Open Access

OBM Hepatology and
Gastroenterology

Original Research

Peptides Obtained by Enzymatic Decomposition of Mackerel Prevent
Nonalcoholic Steatohepatitis in Sprague-Dawley Rats fed a High-fat andHigh-cholesterol Diet
Katsuhisa Omagari 1,2,*, Ayumi Fukuda 1, Machiko Suga 1, Ayumi Ogata 1, Shinta Nishioka 2,
Kazuhito Suruga 1,2, Mayuko Ichimura 3, Koichi Tsuneyama 3
1. Department of Nutrition, Faculty of Nursing and Nutrition, University of Nagasaki, 1-1-1
Manabino, Nagayo-cho, Nagasaki 851-2195 Japan; E-Mails: omagari@sun.ac.jp (K.O.);
m3218001@sun.ac.jp (A.F.); b3214016@sun.ac.jp (M.S.); b3214002@sun.ac.jp (A.O.);
suruga@sun.ac.jp (K.S.)
2. Division of Nutritional Science, Graduate School of Human Health Science, University ofNagasaki, 1-1-1 Manabino, Nagayo-cho, Nagasaki 851-2195 Japan; E-Mail: snishioka@zeshinkai.or.jp (S.N.)
3. Department of Pathology and Laboratory Medicine, Tokushima University Graduate School, 318-15, Kuramoto-cho, Tokushima 770-8503, Japan; E-Mails: ichimura.mayuko@tokushimau.ac.jp (M.I.); tsuneyama.koichi@tokushima-u.ac.jp (K.T.)* Correspondence: Katsuhisa Omagari; E-Mail: omagari@sun.ac.jp; Tel. & Fax: +81-95-813-5201
Academic Editor(s): Tatsuo Kanda
OBM Hepatology and Gastroenterology
2018, volume 2, issue 3
doi:10.21926/obm.hg.1803008

Received: April 9, 2018
Accepted: July 27, 2018
Published: August 4, 2018

Abstract:
Background: The pathogenesis of nonalcoholic steatohepatitis (NASH) is thought to be
multifactorial, and oxidative stress may play an important role in the development of NASH.
Peptides obtained by enzymatic decomposition of mackerel (EMP) including selenoneine wererecently reported to possess antioxidative activity. Thus, EMP is a potential dietary strategy for theprevention of NASH.
Methods: Nine-week-old male Sprague-Dawley rats were fed a high-fat and high-cholesterol (HFC)
diet with or without EMP (HFC supplemented with 1%, 2.5%, or 5% (w/w) EMP) for 9 weeks (n = 6© 2018 by the author. This is an open access article distributed under theconditions of the Creative Commons by Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium or format,
provided the original work is correctly cited.

OBM Hepatology and Gastroenterology 2018; 2(3), doi:10.21926/obm.icm.1803008

7/group). Histopathology and serology were evaluated as well as expressions of genes in the liver
involved in fibrogenesis, inflammation, oxidative stress, and lipid metabolism.
Results: Histologically, the HFC diet with EMP tended to reduce NASH progression compared to
the HFC diet without EMP (NASH diagnostic rate: 80% vs 100%). Serum leptins in rats fed the HFC
diet with EMP tended to be lower than those in rats fed an HFC diet without EMP.
Immunohistochemically, the expression of 4-hydroxynonenal as a marker of oxidative stress in theliver tended to be less evident in rats fed the HFC diet with EMP than without EMP. mRNA levelsof NF-κB tended to be lower in rats fed the HFC diet with EMP than without EMP, whereas mRNAlevels of GPX-1 tended to be higher in rats fed the HFC diet with EMP than without EMP in an
EMP-dose dependent manner. Histological findings and hepatic mRNA expressions indicate thatthe HFC diet containing 1% or 2.5% (w/w) EMP was more likely to reveal a preventive effectagainst NASH progression than the HFC diet with 5% (w/w) EMP.
Conclusions: Our data suggests that EMP containing selenoneine may prevent NASH progression,
presumably through its antioxidative activity. The optimal dose of EMP requires further study.
Keywords
Selenoneine; enzymatic decomposition of mackerel; nonalcoholic steatohepatitis; high-fat andhigh-cholesterol diet; antioxidative activity

1. Introduction
Nonalcoholic fatty liver disease (NAFLD) is currently recognized as the most common cause ofchronic liver disease in many countries. NAFLD consists of two clinical entities: nonalcoholic fatty
liver (NAFL), which is a mostly benign, nonprogressive disease and nonalcoholic steatohepatitis(NASH), which can progress to cirrhosis or even hepatocellular carcinoma. NASH is histologicallycharacterized by hepatic steatosis associated with liver cell injury (ballooning degeneration) andinflammation [1]. The pathogenesis of NASH is thought to be multifactorial, and oxidative stress
may play an important role in the development of NASH [2, 3]. Nutritional factors are commonly
associated with NASH; however, there are few fully proven treatments for NASH, making it
imperative to develop novel dietary strategies that can prevent NASH.
Selenium is an essential micronutrient for humans and animals, and seafood is one of the major
sources of selenium in humans [4, 5]. The antioxidant function of selenium plays protective roles
in various human diseases including prostate, lung, or colon cancers; immunodeficiency; and heartdiseases [6]. Selenoneine is the major form of selenium found in aqueous extracts and is the major
selenium compound in the muscles of fish such as tuna, mackerel, and swordfish [7, 8].
Selenoneine has strong antioxidant properties and a detoxifying function against methylmercury
toxicity [8]. Recently, peptides obtained by enzymatic decomposition of mackerel (EMP)
containing selenoneine were reported to possess antioxidative activity and affect cytokine
secretion such as interleukin-10 (IL-10) [9, 10]. Therefore, this EMP can be a potential dietary
strategy for the prevention of NASH.

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The objective of this study was to evaluate the preventive effect of EMP against NASH
progression in Sprague-Dawley (SD) rats that were fed a high-fat and high-cholesterol diet (HFC;
NASH model) [11]. The mechanisms underlying the effects of EMP, specifically in relation to
fibrogenesis, inflammation, oxidative stress, and cholesterol and lipid metabolism in the liver werealso investigated.
2. Materials and Methods
All procedures performed on the animals were approved by the Animal Use Committee of the
University of Nagasaki, and the animals were maintained in accordance with the University ofNagasaki’s Guidelines for the Care and Use of Laboratory Animals.
2.1 Animals and experimental design
Eight-week-old male Sprague-Dawley (SD) rats were purchased from Japan SLC (Hamamatsu,
Japan) and housed individually in a temperature- and humidity-controlled room (22-24 °C and 5060% relative humidity) with a 12-hour light-dark cycle. After a one-week acclimation period withstandard rodent chow (MF; Oriental Yeast, Tokyo, Japan) and water ad libitum, the rats wererandomly divided into five groups that were fed for 9 weeks as follows: the control group (n = 6)
was fed MF as the normal diet, the HFC group (n = 6) was fed a high-fat and high-cholesterol (HFC)
diet, the EMP1% group (n = 6) was fed a HFC diet supplemented with 1% (w/w) EMP, the EMP2.5%
group (n = 7) was fed a HFC diet supplemented with 2.5% (w/w) EMP, and the EMP5% group (n = 7)
was fed a HFC diet supplemented with 5% (w/w) EMP. The HFC diet was prepared by mixing MF
with 28.75% (w/w) palm oil, 1.25% (w/w) cholesterol, and 0.5% (w/w) sodium cholate [11]. EMP
was provided by L.S Corporation, Tokyo, Japan [12]. Mackerel meat was powdered and heated at
90 °C, and then was digested with proteases from Bacillus subtilis and Aspergillus oryzae. The
hydrolysate was further digested with pancreatin and leucine aminopeptidase. After thesubsequent processes of filtration, separation of oils and fats, concentration, removal of metals,
and finally desiccation, approximately 20 g of EMP powder was obtained from 400 g of mackerel
meat. Ultimately, 100 g of the EMP powder contained 90 g of protein, 0.2 g of lipid, 9.1 g of ash,and 951μg of selenium. The proximate dietary compositions of each diet fed to the rats are shownin Table 1. Daily energy intake and body weight were monitored throughout the study.
At 18 weeks of age, the rats were fasted for 6 hours and sacrificed under anaesthesia withpentobarbital sodium. Blood samples were taken from the inferior vena cava or heart and kept at 20°C until analysis. The epididymal fat pad and liver were removed, washed in cold saline, andweighed. Liver tissues were either placed in 10% neutral buffered formalin or snap frozen in liquid
nitrogen and stored at -80°C.
Table 1 Proximate dietary compositions.
Ingredient\Group
Water (g)Crude protein (g)Crude lipid (g)Crude ash (g)Control
7.90
23.10
5.10
5.80

HFC
5.49
16.05
3.54
4.03

EMP1%
5.46
16.79
3.51
4.08

EMP2.5%
5.42
17.89
3.46
4.16

EMP5%
5.35
19.73
3.37
4.28
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Crude fiber (g)Nitrogen-free extract (g)Palm oil (g)Cholesterol (g)Sodium cholate (g)Total (g)Protein energy ratio (%)
Lipid energy ratio (%)
Carbohydrate energy ratio (%)
Energy (kcal/100g)

2.80
55.30
0.00
0.00
0.00
100.00
25.70
12.77
61.53
359.50

1.95
38.43
28.75
1.25
0.50
100.00
12.63
57.15
30.23
508.60

1.93
38.04
28.46
1.24
0.49
100.00
13.25
56.74
30.01
507.03

1.90
37.45
28.02
1.22
0.49
100.00
14.18
56.13
29.69
504.68

1.85
36.48
27.29
1.19
0.47
100.00
15.76
55.10
29.14
500.76

Control group rats were fed MF diet as a normal diet.
HFC, high-fat and high-cholesterol diet; EMP, peptides obtained by enzymatic decompositionof mackerel; EMP1%, EMP2.5% and EMP5%, HFC diet supplemented with 1%, 2.5% and 5%(w/w) EMP, respectively.

2.2 Serum biochemical analysis
Serum triglyceride (TG), free fatty acid (FFA), total cholesterol (TC), free cholesterol (FC),glucose, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were
determined using Triglyceride E test Wako, NEFA C test Wako, Cholesterol E test Wako, Freecholesterol E test Wako, Glucose C II test Wako, and Transaminase C II test Wako (Wako Pure
Chemical Industries, Osaka, Japan), respectively. Serum insulin, leptin, and adiponectin levels were
measured using a rat insulin enzyme-linked immunosorbent assay (ELISA) kit (Morinaga Instituteof Biological Science Inc., Yokohama, Japan), a mouse/rat leptin ELISA kit (Morinaga Institute of
Biological Science Inc.), and a mouse/rat adiponectin ELISA kit (Otsuka Pharmaceuticals Co., Ltd.,Tokyo, Japan), respectively.
2.3 Hepatic lipid analysis
Hepatic lipids were extracted from the frozen liver using the method of Folch et al. [13]. The
extract was dissolved in isopropanol and analyzed for TG and TC with a kit, as described above.
2.4 Histopathological examinations
Liver tissues stored in 10% neutral-buffered formalin were embedded in paraffin, sectioned at 4
μm, and processed for hematoxylin-eosin (HE) and Azan staining for histopathological examination.
Histological steatosis (0-3), lobular inflammation (0-3), and hepatocyte ballooning (0-2) wereassessed semi-quantitatively to determine the NAFLD activity score (NAS) according to the NASH
Clinical Research Network Scoring System [14]. NAS scores ≥ 5 and ≤ 2 were considered diagnostic
and not diagnostic, respectively, for steatohepatitis. Liver fibrosis (0-4) was also assessed
according to this system [13]. For the immunohistochemical analysis of 4-hydroxynonenal (4-HNE),
deparaffinized liver specimens were incubated with rabbit polyclonal antibodies against 4-HNE(OXIS International, Foster City, CA, USA) to detect oxidative stress-induced lipid peroxidation.
Normal rabbit serum was used as a negative control. EnVision peroxidase was used as the second
antibody (DAKO, Glostrup, Denmark), and 3,3′ diaminobenzidine (DAB) was used as the substrate.
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The intensity of 4-HNE was scored as 0, 1, and 2, indicating no positive staining or slightly positivein the perivenular zone, slightly positive in the periportal zone, and positive staining in
hepatocytes or strongly positive in the periportal zone, respectively. All histopathologicalexaminations were performed by a pathologist (K.T.) who was blinded to the experimental andserological data.
2.5 Quantification of mRNA using real-time polymerase chain reaction
Total RNA from the liver was extracted using RNAiso Plus (Takara Bio, Otsu, Japan) according to
the manufacturer’s instructions. RNA was reverse-transcribed to cDNA templates using acommercial kit (PrimeScript RT Master Mix, Takara Bio). Real-time polymerase chain reaction (PCR)
analysis was performed as described previously [11]. Specific primers were designed using thePrimer-BLAST primer designing tool (National Center for Biotechnology Information [NCBI],
Bethesda, MD, USA) and were synthesized by Greiner Bio-One Japan (Tokyo, Japan) (Table 2).
Table 2 Primer sequences for real-time polymerase chain reaction.
PrimerCOL1A1
TGF-β
α-SMA
TNF-α
MCP-1
IL-1β
IL-6
NF-κB
CYP2E1
HO-1
GPX-1
MnSOD
LXR-α
MTP
FAS
GPAT
CPT-1
AOX

Forward (5’ to 3’)
GCGTAGCCTACATGGACCAA
CTTTGTACAACAGCACCCGC
GCCAAGAAGACATCCCTGAAGTTGATCGGTCCCAACAAGGATCTGTCACGCTTCTGGGCCTGT
CTCCAGTCAGGCTTCCTTGTGGATACCACCCACAACAGACCAGTATGACATCATCAACATGAGAAACGA
CCCATCCTTGGGAACATTTTT
CACAGGGTGACAGAAGAGGCTAAGCTGCTCATTGAGAATGTCGGACCTGCCTTACGACTATGCAGGACCAGCTCCAAGTAGACAAGCTCAAGGCAGTGGTTGCAACATTGACGCCAGTTCCGGCTACCTGAAGGTGAGCCAGAACCTCGGACCCAAATTGC
CCACTGAACAAAACAGAGGTCC

Reverse (5’ to 3’)
AAGTTCCGGTGTGACTCGTGTAGATTGCGTTGTTGCGGTCTGTGGCAGATACAGATCAAGCATTGGGCTACGGGCTTGTCAGGGGCATTAACTGCATCTGGCTGAGGGTCATTCTCCTCACTGTCGAAATGCACAACTCTTTTCTCATTTCCA
CCCCAACCCTCAGCAAGTCGCCAAGGTGCAGTGTGAACAGGGACTCTGGTCTTTGTGTTCCTGAATCTCTTCATTCTTGCCATT
TACTTCTCCTCGGTGACGGAACATCAGTCGGTCGTGG
AGCAGGTACATCGTGGTGTCTTCGAGCCAGTGTCTTCCACAGGTACTCAGACTCCGGGAC
GGCCCCGCAGGTAGATATATTGTCCCAGGGAAACTTCAAAGC

All quantifications were normalized using the housekeeping gene glyceraldehyde 3-phosphate
dehydrogenase (GAPDH). For studies in rats, hepatic expressions of genes involved in fibrosis[collagen type I alpha 1 (COL1A1), transforming growth factor-β (TGF-β), and α-smooth muscle
actin (α-SMA)], inflammation [tumor necrosis factor-α (TNF-α), monocyte chemoattractant
protein-1 (MCP-1), interleukin-1β (IL-1β), interleukin-6 (IL-6), and nuclear factor-κB (NF-κB)+,
oxidative stress [cytochrome P450 family 2 subfamily E polypeptide 1 (CYP2E1), heme oxygenase-1(HO-1), glutathione peroxidase-1 (GPX-1), and manganese superoxide dismutase (MnSOD)],

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cholesterol metabolism [liver X receptor-α (LXR-α)+, and lipid metabolism *microsomal triglyceridetransfer protein (MTP), fatty acid synthase (FAS), glycerol-3-phosphate acyltransferase (GPAT),
carnitine palmitoyltransferase-1 (CPT-1), and acyl-CoA oxidase (AOX)] were quantified. All data
were expressed as a fold change relative to the control group.
2.6 Statistical analysis
All values were expressed as mean ± standard error (SE). Differences between groups were
tested for statistical significance using one-way analysis of variance (ANOVA), followed by
Scheffe’s post hoc test, chi-square test, or Fisher’s exact probability test. Correlations between
two variables were determined by Spearman’s rank correlation coefficient. All analyses were
performed using IBM SPSS statistics software program, version 24 (IBM Co., Somers, NY, USA) on a
Windows computer. A p value of less than 0.05 was considered to be statistically significant.
3. Results
3.1 Cumulative energy intake, body weight, and relative organ weights
During the 9-week study (9 to 18 weeks of age), cumulative energy intake was significantly
higher in the EMP1% group than in the control and EMP5% groups (p = 0.003 and 0.023,
respectively), whereas body weight at 18 weeks of age and weight gain during the 9-week study
were not significantly different among the groups. The liver weight/body weight ratio at 18 weeks
of age was significantly higher in the HFC, EMP1%, EMP2.5%, and EMP5% groups than in thecontrol group (p < 0.001), and this ratio was notably similar between the HFC, EMP1%, EMP2.5%,
and EMP5% groups. The epididymal fat pad weight/body weight ratio was not significantly
different among the groups (Table 3).
Table 3 Cumulative energy intake, body weight, relative organ weights, serum
parameters and hepatic lipid concentrations at 18 weeks of age.
Item\Group

Control(n=6)

Cumulative energy intake (kcal)
Final body weight (g)Body weight gain (g)Liver/body weight (%)
Epididymal fat pad/body weight (%)
Serum triglyceride (mg/dL)
Serum free fatty acid (mEq/L)
Serum total cholesterol (mg/dL)
Serum free cholesterol (mg/dL)
Serum glucose (mg/dL)
Serum insulin (ng/mL)
Serum AST (IU/L)
Serum ALT (IU/L)

5187±86a 5907±165ab 6253±277b 5782±130ab 5426±97a
511±11525±10527±13506±13489±9210±9223±10225±12
208±12
191±7
a
b
b
b
2.8±0.1
5.5±0.2
5.5±0.1
5.4±0.2
5.6±0.2b
1.9±0.1
2.2±0.2
2.2±0.1
2.3±0.1
1.7±0.2
72±8
55±8
52±3
63±5
52±9
0.28±0.06 0.28±0.08 0.28±0.04 0.26±0.04 0.22±0.04
65±5a
91±4ab
83±5ab
92±9ab
95±6b
22±2a
27±2a
26±2a
29±2a
40±3b
134±8
152±14130±9159±15181±456.4±1.3
7.2±1.1
4.6±1.5
5.7±0.9
6.1±1.056±12137±22111±19114±13120±22
a
ab
ab
b
11±1
44±8
51±13
83±8
92±14b

HFC
(n=6)

EMP1%
(n=6)

EMP2.5%
(n=7)

EMP5%
(n=7)

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Serum leptin (ng/mL)
7.4±0.5ab 9.4±1.1b
5.3±0.6ac 5.5±0.4ac 3.6±0.4c
Serum adiponectin (μg/mL)
4.4±0.4
4.0±0.6
3.0±0.4
3.5±0.4
4.0±0.5abbc
c
Hepatic triglyceride (mg/g tissue)
22±4
95±14
127±18151±4150±10c
Hepatic total cholesterol (mg/g tissue) 3.8±0.2a 78.8±9.9b 101.0±12.7b 75.6±4.9b 78.6±12.2b
Values are expressed as means±SE.
abc
Values not sharing the same lowercase letter in a row are significantly different amonggroups (p<0.05). HFC, high-fat and high-cholesterol diet; EMP, peptides obtained by enzymatic
decomposition of mackerel; EMP1%, EMP2.5% and EMP5%, HFC diet supplemented with 1%,
2.5% and 5% (w/w) EMP, respectively; AST, aspartate aminotransferase; ALT, alanine
aminotransferase.

3.2 Serum and hepatic biochemical parameters
At 18 weeks of age, serum TG, FFA, glucose, insulin, AST, and adiponectin levels were notsignificantly different among the groups. Serum TC levels were significantly higher in the EMP5%
group than in the control group (p = 0.039), and serum FC levels were significantly higher in the
EMP5% group than in the control, HFC, EMP1%, and EMP2.5% groups (p < 0.001, 0.005, 0.004, and
0.018, respectively). Serum ALT levels were significantly lower in the control group than in the
EMP2.5% and EMP5% groups (p = 0.001 and <0.001, respectively). Serum leptin levels weresignificantly higher in the HFC group than in the EMP1%, EMP2.5%, and EMP5% groups (p = 0.006,
0.005, and <0.005, respectively); the serum leptin levels were also significantly higher in thecontrol group than in the EMP5% group (p = 0.007). Hepatic TG values were significantly lower inthe control group than in the HFC, EMP1%, EMP2.5%, and EMP5% groups (p = 0.003, <0.001,<0.001, and <0.001, respectively), and were also significantly lower in the HFC group than in the
EMP2.5% and EMP5% groups (p = 0.025 and 0.029, respectively). Hepatic TC values weresignificantly lower in the control group than in the other 4 groups (p < 0.001) (Table 3).
3.3 Histopathological findings of the liver
Representative histopathology of the rat liver and histological assessments are shown in Figure
1 and Table 4. No obvious findings of hepatic steatosis, lobular inflammation, hepatocyte
ballooning, or fibrosis were seen in any of the rats in the control group. In contrast, severe
steatosis (score 3), moderate or severe lobular inflammation (score 2 or 3), and mild or
perisinusoidal and portal/periportal fibrosis (score 1a or 2) were observed in all 6 rats of the HFCgroup, despite no evidence of hepatocyte ballooning. In the EMP1%, EMP2.5%, and EMP5%
groups, severe steatosis (score 3) was observed in 19 (95%) of the rats; moderate or severe lobular
inflammation (score 2 or 3) was observed in 17 (85%) of the rats; and perisinusoidal,
portal/periportal, or bridging fibrosis (score 2 or 3) was observed in 12 (60%) of the 20 rats.
According to the NAS [14], none of 6 rats of the control group (0%), all of 6 rats of the HFC group(100%), 5 of 6 rats of the EMP1% group (83%), 5 of 7 rats of the EMP2.5% group (71%), and 6 of 7
rats of the EMP5% group (86%) were diagnosed with NASH (i.e., having a NAS of 5 of greater).
Representative immunohistochemistry of the rat liver and immunohistochemical assessments
are also shown in Figure 1 and Table 4. Negative staining (score 0) was seen in all 6 rats of thecontrol group, whereas positive staining (score 2) was observed in 5 of 6 rats (83%) in the HFCgroup. In the 20 rats of EMP1%, EMP2.5% or EMP5% group, negative staining (score 0) was
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observed in 5 (25%) rats, and positive staining (score 2) was also observed in 5 (25%) rats. The 4HNE staining score was significantly correlated with the histological lobular inflammation score
and the diagnostic result of NAS (p=0.005 and p=0.001, respectively) (Table 5).

Figure 1 Representative histopathology of the rat liver in the control, HFC, EMP1%,
EMP2.5%, and EMP5% groups at 18 weeks of age. Upper panels: HE-stained sections(original magnification, x100; scale bars=400μm). Middle panels: Azan-stained sections(original magnification, x200; scale bars=200μm). Lower panels: Immunohistochemical
4-HNE-stained sections (original magnification, x100; scale bars=400μm). HFC indicateshigh-fat and high-cholesterol diet; EMP indicates peptides obtained by enzymatic
decomposition of mackerel; EMP1%, EMP2.5%, and EMP5% were fed HFC dietsupplemented with 1%, 2.5%, and 5% (w/w) EMP, respectively.
Table 4 Histopathological and immunohistochemical assessments of the liver at 18 weeks of age.
Item\Group

Score

Control
(n=6)

HFC
(n=6)

EMP1%
(n=6)

EMP2.5%
(n=7)

EMP5%
(n=7)

Steatosis*

0
2
3
0
1
2
3
0
0-2
3-4

6
0
0
6
0
0
0
6
6
0

0
0
6
0
0
5
1
6
0
0

0
0
6
0
1
2
3
6
0
1

0
0
7
0
2
3
2
7
0
2

0
1
6
0
0
5
2
7
0
1

Lobular inflammation*

Hepatocyte ballooning*
NAFLD activity score (NAS)*

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Fibrosis*
4-HNE staining

5-8
0-1
2-3
0
1
2

0
6
0
6
0
0

6
5
1
0
1
5

5
2
4
1
3
2

5
5
2
2
5
0

6
1
6
2
2
3

Values are expressed as the number of rats.
*Scores are according to the NASH Clinical Research Network Scoring System proposed by
Kleiner et al. [14].
HFC, high-fat and high-cholesterol diet; EMP, peptides obtained by enzymatic decompositionof mackerel; EMP1%, EMP2.5% and EMP5%, HFC diet supplemented with 1%, 2.5% and 5%(w/w) EMP, respectively; 4-HNE, 4-hydroxynonenal.

Table 5 Correlations between 4-HNE staining score and histological lobular
inflammation score or NAS score.
Item\4-HNE staining score
Histological lobular inflammation*
Score 0Score 1
Score 2
Score 3
NAFLD activity score (NAS)*
0-2 (non-NASH)
3-4 (borderline)
5-8 (NASH)

0

1

2

6
1
2
2

0
2
5
4

0
0
8
2

6
1
4

0
3
8

0
0
10

Values are expressed as the number of rats.
*Scores are according to the NASH Clinical Research Network Scoring System proposed by
Kleiner et al. [14].
4-HNE, 4-hydroxynonenal; NASH, nonalcoholic steatohepatitis.

3.4 Hepatic mRNA expression
To reveal the effect of EMP on NASH on a molecular level, we evaluated key molecular markers
of fibrosis, inflammation, oxidative stress, and cholesterol and lipid metabolism in the liver. As
shown in Figure 2, the mRNA levels of COL1A1 and α-SMA, which are both involved in fibrogenesis,
tended to be lower in the EMP1% and EMP2.5% groups than in the HFC group; however, themRNA level of TGF-β (which is a key inducer of fibrogenesis), was similar between these groups
and the HFC group. The mRNA levels of TNF-α and IL-1β, which are involved in inflammation,tended to be higher in the EMP groups (EMP1%, EMP2.5%, and EMP5%) than the HFC group in an
EMP-dose dependent manner. The mRNA levels of MCP-1 and IL-6, which are also involved ininflammation, tended to be lower in the EMP1% group and EMP2.5% group compared to the HFCgroup, but were the highest in the EMP5% group. In contrast, the mRNA level of NF-κB, which is acentral regulator of inflammation, tended to be lower in the EMP groups than in the HFC group in

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an EMP-dose dependent manner. The mRNA levels of CYP2E1 and MnSOD, which is a member ofthe cytochrome P450 mixed-function oxidase system and a member of the iron/manganesesuperoxide dismutase family, respectively, did not significantly differ between the HFC group andFigure 2 Hepatic gene expression involved in (A) fibrosis, (B) inflammation, (C)oxidative stress, and (D) cholesterol or lipid metabolism in the control, HFC, EMP1%,

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EMP2.5%, and EMP5% groups (n = 6-7/group) at 18 weeks of age. mRNA levels are
expressed relative to the control group (mean ± SE). abValues not sharing the same
lowercase letter in a row are significantly different among groups (p < 0.05). HFC
indicates high-fat and high-cholesterol diet; EMP indicates peptides obtained byenzymatic decomposition of mackerel; EMP1%, EMP2.5%, and EMP5% were fed HFC
diet supplemented with 1%, 2.5%, and 5% (w/w) EMP, respectively.
EMP groups. The mRNA level of HO-1, which is an inducible enzyme in response to oxidative stress,
tended to be lower in the EMP1% group than in the HFC group. In contrast to this, the mRNA levelof GPX-1, which is an intracellular antioxidant enzyme, tended to be higher in the EMP groups
than in the HFC group in an EMP-dose dependent manner, although the difference was notstatistically significant. The mRNA level of LXR-α, which is involved in hepatic lipid homeostasis,
tended to be lower in the EMP groups than in the HFC group. The mRNA level of MTP, which is a
rate-limiting protein in the synthesis and excretion of very-low-density lipoprotein (VLDL) from theliver, was lower in the EMP1% group than in the control group. The mRNA level of FAS, which is amulti-enzyme protein that catalyzes fatty acid synthesis, tended to be higher in the EMP groups
than in the HFC group in an EMP-dose dependent manner. The mRNA levels of GPAT, CPT-1, and
AOX, which are involved in lipid metabolism, were not significantly different between the HFCgroup and the EMP groups.
The mRNA level of TGF-β was positively correlated with histological hepatic fibrosis (p = 0.001, r= 0.553). The mRNA levels of NF-κB and HO-1 were positively correlated with histological lobular
inflammation of the liver (p = 0.003, r = 0.513 and p = 0.005, r = 0.481, respectively). The mRNAlevel of GPX-1 was negatively correlated with immunohistochemical 4-HNE staining (Figure 3).

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Figure 3 Correlations between hepatic histological findings and gene expressions.
Histological fibrosis score is positively correlated with the relative mRNA level of TGF-β(A). Histological lobular inflammation scores are positively correlated with the relative
mRNA levels of NFκB (B) and HO-1 (C), respectively. Immunohistochemical 4-HNE
staining score is negatively correlated with the relative mRNA level of GPX-1 (D).
*Scores are according to the NASH Clinical Research Network Scoring System proposed by
Kleiner et al. [14].

4. Discussion
The daily doses of EMP in this study were determined based on human doses. The average daily
selenium intake of Japanese people is reported to be approximately 100 μg [15]. Because EMP
contains 951 μg selenium in 100 g, daily doses of EMP1%, 2.5%, and 5% in rats would beequivalent to daily doses of approximately 77, 193, and 385 μg in humans, respectively, after
taking into account the human equivalent dose.
In this study, there were no significant differences in liver/body weight, epididymal fat padweight/body weight, or serum metabolic parameters such as serum glucose, insulin, or
adiponectin at 18 weeks of age between the HFC group and the EMP groups; however, serum
leptin levels were lower in EMP groups than in the HFC group. Leptin possesses a number ofeffects on the immune system and plays an important role in modulating inflammatory responses,
regulation of food intake, and energy expenditure [16]. Indeed, serum leptin levels were
negatively correlated with serum ALT levels in this study (p = 0.003, r = -0.510, data not shown).
No significant difference in epididymal fat pad weight/body weight, serum triglyceride levels, orfree cholesterol levels between the HFC groups and the EMP groups was detected; this indicatesthat the leptin-lowering component of EMP may not largely affect obesity or visceral fat tissue.
Further studies examining the insulin-leptin axis and the role of endotoxins are needed todetermine the mechanism of this effect [16, 17].
The serum FC levels were significantly higher in the EMP5% group than in the control, HFC,
EMP1%, and EMP2.5% groups in this study. The reason for this result is unclear. Lecithincholesterol acyltransferase (LCAT) is synthesized by the liver, and esterifies FC on the surface of
HDL, forming cholesteryl esters [18]. Because the histological hepatic fibrosis was moreprogressive in the EMP5% group, it is conceivable that LCAT synthesis and esterification of FC canbe stagnated.
Histopathological findings of the liver revealed that all 6 rats in the HFC group were diagnosed
with NASH; contrastingly, 4 out of 20 rats (20%) in the EMP groups had borderline scoresaccording to the NASH Clinical Research Network Scoring System [14] and were not diagnosed as
fully-developed NASH in this study. Because the histopathological findings of steatosis andhepatocyte ballooning were similar between the HFC group and the EMP groups, this difference in
the NASH diagnostic rate between the HFC group and the EMP groups (100% vs 80%) was mainly
due to the histopathological lobular inflammation score. Moreover, immunohistochemical staining
intensities of 4-HNE tended to be lower in the EMP groups than the HFC group; the 4-HNE staining
score was significantly correlated with the histological lobular inflammation score and the
diagnostic result of NAS. Oxidative stress seems to be one of the most important mechanisms
leading to hepatic injury in NAFLD, as augmented generation of reactive oxygen species (ROS) can
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induce lipid peroxidation, leading to inflammation [19]. Thus, our results indicate that EMP
reduced oxidative stress, which decreased lobular inflammation and NASH progression.
The TGF‐β pathway is a significant modulator of COL1A1, the type I collagen gene [20,21], while
Smads are a family of proteins that operate downstream of various members of the TGF‐β
superfamily [20]. In this study, the mRNA level of TGF-β was positively correlated with histological
hepatic fibrosis. mRNA levels of TGF‐β in the EMP groups were similar to that in the HFC group,and mRNA levels of COL1A1 and α-SMA tended to be lower in the EMP1% and EMP2.5% groups
than in the HFC group. However, histological hepatic fibrosis was more progressive in the EMP
groups than in the HFC group. The reason for these discrepancies is not clear but may be
attributable to the non-Smad signaling pathways [22].
Antioxidants can inhibit NF-κB activation [23]. In this study, the mRNA levels of NF-κB werepositively correlated with histological lobular inflammation of the liver and tended to be lower inthe EMP groups (EMP1%, EMP2.5%, and EMP5%) than in the HFC group in an EMP-dose
dependent manner. In contrast, the mRNA level of the intracellular antioxidant enzyme GPX-1 was
negatively correlated with immunohistochemical 4-HNE staining and tended to be higher in the
EMP groups than in the HFC group in an EMP-dose dependent manner, although the difference
was not statistically significant. These results indicate that the lobular inflammation in EMP groups
can be reduced by NF-κB suppression via GPX-1 expression. However, the histological lobular
inflammation score tended to be lower in the EMP1% and EMP2.5% groups but not in the EMP5%
group. The reason for this discrepancy is not obvious, but inflammation-related cytokines such as
TNF-α, MCP1, IL-1β, and IL-6 in the EMP5% group can be induced by non-NF-κB signaling pathways,
which may offer an explanation [24].
Our results from the histological fibrosis score, NAS score, and hepatic mRNA expressions
indicate that the EMP5% group did not reveal a preventive effect on NASH progression.
Supranutritional selenium intake or high plasma selenium concentration was reported to be
associated with type 2 diabetes or insulin resistance [8, 25, 26]. Selenium supplementationinduces selenoproteins such as intracellular antioxidant enzyme GPX-1 and selenium transporter
selenoprotein P, which are both implicated in insulin resistance due to the impairment of insulin
signaling in the liver and skeletal muscle [8, 25, 27-29]. In this study, the mRNA level of HO-1,
which is an inducible enzyme in response to oxidative stress, was positively correlated withhistological lobular inflammation of the liver and tended to be lower in the EMP1% group but
higher in the EMP5% group when compared to the HFC group. ROS can induce lipid peroxidation,
leading to fibrogenesis through the activation of stellate cells, and also inhibit hepatocyte
secretion of VLDL, which induces liver fat accumulation [19]. Malaguarnera et al. reported that asignificant correlation between the increased levels of HO-1 and lipid peroxidation as well as theincreased HO-1 expression reflected the severity of NASH [30]. Therefore, 5% (w/w)
supplementation of EMP to a HFC diet was an excessive dose for SD rats in the prevention of NASH.
5. Conclusions
Our results demonstrate that EMP may possess a preventive effect on NASH progression,
presumably through its antioxidative stress activity. The EMP1% and 2.5% groups, which were fed
a HFC diet supplemented with 1% and 2.5% (w/w) EMP for 9 weeks, respectively, were more

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effective in prevention than the EMP5% group, which was fed a HFC diet supplemented with 5%(w/w) EMP for 9 weeks. The optimal dose of EMP should be further researched in future studies.
Acknowledgments
The authors thank Mr. Satoshi Matsumoto, L.S Corporation, for providing peptides obtained byenzymatic decomposition of mackerel and for his helpful advice.
Author Contributions
Katsuhisa Omagari created the study design, adjusted results, and finalized the manuscript.
Ayumi Fukuda carried out the experiments and performed the data analysis.
Machiko Suga carried out the experiments and performed the data analysis.
Ayumi Ogata carried out the experiments and performed the data analysis.
Shinta Nishioka performed the data interpretation and revised the manuscript.
Kazuhito Suruga carried out PCR for mRNA expression and performed the data interpretation.
Mayuko Ichimura carried out the experiments and data collection.
Koichi Tsuneyama performed the histopathological assessment and corrected the manuscript.
Funding
This work was supported by research funds from the University of Nagasaki and a research
grant from L.S Corporation.
Competing Interests
Katsuhisa Omagari received a research grant from L.S Corporation for this work. The otherauthors have declared that no competing interests exist.
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