examination, we had confirmed that feeding of a CDAA diet for one week induced nothing

other than histological steatosis and increases in plasma aminotransferases (unpublished

observation). To examine the therapeutic, rather than preventive, anti-fibrotic effect of an


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Nrf2-activator, we used the delayed-administration regimen with NK-252 as a representative

agent, a regimen that consisted of four-week administration of the drug after six-weeks of

pre-feeding with a rats on a six-week CDAA diet before administration (pre-CDAA control)

already showed a fibrosis score of at least 2, with slight fibrosis frequently present in the

centrilobular and periportal area (Fig. 7A). They also displayed approximately four-fold

augmentation of the liver fibrosis area compared with pre-naive rats (3.39%, 0.86%,

respectively, Fig. 7B). Four-week administration of NK-252 resulted in substantial

retardation of the progression of fibrosis compared with CDAA control rats, as reflected in

both the fibrosis score (median score-3) and fibrosis area (5.20%) (Fig. 7A, B). In addition,

infiltration of inflammatory cells in the peribiliary region was almost completely inhibited by

the administration of NK-252 (Supplemental Table 2).


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Discussion

Published studies using knockout or knockdown animals have demonstrated a relationship

between NASH and the Keap1-Nrf2 system. That is, loss of Nrf2 leads to rapid onset and

exacerbation of steatohepatitis in animal models of NASH (Chowdhry et al., 2010, Sugimoto

et al., 2010). In contrast, loss of Keap1, with enhanced expression of Nrf2, attenuates

hepatic steatosis (Zhang et al., 2010). However, potential contribution of Nrf2 to progression

of fibrosis remains unknown. In addition, some chemical Nrf2-activators have been reported

to prevent obesity and insulin resistance (Shin et al., 2009, Yu et al., 2011) —the disorders

that underlie NASH—while there is still no clear-cut evidence whether Nrf2-activators are

clinically effective in NASH.

Among prototypical Nrf2-activators, OPZ, previously studied in a phase II trial of patients

with liver fibrosis or cirrhosis (Kim et al., 2011), was assumed to be a representative tool to

investigate the efficacy of chemical Nrf2-activators on NASH. However, OPZ may have

various target molecules other than Keap1 (Kang et al., 2002, Brooks et al., 2009)

presumably due to its mechanism of action, i.e., reacting with the sulfhydryl groups of

cysteines. Thus, OPZ probably has various effects independent of Nrf2 activation and OPZ

alone appears to be insufficient to confirm our hypothesis. To create a more specific

Nrf2-activator, we designed and synthesized a novel biaryl urea compound NK-252, with no


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sulfhydryl reactive group. In cell-based assays, NK-252 was found to have both Nrf2

activation effects and an antioxidant capability greater than those of OPZ. More surprising is

that an in vitro BIAcore™ binding study revealed that NK-252 interacts directly with the

Keap1-DC domain containing the Nrf2-binding site, whereas OPZ does not. This result

indicates that NK-252 activates Nrf2 through a unique mechanism of action, probably in a

more specific manner than prototypical Nrf2-activators like OPZ. Although further verification

is needed, including structural analysis of binding of NK-252 to Keap1 (e.g. X-ray

crystallography), it is possible that NK-252 represents the first chemical compound that

competitively inhibits the binding of Nrf2 to Keap1. Regarding drug discovery, it appears that

the OPZ-based design of Nrf2-activators may not be achievable because some

modifications of its chemical structure are thought to result in interaction with cysteine

residues in proteins other than Keap1. However, the NK-252-based design seems to

represent a more feasible approach to the production of specific Nrf2-activators.

The CDAA-diet model has been developed as a nutritional animal model of NASH

representing the progression of NASH that occurs when associated with worsening

oxidative damage (Nakae et al., 1992). In this study, rats on a CDAA diet exhibited an

increased level of protein oxidation in the liver, as occurs in patients with NASH (Valenti et

al., 2008); this model was therefore assumed to be the most appropriate for evaluating the


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efficiency of Nrf2-activators. OPZ and NK-252 commonly attenuated development of fibrosis

in rats on a CDAA diet in both qualitative (histopathological scoring) and quantitative

(assessment of fibrosis area) terms, likely due to suppression of HSCs activation, which play

a pivotal role in fiber synthesis and degradation. Dose-dependency of NK-252 was observed

for Nrf2-activating, antioxidant and anti-fibrotic properties, which suggests some correlation

between Nrf2 activation, suppression of oxidative stress and attenuation of liver fibrosis.

Protein carbonyl measurement did not detect sufficient antioxidant property of OPZ to

explain its anti-fibrotic property, which indicates at least a partial contribution by the

nonspecific effect of OPZ discussed above. However, the possibility remains that other

markers of oxidative stress might reveal its antioxidant property more precisely.

Moreover, the delayed-administration NK-252 regimen demonstrated that it can prevent

further progression of established fibrosis. This finding supports the possibility that

Nrf2-activators delay the development of liver fibrosis in patients with NASH. In this study,

we did not demonstrate recovery from fibrosis with NK-252 administration, compared with

baseline. However, further investigations using a different regimen or other animal models

are necessary to confirm whether or not Nrf2-activators can reverse liver fibrosis

The cell-based assay revealed the preventive effects of OPZ and NK-252 against

H2O2-induced cytotoxicity in the Huh-7 cells, which originate from hepatocytes. Therefore,


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the in vivo hepatoprotective effects of these compounds in the liver of rats on a CDAA diet

were likely to be due to their direct actions on hepatocytes. It is generally accepted that

there is sequential crosstalk between hepatocytes, inflammatory cells and HSCs. Hepatic

injury causes accelerated recruitment of inflammatory cells (e.g., Kupffer cells) and their

pro-fibrogenic cytokine secretion stimulates extracellular matrix synthesis (ECM) synthesis

by activating HSCs. In this study, OPZ and NK-252 were found to block the infiltration of

inflammatory cells, probably due to attenuation of hepatic injury. Furthermore, there is a

possibility that the attenuation of fibrosis seen with these compounds was also attributable

to their hepatoprotective properties.

HSCs may also be a direct target of Nrf2-activators. It is widely assumed that persistent

auto-/paracrine stimulation of activated HSCs by TGF-β1, a potent pro-fibrogenic cytokine,

is the key mechanism in liver fibrogenesis. TGF-β1 has been reported to activate SMAD

family of transcription factors, which lead to induction of fibrogenic genes expression

including COL1A1 and TIMP-1 (Verrecchia et al., 2001) and autoinduction of TGF-β1

(Roberts et al., 2006). TIMP-1, an inhibitor of ECM-degrading proteases, not only prevents

the degradation of ECM, but also inhibits the apoptosis of activated HSCs (Murphy et al.,

2002), resulting in the synergistic inductuion of ECM synthesis. These previous findings,

taken together with our analysis of gene expression, suggest that Nrf2-activators modulate


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TGF-β1-mediated fiber synthesis and degradation in the HSCs of rats on a CDAA-diet.

Moreover, published studies have reported cross-talk between TGF-β1 and ROS signaling

in HSCs. TGF-β1 induces the accumulation of H2O2, and this ROS act as intracellular signal

mediator of the pro-fbrogenic action of TGF-β1 (De et al., 1999, García-Trevijano et al.,

1999), which suggests that ROS stimulates induction of fibrogenic genes expression

including TGF-β1 itself. Therefore, once ROS has stimulated TGF-β1 signal transduction, a

vicious circle between ROS and TGF-β1 presumably promotes HSCs activation and ECM

synthesis. Nrf2-activators with antioxidant properties may prevent progression of liver

fibrosis through the interruption of this vicious circle. On examination of the molecular

mechanism underlying anti-fibrotic properties of Nrf2-activators on HSCs activation, a

further contrasting point is that another prototypical Nrf2-activator, sulforaphane, has

recently been reported to suppress the TGF-β signaling pathway by inhibiting

phosphorylation and transcriptional activation of SMAD3, leading to a reduction in

TGF-β-induced expression of fibrogenic genes (Oh et al., 2012). Although the contributions

made by their anti-fibrotic effects remain unclear, OPZ and NK-252 may also have the same

effect.

There are many animal models of NAFLD, but most are limited as they either simply

demonstrate obesity/insulin resistance-metabolic syndrome-related phenotypes (e.g., fatty


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liver) without replicating the progressive stages of liver pathology exhibited by patients with

NASH (e.g., steatohepatitis and fibrosis), or they show the typical pathology of NASH but the

etiology is not the metabolic syndrome. We therefore need to establish different models that

can be used for appropriate evaluation of target compounds. The CDAA-diet model is

assumed to fall into the latter category, as our preliminary examination revealed that this

model did not show any body weight gain and exhibited markedly reduced levels of

triglycerides in the plasma throughout the 10-weeks study period (unpublished observation),

likely due to the blockage of very-low-density lipoprotein (VLDL) secretion induced by

CDAA-diet (Kawaguchi et al., 2004, Fon and Rozman, 2011).These results indicate that the

metabolic profile of CDAA-diet model is generally not the same or partially converse of

human NASH. In this study, we did not detect any histological changes of fat accumulation

induced by administration of NK-252 or OPZ. However, there is possibility that such

anti-steatotic effects would be detectable using another animal model.

Collectively, these studies revealed that OPZ and NK-252 attenuate the progression of

fibrosis and indicated that their mechanism of action involves both a direct action on HSCs,

and hepatoprotective and anti-inflammatory properties. In other words, Nrf2-activators

probably counteract NASH-related fibrosis in an additive manner. Using Nrf2-activators with

independent mechanisms of action, we demonstrated that the activation of Nrf2 is


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responsible for their anti-fibrotic effects. It has been reported in a model based on a high-fat

diet that rosiglitazone, a peroxisome proliferator-activated receptor (PPAR)-γ ligand,

protects against liver fibrosis in concert with induction of hepatic NQO1 gene expression

(Gupte et al., 2010). Meanwhile, our results provide the first evidence that Nrf2 activation

alone is sufficient to attenuate the progression of NASH-related fibrosis. The strategy of Nrf2

activation is expected to present new opportunities for treatment of NASH with hepatic

fibrosis. Taking into consideration the efficacy of Nrf2-activators against the metabolic

syndrome (Shin et al., 2009, Yu et al., 2011), Nrf2-activators may act as potential

therapeutic agents for patients at any stage of NASH as it progresses.


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Authorship Contributions

Participated in research design: Asaoka, Yoshizawa, Aoki, Noda, Yamada, Kaino, Mochizuki,

and Shimozono.

Conducted experiments: Asaoka, Yoshizawa, Aoki, Noda, and Shimozono.

Contributed new reagents or analytic tools: Aoki, and Noda.

Performed data analysis: Asaoka, and Shimozono.

Wrote or contributed to the writing of the manuscript: Shimozono.


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References

Blight KJ, McKeating JA, Rice CM (2002) Highly permissive cell lines for subgenomic and

genomic hepatitis C virus RNA replication. J Virol 76: 13001-13014.

Brooks SC 3rd, Brooks JS, Lee WH, Lee MG, Kim SG (2009) Therapeutic potential of

dithiolethiones for hepatic diseases. Pharmacol Ther 124: 31-43.

Brunt EM (2001) Nonalcoholic steatohepatitis: definition and pathology. Semin Liver Dis 21:

3-16.

Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, Charlton M, Sanyal AJ

(2012) The diagnosis and management of non-alcoholic fatty liver disease: practice

Guideline by the American Association for the Study of Liver Diseases, American College

of Gastroenterology, and the American Gastroenterological Association. Hepatology 55:

2005-2023.

Chowdhry S, Nazmy MH, Meakin PJ, Dinkova-Kostova AT, Walsh SV, Tsujita T, Dillon JF,

Ashford ML, Hayes JD (2010) Loss of Nrf2 markedly exacerbates nonalcoholic

steatohepatitis. Free Radic Biol Med 48: 357-371.

Comar KM, Sterling RK (2006) Review article: Drug therapy for non-alcoholic fatty liver

disease. Aliment Pharmacol Ther 23: 207-215.

De Bleser PJ, Xu G, Rombouts K, Rogiers V, Geerts A (1999) Glutathione levels discriminate


at ASPE

T Journals on A

pril 4, 2019m


Dow

nloaded from


MOL #84269 30

between oxidative stress and transforming growth factor-beta signaling in activated rat

hepatic stellate cells. J Biol Chem 274: 33881-33887.

Fon Tacer K, Rozman D (2011) Nonalcoholic Fatty liver disease: focus on lipoprotein and

lipid deregulation. J Lipids 2011: 783976.

García-Trevijano ER, Iraburu MJ, Fontana L, Domínguez-Rosales JA, Auster A,

Covarrubias-Pinedo A, Rojkind M (1999) Transforming growth factor beta1 induces the

expression of alpha1(I) procollagen mRNA by a hydrogen

peroxide-C/EBPbeta-dependent mechanism in rat hepatic stellate cells. Hepatology 29:

960-970.

Gupte AA, Liu JZ, Ren Y, Minze LJ, Wiles JR, Collins AR, Lyon CJ, Pratico D, Finegold MJ,

Wong ST, Webb P, Baxter JD, Moore DD, Hsueh WA (2010) Rosiglitazone attenuates

age- and diet-associated nonalcoholic steatohepatitis in male low-density lipoprotein

receptor knockout mice. Hepatology 52: 2001-2011.

Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K,

Hatayama I, Yamamoto M, Nabeshima Y (1997) An Nrf2/small Maf heterodimer mediates

the induction of phase II detoxifying enzyme genes through antioxidant response

elements. Biochem Biophys Res Commun 236: 313-322.

Itoh K, Tong KI, Yamamoto M (2004) Molecular mechanism activating Nrf2-Keap1 pathway


at ASPE

T Journals on A

pril 4, 2019m


Dow

nloaded from


MOL #84269 31

in regulation of adaptive response to electrophiles. Free Radic Biol Med 36: 1208-1213.

Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, Yamamoto M (1999) Keap1

represses nuclear activation of antioxidant responsive elements by Nrf2 through binding

to the amino-terminal Neh2 domain. Genes Dev 13: 76-86.

Kang KW, Kim YG, Cho MK, Bae SK, Kim CW, Lee MG, Kim SG (2002) Oltipraz regenerates

cirrhotic liver through CCAAT/enhancer binding protein-mediated stellate cell inactivation.

FASEB J 16: 1988-1990.

Kawaguchi K, Sakaida I, Tsuchiya M, Omori K, Takami T, Okita K (2004) Pioglitazone

prevents hepatic steatosis, fibrosis, and enzyme-altered lesions in rat liver cirrhosis

induced by a choline-deficient L-amino acid-defined diet. Biochem Biophys Res Commun

315: 187-195.

Kim SG, Kim YM, Choi JY, Han JY, Jang JW, Cho SH, Um SH, Chon CY, Lee DH, Jang JJ,

Yu E, Lee YS (2011) Oltipraz therapy in patients with liver fibrosis or cirrhosis: a

randomized, double-blind, placebo-controlled phase II trial. J Pharm Pharmacol 63:

627-635.

Kobayashi M, Li L, Iwamoto N, Nakajima-Takagi Y, Kaneko H, Nakayama Y, Eguchi M,

Wada Y, Kumagai Y, Yamamoto M (2009) The antioxidant defense system Keap1-Nrf2

comprises a multiple sensing mechanism for responding to a wide range of chemical


at ASPE

T Journals on A

pril 4, 2019m


Dow

nloaded from


MOL #84269 32

compounds. Mol Cell Biol 29: 493-502.

Koek GH, Liedorp PR, Bast A (2011) The role of oxidative stress in non-alcoholic

steatohepatitis. Clin Chim Acta 412: 1297-1305.

Konwinski RR, Haddad R, Chun JA, Klenow S, Larson SC, Haab BB, Furge LL (2004)

Oltipraz, 3H-1,2-dithiole-3-thione, and sulforaphane induce overlapping and protective

antioxidant responses in murine microglial cells. Toxicol Lett 153: 343-355.

Kwak MK, Itoh K, Yamamoto M, Sutter TR, Kensler TW (2001) Role of transcription factor

Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer

chemoprotective agent, 3H-1, 2-dimethiole-3-thione. Mol Med 7: 135-145.

Kwak MK, Wakabayashi N, Itoh K, Motohashi H, Yamamoto M, Kensler TW (2003)

Modulation of gene expression by cancer chemopreventive dithiolethiones through the

Keap1-Nrf2 pathway. Identification of novel gene clusters for cell survival. J Biol Chem

278: 8135-8145.

Murphy FR, Issa R, Zhou X, Ratnarajah S, Nagase H, Arthur MJ, Benyon C, Iredale JP

(2002) Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of

metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition:

implications for reversibility of liver fibrosis. J Biol Chem 277: 11069-11076.

Nakae D, Yoshiji H, Mizumoto Y, Horiguchi K, Shiraiwa K, Tamura K, Denda A, Konishi Y


at ASPE

T Journals on A

pril 4, 2019m


Dow

nloaded from


MOL #84269 33

(1992) High incidence of hepatocellular carcinomas induced by a choline deficient

L-amino acid defined diet in rats. Cancer Res 52: 5042-5045.

Nioi P, McMahon M, Itoh K, Yamamoto M, Hayes JD (2003) Identification of a novel

Nrf2-regulated antioxidant response element (ARE) in the mouse NAD(P)H:quinone

oxidoreductase 1 gene: reassessment of the ARE consensus sequence. Biochem J 374:

337-348.

Oh CJ, Kim JY, Min AK, Park KG, Harris RA, Kim HJ, Lee IK (2012) Sulforaphane attenuates

hepatic fibrosis via NF-E2-related factor 2-mediated inhibition of transforming growth

factor-β/Smad signaling. Free Radic Biol Med 52: 671-682.

Padmanabhan B, Tong KI, Ohta T, Nakamura Y, Scharlock M, Ohtsuji M, Kang MI,

Kobayashi A, Yokoyama S, Yamamoto M (2006) Structural basis for defects of Keap1

activity provoked by its point mutations in lung cancer. Mol Cell 21: 689-700.

Reisman SA, Aleksunes LM, Klaassen CD (2009a) Oleanolic acid activates Nrf2 and

protects from acetaminophen hepatotoxicity via Nrf2-dependent and Nrf2-independent

processes. Biochem Pharmacol 77: 1273-1282.

Reisman SA, Buckley DB, Tanaka Y, Klaassen CD (2009b) CDDO-Im protects from

acetaminophen hepatotoxicity through induction of Nrf2-dependent genes. Toxicol Appl

Pharmacol 236: 109-114.


at ASPE

T Journals on A

pril 4, 2019m


Dow

nloaded from


MOL #84269 34

Roberts AB, Tian F, Byfield SD, Stuelten C, Ooshima A, Saika S, Flanders KC (2006)

Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor

suppression and metastasis. Cytokine Growth Factor Rev 17: 19-27.

Sanyal AJ, Chalasani N, Kowdley KV, McCullough A, Diehl AM, Bass NM,

Neuschwander-Tetri BA, Lavine JE, Tonascia J, Unalp A, Van Natta M, Clark J, Brunt EM,

Kleiner DE, Hoofnagle JH, Robuck PR (2010) Pioglitazone, vitamin E, or placebo for

nonalcoholic steatohepatitis. N Engl J Med 362: 1675-1685.

Shin S, Wakabayashi J, Yates MS, Wakabayashi N, Dolan PM, Aja S, Liby KT, Sporn MB,

Yamamoto M, Kensler TW (2009) Role of Nrf2 in prevention of high-fat diet-induced

obesity by synthetic triterpenoid CDDO-imidazolide. Eur J Pharmacol 620: 138-144.

Sies H (1997) Oxidative stress: oxidants and antioxidants. Exp Physiol 82: 291-295.

Sreekumar R, Rosado B, Rasmussen D, Charlton M (2003) Hepatic gene expression in

histologically progressive nonalcoholic steatohepatitis. Hepatology 38: 244-251.

Sugimoto H, Okada K, Shoda J, Warabi E, Ishige K, Ueda T, Taguchi K, Yanagawa T,

Nakahara A, Hyodo I, Ishii T, Yamamoto M (2010) Deletion of nuclear factor-E2-related

factor-2 leads to rapid onset and progression of nutritional steatohepatitis in mice. Am J

Physiol Gastrointest Liver Physiol 298: G283-294.

Tong KI, Katoh Y, Kusunoki H, Itoh K, Tanaka T, Yamamoto M (2006) Keap1 recruits Neh2


at ASPE

T Journals on A

pril 4, 2019m


Dow

nloaded from


MOL #84269 35

through binding to ETGE and DLG motifs: characterization of the two-site molecular

recognition model. Mol Cell Biol 26: 2887-2900.

Valenti L, Rametta R, Dongiovanni P, Maggioni M, Fracanzani AL, Zappa M, Lattuada E,

Roviaro G, Fargion S (2008) Increased expression and activity of the transcription factor

FOXO1 in nonalcoholic steatohepatitis. Diabetes 57: 1355-1362.

Vernon G, Baranova A, Younossi ZM (2011) Systematic review: the epidemiology and

natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in

adults. Aliment Pharmacol Ther 34: 274-285.

Verrecchia F, Chu ML, Mauviel A (2001) Identification of novel TGF-beta /Smad gene targets

in dermal fibroblasts using a combined cDNA microarray/promoter transactivation

approach. J Biol Chem 276: 17058-17062

Yu Z, Shao W, Chiang Y, Foltz W, Zhang Z, Ling W, Fantus IG, Jin T (2011) Oltipraz

upregulates the nuclear factor (erythroid-derived 2)-like 2 [corrected] (NRF2) antioxidant

system and prevents insulin resistance and obesity induced by a high-fat diet in C57BL/6J

mice. Diabetologia 54: 922-934.

Zhang YK, Yeager RL, Tanaka Y, Klaassen CD (2010) Enhanced expression of Nrf2 in mice

attenuates the fatty liver produced by a methionine-and choline-deficient diet. Toxicol Appl

Pharmacol 245: 326-334.


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Figure Legends

Fig. 1. Chemical structures of OPZ and NK-252.

Fig. 2. OPZ and NK-252 activate NQO1-ARE, which are inhibited by Nrf2 knock-down.

Huh-7.5 cells with stable expression of an ARE-luciferase reporter gene were transfected

with negative control (NC) siRNA or Nrf2 siRNA. After transfection (30 h), cells were treated

for 16 h with 0.3-30 μmol/L OPZ or NK-252. The average luciferase activity value for control

cells with NC siRNA transfection was arbitrarily set to 100%, and all other results were

expressed relative to this. The results shown represent the means ± SD from duplicate

assays for OPZ (A) and NK-252 (B).

Fig. 3. OPZ and NK-252 have protective effects against H2O2-induced cytotoxicity. Huh-7

cells were treated for 24 h with 0.3-30 μmol/L OPZ or NK-252, and then cells were washed

and treated for 24 h with 1 mmol/L H2O2. The average cell viability value for control cells

without H2O2 was arbitrarily set to 100%, and all other results were expressed relative to this.

The results shown represent the means ± SD from triplicate assays for OPZ (A) and NK-252

(B).

Fig. 4. NK-252 but not OPZ interacts directly with the Keap1-DC domain which contains the

Nrf2-binding site. Interaction between Keap1-DC domain and OPZ or NK-252 was

determined by the BIAcore™ assay. 12.5−100 μmol/L OPZ or NK-252 were applied to a


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Keap1-DC-bounded sensor chip. The resonance units reflect the number of attached

molecules. Each line indicates the resonance unit curve of 12.5, 25, 50, 100 μmol/L

compounds with Keap1-DC, respectively from singlet assays for OPZ (A) and NK-252 (B).

Fig. 5. OPZ and NK-252 exert histopathological anti-fibrotic efficacies in the liver of NASH

model rats. (A-E) Histopathological evaluation was performed with the liver of rats fed a diet

of CRF-1 for 10 weeks (naive), rats fed a diet of CDAA for 10 weeks (CDAA control), or rats

on a CDAA diet with nine-week administration of OPZ or NK-252 (CDAA+OPZ,

CDAA+NK-252_low or CDAA+NK-252_high). (A-C) Serial or near-serial sections stained

with H&E (A) Sirius red (B) and α-SMA (C) (scale bar, 1 mm). (D) Histopathological fibrosis

scores were evaluated with the modified Brunt’s method consisting of score-1 to -4. (E)

Quantitative evaluation of Sirius red-positive fibrosis area. The results shown represent

individual scores or means ± SEM (N=3-8). † P < 0.05, and †† P < 0.01 between each

compound administration group versus CDAA control (Steel’s test).

Fig. 6. OPZ and NK-252 upregulate NQO1 gene expression, exert antioxidant property and

downregulate fibrogenic gene expression in the liver of rats on a CDAA diet. (A, C-E) Total

RNA was isolated from the liver of rats described in the legend of Fig. 5, and then real-time

qPCR was performed. The results shown represent means ± SEM (N=3-8) of the gene

expression of NQO1 (A), TGF-β1 (C), COL1A1 (D) and TIMP-1 (E) relative to those of naive


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rats. (B) Protein carbonyl derivatives in each liver homogenate of rats described in the

legend of Fig. 5 were detected by an ELISA assay. The results shown represent means ±

SEM (N=3-8) of nanomole of carbonyl derivatives per milligram of total proteins assayed.* P

< 0.05, ** P < 0.01, and *** P < 0.001 between each compound administration group versus

CDAA control (Dunnett’s test). † P < 0.05, and †† P < 0.01 between each compound

administration group versus CDAA control (Steel’s test).

Fig. 7. NK-252 prevents further progression of established fibrosis in the liver of NASH

model rats. (A, B) Histopathological evaluation was performed with the liver of rats on a

six-week CRF-1 diet rats (pre-naive), rats on a six-week CDAA diet (pre-CDAA control), rats

on a 10-week CRF-1 diet (naive), rats on a 10-week CDAA diet (CDAA control), or rats on a

CDAA diet with four-week administration of NK-252 (CDAA+NK-252_DA). The procedure

was described in the legend of Figure 5. The results shown represent fibrosis score (A) and

means ± SEM (N=3-8) of rate of liver fibrosis area (B). †P < 0.05 between each compound

administration group versus CDAA control (Steel’s test).


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Tables

Table 1

Weighing data and biochemical data

Group N

Age

(week)

Body weight

(g)

Liver wet weight

(g)

Plasma ALT

(IU/L)

Plasma AST

(IU/L)

naive 3 16 259.3 ± 3.5 7.57 ± 0.29 28.0 ± 2.2 104.0 ± 11.1

CDAA control 9 16 212.4 ± 3.7 10.32 ± 0.20 170.7 ± 9.6 344.4 ± 17.3

CDAA+OPZ 8 16 227.0 ± 3.4 * 13.88 ± 0.3 *** 152.6 ± 8.1 256.8 ± 14.4 ***

CDAA+NK-252_low 8 16 216.5 ± 3.2 12.25 ± 0.18 *** 164.4 ± 11.8 285.3 ± 11.8 *

CDAA+NK-252_high 8 16 226.4 ± 4.2 * 14.09 ± 0.37 *** 120.8 ± 3.2 ** 238.5 ± 9.6 ***

pre-naive 3 12 245.0 ± 0.9 8.63 ± 0.23 22.0 ± 0.8 94.0 ± 18.5

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

Molecular Pharm

acology Fast Forward. Published on A

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I: 10.1124/mol.112.084269

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pre-CDAA control 9 12 198.9 ± 2.9 12.94 ± 0.24 245.0 ± 16.1 346.3 ± 9.2

CDAA+NK-252_DA 7 16 223.7 ± 3.6 13.27 ± 0.36 *** 150.4 ± 10.6 219.0 ± 12.5 ***

* P < 0.05, ** P < 0.01, and *** P < 0.001 between each compound administration group versus CDAA control (Dunnett's test)

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

Molecular Pharm

acology Fast Forward. Published on A

pril 16, 2013 as DO

I: 10.1124/mol.112.084269

at ASPET Journals on April 4, 2019 molpharm.aspetjournals.org Downloaded from



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Nrf2-activators Attenuate the Progression of Nonalcoholic

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