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Non-alcoholic steatohepatitis onset of mechanisms under
diabetic background and treatment strategies
Thesis for the degree of Doctor of Philosophy (PhD)
by
MST. REJINA AFRIN
Department of Clinical Pharmacology
Faculty of Pharmaceutical Sciences
Niigata University of Pharmacy and Applied Life Sciences
Niigata, Japan
Supervised by
Professor Kenichi Watanabe, M.D., Ph.D.,
Reviewed by:
Professor Kenji Suzuki, M.D., Ph.D.,
Professor Toshiyuki Sakamaki, Ph.D.,
Professor Kazuyuki Ueno, Ph.D.,
February 2017
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
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DEDICATED TO
My beloved parents, brother, husband, respectable
teachers and specially to my supervisor
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CONTENTS
Page No.
ACKNOWLEDGEMENT………………………………………………….5
ABBREVIATIONS…………………………………………………………7
ABSTRACT…………………………………………………………………12
INTRODUCTION………………………………………………………….18
Background…………………………………………………………………19
Definitions…………………………………………………………………..20
NASH-HCC………………………………………………………………… 21
Histopathology of NASH………………………………………………….22
Factors associated with NASH…………………………………………. 26
Pathophysiology of NASH…………………………………………………31
Epidemiology and prevalence of NASH……………………………… 42
Treatment for NASH………………………………………………………43
Curcumin……………………………………………………………………44
Le Carbone…………………………………………………………………. 46
SCOPE OF STUDY..............................................................................48
CHAPTER ONE…………………………………………………………… 50
Introduction…………………………………………………………………51
Materials and methods……………………………………………………54
Results……………………………………………………………………… 58
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Discussion……………………………………………………………………..71
Conclusions………………………………………………………………… 79
CHAPTER TWO…………………………………………………………… 80
Introduction………………………………………………………………… 81
Materials and methods…………………………………………………… 83
Results…………………………………………………………………………88
Discussion…………………………………………………………………… 102
CHAPTER THREE………………………………………………………….108
Introduction………………………………………………………………… 109
Materials and methods…………………………………………………… 111
Results…………………………………………………………………………116
Discussion……………………………………………………………………..130
CHAPTER FOUR…………………………………………………………….138
Introduction…………………………………………………………………..139
Materials and Methods……………………………………………………..140
Results…………………………………………………………………………143
Discussion…………………………………………………………………….154
SUMMARY………………………………………………………………….. 159
REFFERENCES……………………………………………………………..161
LIST OF PUBLISHED ARTICLES……………………………………….192
FELLOWSHIP………………………………………………………………. 198
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ACKNOWLEDGEMENT
All my gratitude to almighty ALLAH.
I would like to express my best regards profound gratitude,
indebtedness and deep appreciation to my honorable and beloved supervisor
Professor Kenichi Watanabe, Department of Clinical Pharmacology, Niigata
University of Pharmacy and Applied Life Science (NUPALS), for his constant
supervision, expert guidance, enthusiastic encouragement and never-ending
inspiration throughout the entire period of my research work as well as to
prepare this dissertation. I really owe him forever for giving me such an
opportunity to work in close association with him.
I would like to express my heartfelt thanks to Dr. Meilei Harima, for
her valuable suggestions and inspiration during the course of my research
work.
I am extremely thankful to NUPALS and MEXT: The Ministry of
Education, Culture, Sports, Science and Technology of Japan for providing
me Japanese Government Scholarship (Monbukagakusho) during my study
in Japan.
I offer my special thanks to Dr. Somasundaram Arumugum, for his
active help, and enthusiastic encouragement throughout the entire period of
my research work. Special indebtedness and gratitude to Dr. Hiroyuki
Yoneyama, Executive Director, Stelic Institute of Regenerative Medicine,
Tokyo for his wise advice, constructive criticism and valuable suggestions
during the period of my research work.
I am highly and cordially grateful to our collaborator, Professor Kenji
Suzuki, Department of Gastroenterology, Niigata University of Medical and
Dental Sciences, Niigata City, Japan; Professor Kazuyuki Ueno, Department
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
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of Pharmaceutical Sciences, NUPALS, Niigata; and Professor Toshiyuki
Sakamaki, Department of Public Health, NUPALS, Niigata for their
sincerely reviewing my thesis paper and support.
I wish to express my gratefulness to Professor Mir Imam Ibne Wahed,
Department of Pharmacy, University of Rajshahi, Bangladesh for his
valuable suggestions, affection, and constant inspiration during the course of
my research work. I cannot find words to express my gratitude to my
supervisor during the M. Pharm thesis work, Professor Golam Sadik,
Department of Pharmacy, University of Rajshahi, Bangladesh, who
introduces me with research and makes me interested and patient in
research works. I am extremely thankful to Dr. Aktar Ali, Director,
Biomedical and Obesity Research Core, University of Nebraska, Lincoln,
USA for introducing me with Professor Kenichi Watanabe and his laboratory.
I convey my heartiest thanks to my lab mates, Dr, Vigneshwaran
Pichaimani, Vengadeshprabhu Karuppagounder, Remya Sreedhar, Mayumi
Nomoto, Shizuka Miyashita, and Hiroshi Suzuki for their encouragement and
kind cooperation throughout the research work.
I would like to express my eternal appreciations towards my husband
Md. Azizur Rahman, Graduate Student of Department of Immunology and
Medical Zoology, Faculty of Medicine, Niigata University Graduate School of
Medical and Dental Sciences, Niigata City, Japan for his continuous support
and sincere pray for making my dream come true.
Last but not be least, I wish to express my sincere gratitude and
heartfelt obligation to my beloved and respected parents Md. Abdur Razzak,
and Mst. Sabina Yasmin, and my younger brother Md. Sabbir Hossain for
their moral and financial support, constant encouragement and never ending
affection and blessing when they were needed most.
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ABBREVIATIONS
ACC, Acetyl-CoA Carboxylase;
ADRP, adipose differentiation related protein;
AdipoR, adiponectin receptor;
ALP, alkaline phosphatase;
ALT, alanine aminotransferase;
AMPK, Adenosine monophosphate-activated protein kinase;
ANOVA, One way analysis of variance;
ASK, apoptosis signal regulating kinase;
AST, aspartate aminotransferase;
ATF, activating transcription factor;
BCA, Bicinchoninic acid;
BCL, B-cell lymphoma;
CCL, chemokine (C-C motif) ligand
CD, Cluster of differentiation;
C/EBPβ, CCAAT/enhancer binding protein beta;
ChREBP, Carbohydrate-responsive element-binding protein;
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CTGF, connective tissue growth factor;
CYP, cytochrome P450;
DAMPs, Damage-associated molecular patterns;
DCP, Des-gamma-carboxy prothrombin;
DM, Diabetes Mellitus;
eIF2α, eukaryotic initiation factor 2 alpha;
ERK, extracellular-signal-regulated kinases;
ERS, endoplasmic reticulum stress,
FAS, Fatty acid synthase;
FFA, Free fatty acid;
FC, Free cholesterol;
GAPDH, Glyceraldehyde-3-phosphate dehydrogenase;
GLUT, Glucose transporter type;
GRP78, glucose regulated protein 78;
HCC, hepatocellular carcinoma;
H&E, hematoxylin and eosin;
HFD, high fat diet;
HFE, Human hemochromatosis protein;
HMGB, high mobility group box;
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HO-1, Heme-oxygenase-1;
IFN, interferon;
IL, interleukin;
IR, Insulin Resistance;
IRE1α, inositol requiring enzyme 1 alpha;
IP10, interferon inducible protein 10;
IκB, nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor;
JNK, c-Jun-N-terminal kinase;
KC, Kupffer cells;
LC, Le Carbone;
MAPK, Mitogen-activated protein kinase;
MMP, Matrix metalloproteinase;
MT, Masson trichrome;
MCD, methionine- and choline-deficient;
NAFLD, non-alcoholic fatty liver disease;
NASH, non-alcoholic steatohepatitis;
NF-κB, nuclear factor-κB;
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Nrf2, nuclear factor-erythroid 2-related factor-2;
Ox-LDL-R1, oxidized low-density lipoprotein receptor-1;
PAMPs, Pathogen-associated molecular patterns;
PERK, double stranded RNA depended protein kinase-like endoplasmic
reticulum kinase;
PEPCK, Phosphoenolpyruvate carboxykinase;
PNPLA3, Patatin-like phospholipase domain-containing protein 3;
PGC1α, Peroxisome proliferator-activated receptor γ coactivator-α;
PPAR, Peroxisome proliferator-activated receptor;
RAGE, Receptor for advanced glycation end products;
ROS, reactive oxygen species;
SCD, Stearoyl-CoA desaturase;
SD, Sprague-Dawely;
SEM, standard error of mean;
SFA, Saturated fatty acid;
SIRT, Sirtuin;
SREBP, sterol regulatory element binding protein isoform;
STZ, Streptozotocin;
TC, total cholesterol;
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TG, triglyceride;
Timp, Tissue inhibitor of metalloproteinases
TLR, toll like receptor;
TNF, tumor necrosis factor;
TRAF, TNF receptor –associated factor 2;
UPR, unfolded protein response.
VEGF, vascular endothelial growth factor
XBP, X-box binding protein
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ABSTRACT
Non-alcoholic fatty liver disease (NAFLD) is the buildup of extra fat in
liver cells due to cause other than excessive alcohol use. Non-alcoholic
steatohepatitis (NASH) is a progressive form of NAFLD, characterized by
necro-inflammation, lipid accumulation and fibrosis. NASH is an increasingly
common chronic liver disease with worldwide distribution and a major health
problem, owing to its close association with diabetes, obesity and the metabolic
syndrome. NASH acts as a significant risk factor for the development of
cirrhosis and hepatocellular carcinoma (HCC), a primary malignancy in liver
and the third leading cause of cancer-related deaths worldwide. Little is known
about the factors responsible for the transition of steatosis to steatohepatitis
and its progression in NAFLD/NASH. Perpetuate liver inflammation
represents a crucial aspect in NASH pathogenesis and has also been a critical
factor in the progression to HCC. Understanding the molecular mechanisms
linking hepatocytes necrosis to inflammation and progressive liver damage is,
therefore, critical to the development of novel therapeutic strategies for NASH-
HCC. Activation of High-mobility group box 1 (HMGB1), a DNA-binding
nuclear protein is an inflammatory cytokine, correlates positively with disease
states mediated by inflammatory stimuli, including, liver ischemia-reperfusion
injury, liver transplantation, cancer and HCC. Whether HMGB1 plays any role
in the novel NASH-HCC model is not well addressed. Once HMGB1 activated,
binds to cell surface receptors, including toll like receptor (TLR) 2, TLR4 then
enhance the nuclear factor κB (NF-κB), extracellular signal-regulated kinase
(ERK) signaling, and thereby triggering a variety of inflammatory cytokines in
the target cells. It is proposed that NF-κB acts as a central link between
hepatic injury, fibrosis, and HCC. Since NASH is considered as a hepatic
manifestation of the metabolic disorder, it is well established that the sirtuin
1 (SIRT1) -AMP-activated protein kinase (AMPK) axis, act as a central
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signaling system for controlling the lipid metabolism pathway. The activation
of this signaling in several metabolic tissues, including liver has been proposed
to increase the rate of fatty acid oxidation and restrain the lipogenesis mostly
by modulating the activity of peroxisome proliferator-activated receptor
(PPAR) γ coactivator-α (PGC-1α) /PPARα or SREBP-1c through distillation and
phosphorylation, respectively. Apart from energy control and lipid metabolism,
AMPK is implicated in multiple processes such as apoptosis, autophagy, and
senescence, which may be promising target for the treatment of NASH-HCC.
The absence of an effective pharmacological therapy for NASH is a major
incentive for research into novel therapeutic approaches for this condition.
Curcumin, a natural phenolic compound, has a wide spectrum of therapeutic
effects such as antitumor, anti-inflammatory, anti-cancer and so on. On the
other hand, Le Carbone (LC) is a charcoal supplement, enriched with dietary
fibers, having AMPK-SIRT1 enhancing activity. The study aimed to
investigate the underlying mechanisms of curcumin and LC to protect liver
damage and progression of NASH in a novel NASH-HCC mouse model.
In the present study, I have used two animal models; type 1 diabetic rats,
and experimental NASH-HCC mice to investigate the mechanisms of hepatic
complications in diabetes and in NASH-HCC along with their treatment
options.
First, I investigated the role of curcumin in the liver of experimental
type 1 diabetic rats. For this study, I used male Sprague-Dawley rats and
experimental diabetes was induced by a single intraperitoneal injection of
streptozotocin (STZ) at a dose of 55 mg/kg body weight and 3 weeks after
confirmation of diabetes the rats were divided into two groups; diabetic
control group (n=10), treated with vehicle and curcumin group (n=10),
diabetic rats treated with curcumin (suspended in 1% gum Arabic) was at a
dose of 100 mg/kg/day by oral gavage and continued for 8 weeks. The blood
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
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glucose levels and rats weights were measured in each week during the
experiment. On the day of sacrifice the blood and liver tissues were collected
from the rats of each group. Plasma was separated from the blood to estimate
the triglycerides (TG), total cholesterol (TC), and alanine amino transferase
(ALT). The excised liver tissues were assessed by histopathology,
immunohistochemistry and Western blot analysis. My findings have
demonstrated that after 11 weeks of diabetes induction, diabetic rats
exhibited hepatic dysfunction, as evidence by elevated plasma ALT levels and
the histopathological changes which appeared fatty liver, cell hypertrophy
and increased ratio of liver to body weight. Blood glucose levels and plasma
TG, TC levels were also significantly elevated in diabetic rats. Hyperglycemia
increased the hepatic glucose regulated protein 78, activated the sub-arm of
the unfolded protein response (UPR) signaling protein such as phospho -
double stranded RNA depended protein kinase – like ER kinase (PERK),
inositol requiring enzyme1α, activating transcription factor (ATF)6α in the
diabetic rats, which suggest there is an increased endoplasmic reticulum (ER)
stress. Moreover, ER stress related apoptotic marker proteins such as C/EBP
homologous protein (CHOP), tumor necrosis factor (TNF) receptor- associated
factor (TRAF) 2, apoptosis signal-regulating kinase (ASK)1, phospho-p38
mitogen activated protein kinase (p-p38MAPK) and inflammatory cytokines
TNFα, interleukin (IL)-1β, TLR4 liver macrophage ED1 were significantly
elevated in the liver tissues of diabetic rats. Apoptotic and anti-apoptotic
signaling proteins such as cleaved caspase-3 and bcl2, were significantly
increased and decreased respectively in diabetic rats and curcumin treatment
prevented all of these alterations. Interestingly, curcumin treatment for 8
weeks could ameliorate all of these alterations with a significant result and
improved the liver function in diabetic rats.
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In my next study, I have used a novel NASH-HCC mouse model under
diabetic background to investigate the effect of curcumin treatment and
dietary supplement, Le Carbone (LC). To induce this model neonatal
C57BL/6 male mice were exposed to low-dose STZ and were fed a HFD from
the age of 4 weeks to 14 or 16 weeks. The same dose of curcumin (100 mg/kg)
was given daily by oral gavage, started at the age of 10 weeks and continued
until 14 weeks along with HFD feeding. LC suspension was administered
orally using 5 mg/mouse/day as a prevention, from the age of 6 weeks and
continued until 16 weeks of age along with HFD feeding. Each week body
weight and the blood glucose levels were checked in every group. At the end
of the experiment, blood samples and livers were collected. Serum was
separated from the blood for the estimation of TG, TC, amino transferases
(ALT, AST), alkaline phosphatase (ALP) test. The liver tissues were used for
histopathological and Western blot protein analysis. NASH mice developed
steatohepatitis by a significant elevation of serum aminotransferases, with
high NAFLD activity score and fibrosis. In the NASH mice, blood glucose
levels and serum TG, TC levels were also elevated. This model has no effect
on body weight changes in all the experimental groups but increased the
ratio of liver weight to body weight in NASH mice significantly than normal
mice. Along with these, the liver of the NASH group showed pale yellow color,
swelling and granular surface with tumor protruding. In case of curcumin
treatment, all of these abnormalities were significantly lesser than the NASH
mice and no tumor protrusion was found. In addition, curcumin treatment
markedly reduced the hepatic protein expression of oxidative stress, pro-
inflammatory cytokines, including interferon (IFN) γ, IL-1β and IFN γ-
inducible protein 10, which were elevated in NASH mice. Furthermore,
curcumin treatment significantly reduced the cytoplasmic translocation of
HMGB1 and the protein expression of TLR4. Nuclear translocation of NF-κB
was also dramatically attenuated by the curcumin in NASH liver. Curcumin
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treatment effectively reduced the progression of NASH by suppressing the
protein expression of glypican-3, vascular endothelial growth factor, and
prothrombin, which reveal HCC in the NASH liver. Our data suggest that
curcumin reduces the progression of NASH to HCC and liver damage, which
may act via inhibiting HMGB1-NF-κB translocation.
Administration of LC also improved the histopathological changes in
NASH liver. Furthermore, LC suspension prevented the lipogenesis and
promoted fatty acid oxidation in the NASH liver by significantly increasing
hepatocytes protein expression of peroxisome proliferator-activated receptor
(PPAR) α in comparing with NASH mice. The reduced hepatic protein
expression of phospho-AMPKα and SIRT1 were significantly elevated by the
LC suspension when compared to NASH mice. In addition, fibrosis (collagen
4, MMP-9) and oxidative stress (p47phox) were also markedly reduced and
the anti-oxidant HO-1, Nrf2 expression were increased in LC treated group in
comparison to NASH liver.
After that, to stand the reason behind considering this novel NASH-
HCC model under diabetic background for my experiment, I have compared
the pathophysiology between DM rats and NASH mice. I have found that DM
and NASH have shown similar pathogenic abnormalities both in liver and
serum, except the hepatic protein expression of glypican-3, which was
significantly elevated in NASH group only. From the other studies and my
data, it is confirmed that glypican-3 is a potential liver cancer therapeutic
target as it is over-expressed in HCC but not expressed or expressed at a low
levels in normal and DM liver tissue.
My results suggest that the novel NASH-HCC model was associated
with hyperglycemia, lipogenesis, inflammation, fibrosis, oxidative stress and
tumorigenesis, which maintained a sequential progression of NASH from
NAFLD to HCC under diabetic background. Curcumin treatment could
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ameliorates all of these abnormalities might be through modulation of
HMGB1-NF-κB translocations and protect the diabetic liver by reducing ER
stress with its anti-hyperglycemic properties. Where, Le Carbone could
ameliorate the oxidative stress, lipogenesis and protect the liver function
through activation of the AMPK-SIRT1 pathway. Collectively, my present
study provides the data to support the role of curcumin and LC in
ameliorating the progression of NASH in NASH-HCC mice.
Keywords: AMPKα; Diabetes; Endoplasmic reticulum stress; Fibrosis;
Hepatocellular carcinoma; HMGB1; NF-κB ; Non-alcoholic steatohepatitis;
PPARα.
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INTRODUCTION
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INTRODUCTION
1. Background
Steatohepatitis is a type of liver disease, characterized by inflammation of
the liver with concurrent fat accumulation in the liver classically seen in
alcoholics as a part of alcoholic liver disease. Frequently it is found in people
with diabetes and obesity and is related to metabolic syndrome. When not
associated with excessive alcohol intake, it is referred to as nonalcoholic
steatohepatitis (NASH), and is the progressive form of the relatively benign
non-alcoholic fatty liver disease (NAFLD) [1].
The term NASH, coined by Dr. Ludwig and his colleagues in 1980 to describe
the biopsy findings in 20 middle aged patients with steatohepatitis in the
absence of significant alcohol consumption, has served the field well by
bringing attention to this entity and promoting further research [2]. Although
the paper by Ludwig et al. is often referred to as the first report of NASH, the
histopathological features seen in NASH were described earlier [3], [4]. Over
the years several names have been used to describe this condition: diabetic
hepatitis [5], non-alcoholic steatonecrosis, alcohol-like liver disease in the
non-alcoholic [6], non-alcoholic fatty hepatitis, fatty liver hepatitis [7], bright
liver syndrome [8], and non-alcoholic steatosis syndromes [9]. There is a
strong association between the occurrence of fatty liver and insulin resistance
(IR), one of the core features of the metabolic syndrome [10].
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During the last three decades a large number studies have challenged the
nature of NASH. Some patients with this condition progress to liver cirrhosis
and hepatocellular carcinoma (HCC) [11]. These observations have spurred
an immense interest among scientists all over the world. In this time more
than 200 articles have published investigating different aspects of this
intriguing condition.
1.2. Definitions
First, NAFLD is the buildup of extra fat or triglyceride inside the hepatocytes
that is not caused by alcohol. It is normal for the liver to contain some fat.
Traditionally, if more than 5% - 10% percent of the liver’s weight is fat, then
it is called a fatty liver (steatosis).
NASH is a severe and progressive form of NAFLD result in liver
inflammation and damage, characterized by hepatocyte ballooning,
necroinflammation, and often associated with fibrosis. It is a significant form
of chronic liver disease both in adults and children. The natural history of
NASH ranges from indolent to end-stage liver disease. NASH is a common,
often “silent” liver disease. Most people with NASH feel well and are not
aware that they have a liver problem. Nevertheless, it can be severe and can
lead to cirrhosis, in which the liver is permanently damaged and scarred and
no longer able to work properly. Where, NASH revealed as an major cause of
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virus independent HCC, which may account for a large proportion of HCC in
developing countries [12].
HCC, called malignant hepatoma, is the most common type of liver cancer.
The fastest growing cause of cancer-related death is HCC, which is at least
partly attributable to the rising prevalence of NASH. Most cases of HCC are
secondary to either a viral infection (hepatitis B or C) or cirrhosis [11].
1.3. NASH and HCC
NASH can progress to cirrhosis and its related complications. Growing
evidence suggests that NASH accounts for a large proportion of idiopathic or
cryptogenic cirrhosis, which is associated with the typical risk factors for
NASH. HCC is a rare, although important complication of NAFLD. Diabetes
and obesity have been established as independent risk factors for the
development of HCC [11]. New evidence also suggested that hepatic iron
deposition increases the risk of HCC in NASH derived cirrhosis. Multiple
case reports and reviews of HCC in the setting of NASH support the
association of diabetes and obesity with the risk of HCC, as well as suggest
age and advanced fibrosis as significant risks. Insulin resistance and its
subsequent inflammatory cascades that is associated with the development of
NASH appear to play a significant role in the pathogenesis of HCC. The
complications of NASH such as cirrhosis and HCC, are expected to increase
with the growing epidemic of diabetes and obesity [13].
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Fig.1: The spectrum of liver diseases ranging from hepatic steatosis (simple
intrahepatic accumulation of lipid droplets) through steatosis with
inflammation and fibrosis (i.e., non-alcoholic steatohepatitis, NASH) to
cirrhosis and HCC.
1.4. Histopathology of NASH
NASH is a disorder that is histologically characterized by steatosis and
lobular hepatitis with necrosis or ballooning degeneration and fibrosis. The
different parts of this spectrum are probably best regarded as parts of
histological continuum. The histopathological hallmark of NAFLD is
macrovesicular steatosis, which predominantly affects the perivenular
regions, and it can extend to a panacinar distribution in severe cases (Figure
2).
NASH
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Figure-2: Pronounced macrovesicular steatosis (Hematoxylin and eosin).
Figure -3: Hepatocellular steatosis, hypertrophy and inflammation.
Macrovesicular steatosis (dotted line arrow): large lipid droplets are present
in hepatocytes; microvesicular steatosis (bold arrow): small lipid droplets are
present in hepatocytes. Hypertrophy (open arrow): the representative cell is
much larger than the surrounding steatotic hepatocytes but has the same
cytoplasmic characteristics. Clusters (aggregates) of inflammatory cells
(within circles). Hematoxylin and eosin; magnification 200x.
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Figure-4: A Masson trichrome stain shows perivenular/pericellular ("chicken
wire") fibrosis (blue area) in nonalcoholic steatohepatitis (NASH) (200×
magnification).
Figure-5: NASH cirrhosis (Masson trichrome staining).
When the hepatic steatosis is accompanied by features of necroinflammation,
ballooning of hepatocytes, and the presence of Mallory-Denk bodies; the diagnosis
of NASH can be made. Briefly, the two key features of NASH, steatosis and
inflammation, were categorized as follows: steatosis was determined by
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analyzing hepatocellular vesicular steatosis, i.e. macrovesicular steatosis and
microvesicular steatosis separately, and by hepatocellular hypertrophy as
defined in Figure 3. Inflammation was scored by analyzing the amount of
inflammatory cell aggregates (Figure 3) [14].
Fibrosis is considered as a feature of steatohepatitis, the typical pattern of
fibrosis of NAFLD is a perisinusoidal and /or pericellular distribution (Figure
4). Patients with NASH, mainly those with advanced fibrosis (bridging fibrosis), are
at higher risk for developing decompensated cirrhosis (Figure 5), HCC.
The different histological definitions have been used by different authors [15]. The
scoring system of NASH for rodent animal developed by Liang et al. [14] has been
shown in Table 1. It is unifies the lesions of steatosis and necroinflammation into a
“grade” and those of fibrosis into a “stage” [16].
Table 1: Grading of the histopathological lesions in NASH according to Liang et al
[14].
Histological features Score
0 1 2 3
Steatosis:
Macrovesicular steatosis <5% 5-33% 33-66% >66%
Microvesicular steatosis <5% 5-33% 33-66% >66%
Hypertrophy <5% 5-33% 33-66% >66%
Inflammation:
Number of inflammatory focci/field
<0.5
0.5-1.0
1.0-2.0
>2.0
Where, steatosis and hypertrophy are evaluated at 40× to 100× magnification, and
inflammation is evaluated at 100× magnification.
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Table 2 Staging of the fibrosis in NASH according to Brunt [16].
Fibrosis Stage
Stage 1
Zone 3 perisinusoidal/pericellular fibrosis; focally or extensively present.
Stage 2
Zone 3 perisinusoidal/pericellular fibrosis with focal or extensive periportal
fibrosis.
Stage 3
Zone 3 perisinusoidal/pericellular fibrosis and portal fibrosis with focal or
extensive bridging fibrosis.
Stage 4
Cirrhosis
To evaluate fibrosis 40× magnification is used.
Using multiple logistic regression the NAFLD activity score (NAS) was constructed.
The NAS is the unweighted sum of steatosis, lobular inflammation, and
hepatocellular ballooning scores. NASH was defined as a NAS of ≥5, “borderline
NASH” as a NAS of 3 or 4 “not NASH” as a NAS of <3.
1.5. Factors associated with NASH
As more contributing factors are continuously identified, a more complex
picture of NASH pathogenesis is emerging. Obesity, diabetes mellitus (DM),
hyperlipidemia, metabolic syndrome, and IR have been established as risk
factors for NAFLD and its progressive form NASH.
1.5.1. Genetic factors
Recent studies have demonstrated that polymorphisms of a number of
candidate genes, including those encoding for immunoregulatory proteins,
proinflammatory cytokines and fibrogenic factors, could influence the
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appearance of NASH and the eventual development of liver fibrosis in
patients with NAFLD [17]. Genetic variations can explain susceptibility to
develop NASH in patients with obesity and/or diabetes. Moreover, it has been
hypothesized that genetic factors influence fibrosis progression. The role of
HFE C282Y heterozygosity and iron accumulation are unclear, since studies
have yielded contradictory results [18]. A significant association with a SNP
was identified in patatin-like phospholipase domain-containing 3 (PNPLA3)
on chromosome 22. Variations of PNPLA3 are associated with histological
severity in patients with NASH [19].
1.5.2. Epigenetics
Epigenetic changes consist in modifications at the transcriptional level
affecting gene expression and phenotype. A number of epigenetic aberrations
have been associated with NAFLD pathogenesis, causing alterations in lipid
metabolism, IR, dysfunction of endoplasmic reticulum (ER) and
mitochondria, oxidative stress and inflammation [20]. The different
epigenetic pathways potentially involved in NAFLD. Aberrant DNA
methylation is a major epigenetic process in NAFLD development and
progression to NASH [21].
1.5.3 Dietary Factors
Lifestyle changes focusing on weight loss remain the keystone of NAFLD and
NASH treatment [22]. Recent reports indicate that lifestyle modifications
based on decreased energy intake and/or increased physical activity during
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6–12 months cause improvement in biochemical and metabolic parameters
and reduce steatosis and inflammation [23]. Conversely, increased
consumption of sugar-sweetened food and beverages has been associated with
NAFLD development and progression. High intake of fructose, used as food
and drink sweetener, is implicated in NAFLD pathogenesis through several
mechanisms. In addition, a fructose-enriched diet contributes to induce liver
fibrosis in animal models of NASH [24]. Via the portal vein, dietary fructose
reaches the liver in high concentrations, exerting a lipogenic action by
activation of the transcription factors sterol regulatory element binding
protein isoform (SREBP)1 and Carbohydrate-responsive element-binding
protein (ChREBP) and subsequent induction of acetyl-CoA carboxylase (ACC)
1, fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1) [25].
Dietary iron overload has been recently implicated in NASH pathogenesis. A
study by Handa et al. [26] shows that dietary iron excess leads to a severe
NASH phenotype in an obese, diabetogenic mouse model characterized by
oxidative stress, inflammation and ballooning. Moreover, emerging evidence
indicates that hepatic copper (Cu) deficiency is associated with NAFLD
development and progression. In an experimental rat model, a Cu deficient
diet coupled with high sucrose intake provoked NASH, even in the absence of
obesity or severe steatosis.
1.5.4. Obesity and DM
The liver plays a central and crucial role in the regulation of carbohydrate
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metabolism. Its normal functioning is essential for the maintenance of blood
glucose levels and of a continued supply to organs that require a glucose
energy source. This central role for the liver in glucose homeostasis offers a
clue to the pathogenesis of glucose intolerance in liver diseases. Hepatic fat
accumulation is a well-recognized complication of DM with a reported
frequency of 40–70%. Hepatic fat accumulation in type 1 DM may be due to
lipoprotein abnormalities, hyperglycemia-induced activation of ChREBP and
SREBP 1c, with subsequent intrahepatic fat synthesis or combination of
these mechanisms [27]. Fat is stored in the form of triglyceride (TG) and may
be a manifestation of increased fat transport to the liver, enhanced hepatic
fat synthesis, and decreased oxidation or removal of fat from the liver. In
patients with DM and steatohepatitis, Mallory bodies such as those seen in
alcoholic liver disease may be seen. NASH has been associated most
commonly with obese women with diabetes, but the disease is certainly not
limited to patients with this clinical profile [28]. There is certainly a higher
prevalence in type 2 diabetic patients on insulin [7]. NASH should be
considered as a cause for chronically elevated liver enzymes in asymptomatic
diabetic patients particularly if they are obese and have hyperlipidemia
[29]. In type 2 diabetic patients with or without obesity, up to 30% have fat
with inflammation, 25% have associated fibrosis, and 1–8% have cirrhosis
[30], [31], [32]. In an animal model of type 1 DM, there is a high incidence of
perisinusoidal hepatic fibrosis, while in humans perisinusoidal fibrosis often
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parallels with diabetic microangiopathy [33]. There is an increased incidence
of cirrhosis in diabetic patients, and, conversely, at least 80% of patients with
cirrhosis have glucose intolerance [34], [35]. The reported prevalence of
cirrhosis in diabetes varies widely. Diabetes increases the risk of
steatohepatitis, which can progress to cirrhosis.
Large population-based cohort studies from Sweden, Denmark, and Greece
demonstrated a 1.86-fold to 4- fold increase in risk of HCC among patients
with diabetes, which is closely associated with obesity and NAFLD [36]. In a
larger longitudinal study, using the same group of diabetic patients and
nondiabetic controls over 10-15 years, it was found that the incidence of HCC
was increased more than two-fold among diabetic patients with higher
increase among those with longer duration of follow-up. The risk for HCC
was attributable to diabetes, and could not be explained by the presence of
underlying liver disease or other risk factors. Diabetes is clearly established
as an independent risk factor for HCC. NAFLD was found in approximately
38% of cases and possibly contributed to a higher number of cases given the
fact that all of the patients had established diabetes [13].
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1.6. Pathophysiology of NASH
The physiopathology of NAFL and NASH and their progression to cirrhosis
involve several parallel and interrelated mechanisms, such as, IR, abnormal
lipid metabolism, lipotoxicity, inflammation, oxidative stress, mitochondrial
dysfunction, altered production of cytokines and adipokines, gut dysbiosis and
ER stress (Figure 6).
Figure 6: Outline of the pathogenesis of NASH. Signals generated inside the
liver as a consequence of increased lipid accumulation, together with signals
derived from extrahepatic organs cooperate to induce inflammation and
fibrosis. FFA, free fatty acids; PAMPs, pathogen-associated molecular
patterns; ER, endoplasmic reticulum; ROS, reactive oxygen species; HSC,
hepatic stellate cell.
1.6.1 Increased Lipogenesis and lipotoxicity
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Steatohepatitis develops only in a fraction of patients with NAFLD. From a
pathophysiological standpoint NASH develops when the excess lipid
accumulation results in hepatic lipotoxicity. A critical concept is that
increased hepatic fat does not invariably result in hepatocellular damage,
and understanding the mechanisms leading to lipotoxicity is of pivotal
importance for the comprehension of this field. Anyhow, fat accumulation
appears to be a prerequisite for the development of NASH.
Fat accumulates in the liver mainly in the form of TG, although several other
lipid species are present. Human and animal studies have shown that
accumulation of TG in NAFLD is the result of the expansion of the
intrahepatic pool of free fatty acids (FFA). FFA influx is dependent on: a) the
amount of FFA released by the adipose tissue due to IR and excessive
lipolysis; b) dietary fat via chylomicron metabolism; c) de novo lipogenesis
(Figure 7).
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Figure 7: Central role of free fatty acids (FFA) in the pathogenesis of NAFLD.
In the presence of insulin resistance, the majority of FFA reaching the liver
derive from adipose tissue lipolysis. Lipids from the diet or de novo
lipogenesis also contribute to expand the FFA pool. In the hepatocyte, FFA
may be oxidized at mitochondrial and extra-mitochondrial sites, or
incorporated in triglycerides. These in turn may be accumulated in lipid
droplets, leading to steatosis or secreted in the circulation as VLDL, leading
to a proatherogenic lipid pattern.
These lipids, and in particular saturated fatty acid (SFA), can activate a
variety of intracellular responses resulting in lipotoxic stress in the ER and
mitochondria, respectively. As a consequence, apoptosis occurs which
represents a key pathogenic feature of NASH. FFA and free cholesterol (FC)
implicated in NASH have been explored experimentally, mostly in dietary
studies. Such studies demonstrate the unequivocal potential of such lipid
molecules to kill cells of hepatocyte lineage, by directly or indirectly
activating c-Jun-N-terminal kinase (JNK) and the mitochondrial/lysosomal
cell death pathway, [37] and also to stimulate pro-inflammatory signaling via
nuclear factor kappa B (NF-κB) and JNK/activator protein 1 (AP-1).
Lipotoxicity drives the development of progressive hepatic inflammation and
fibrosis in a subgroup of patients with NAFLD, causing NASH and even
progression to cirrhosis and HCC.
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1.6.2. Mitochondrial dysfunction and oxidative stress
Oxidative stress has been recognized as a major factor in the pathogenesis of
NASH. Based on the evidence that a high amount of intracellular reactive
oxygen species (ROS) are generated in mitochondria and ROS overproduction
is elicited in the presence of respiratory chain disruption, mitochondrial
impairment has been suggested as a main event in NASH development [38].
Along these lines, structural and functional defects in mitochondria have
been reported in patients with NASH [38]. Several mechanisms contribute to
mitochondrial impairment and subsequent hepatic cell injury during NASH,
mainly associated with lipotoxicity. An adaptive mechanism dependent on
the histone deacetylases sirtuin (SIRT)1 and SIRT3, aimed to enhance
mitochondrial activity and hepatic β-oxidation [39].
Cytochrome P450 (CYP) 2E1 promotes oxidative stress, inflammation and
protein modifications, by hydrolyzing molecules such as fatty acids and
ethanol into toxic metabolites, including ROS, which cause respiratory chain
disruption and mitochondrial damage, resulting in hepatocyte injury and
progression to NASH [40].
1.6.3. Inflammation
Inflammation represents a crucial aspect in NASH pathogenesis. Overload of
toxic lipids, mainly FFA, causes cellular stress and induces specific signals
that trigger hepatocyte apoptosis, the prevailing mechanism of cell death in
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NASH, correlating with the degree of liver inflammation and fibrosis [41].
Different types of immune cells are recruited and/or activated to the site of
injury, contributing to NAFLD development and progression. Kupffer Cell
(KC) activation is critical in NASH and precedes the recruitment of other
cells [42]. Lanthier et al. [43] have shown that KC depletion increases insulin
sensitivity and ameliorates inflammation and fibrosis. Differentiation of KCs
towards a pro-inflammatory phenotype is principally driven by pathogen-
associated molecular patterns (PAMPs) that, interacting with toll-like
receptor (TLR)s, induce the secretion of various cytokines, such as interleukin
(IL)-1β, IL-12, tumor necrosis factor (TNF)-α, chemokine (C-C motif) ligand
(CCL)2 and CCL5, concurring to further hepatocyte damage and release of
damage-associated molecular patterns (DAMPs). DAMPs, in turn, act on
TLRs amplifying KCs activation and inflammation.
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Figure-8: Pivotal role of activated Kupffer cells in the pathogenesis of
nonalcoholic steatohepatitis and fibrogenesis. Increased levels of
lipoipolysaccaride (LPS) and lipotoxic lipid products lead to activation of
Kupffer cells, which release chemotactic factors (e.g. CCL2), and
proinflammatory cytokines, and generate oxidative stress-related products
including reactive oxygen species (ROS). These factors contribute to
hepatocyte injury, which in turn, through danger signals, cause further
activation of toll-like receptors (TLRs). In addition, inflammation and
damage contribute to hepatic stellate cell activation and fibrosis.
1.6.4. Pattern recognition receptors and the inflammasomes
TLRs are highly conserved receptors that recognize endogenous danger
signals, such as molecules released by damaged cells DAMPs or exogenous
danger signals, as gut-derived PAMPs [44]. Due to the high liver exposure to
danger signals via the portal system, TLR-induced pathways play a central
role in activation of hepatic cells, primarily KC, but also hepatocytes and
HSC. As pattern recognition receptors (PRR), TLRs act as defense
mechanism, but are also implicated in the pathogenesis of NASH.
Importantly, inhibition of TLR2 signaling prevents insulin resistance in HFD
mice [45], whereas TLR2-deficient mice fed high fat-diet (HFD) display
reduced levels of inflammatory cytokines and do not develop NASH [46].
The crucial role of TLR4 in NAFLD pathogenesis has been demonstrated in
TLR4-deficient mice, that display lower levels of inflammatory mediators and
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fail to develop NAFLD or IR [47]. ROS production and subsequent activation
of the unfolded protein response (UPR) are also induced in TLR4-activated
KCs, representing an additional mechanism triggered by TLRs in NAFLD
progression. TLR4-mediated inflammatory response can also be elicited by
DAMPs released by necrotic cells, such as high mobility group box 1
(HMGB1) or phospholipids. These molecules stimulate monocyte and KCs to
secrete inflammatory mediators (Figure 9). HMGB1 can be regulated from
activated immune cells and translocation to cytosol and extracellular
mediates liver injury then also promotes HCC. It is noteworthy that, in the
presence of high glucose, TLR4 activation and downstream signaling can be
triggered by FFA [48], clarifying, at least in part, the mechanism by which
saturated fatty acids, frequently enhanced in plasma of obese patients, have
toxic effects. HMGB1 is a constitutively expressed nuclear protein that
induces transcriptional activation, and is released in response to different
stimuli, such as PAMPs and DAMPs. HMGB1 interacts with a broad
spectrum of receptors [TLR4, TLR2, TLR9, and receptor for advanced
glycation end products (RAGE)] exerting proinflammatory actions in complex
with other factors, as single stranded DNA, LPS and IL-1β [49].
An important role in NASH pathogenesis has been recently ascribed to the
nucleotide oligomerization domain (NOD)-like receptors (NLRs). NLR
activation in response to DAMPs or PAMPs leads to the assembly of
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inflammasome. Activation of inflammasome, mediated by PRRs via NF-κB,
can be induced by a broad spectrum of signals, such as uric acid, ROS, ATP
and mitochondrial DNA, and results in secretion of mature IL-1 and IL-18.
These cytokines, acting on different cell types, elicit inflammatory signals in
liver as well as in the adipose tissue and intestine, triggering steatosis,
insulin resistance, inflammation and cell death [50].
Figure 9: Inflammasomes and the liver. In steatosis, hepatic damage leads to
generation of damage-associated molecular pattern (DAMPs), while
alterations in microbiota lead to increased availability of pathogen-associated
molecular patterns (PAMPs). DAMPs and PAMPs act on receptors localized
on liver cells leading to activation of different inflammasomes and release of
cytokines implicated in NASH. NLRP3: NOD-like receptor family, pyrin
domain containing 3; AIM2: Absent in melanoma 2.
Inflammasomes play an important role in NAFLD development and
progression to NASH, found both in humans and animal models. NLRP3, an
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inflammasome has been reported to activate in several diet induced
steatohepatitis, as well as following methionine choline deficient (MCD),
HF/HC/HS feeding [51]. Moreover, NLRP3 gain of function correlates with
liver fibrosis.
1.6.5. ER stress
ER stress has been implicated in a number of liver diseases, including NASH.
ER dysfunction, ATP depletion or other stimuli induce the UPR, an adaptive
mechanism directed to avoid luminal accumulation of defective proteins and
apoptosis initiation.
Pathways activated by cellular response to ER stress involve JNK, an
activator of inflammation and apoptosis implicated in NAFLD progression to
NASH [52] and SREBP-1c, which induces liver fat accumulation, worsening
ER stress. In vitro studies showed that exposure of hepatic cells to a lipotoxic
concentration of palmitate, a SFA, is associated with ER calcium depletion,
ROS accumulation and apoptosis [53]. The pathways associated with ER
stress shown in figure 10.
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Figure-10: Mammalian unfolded protein response (UPR) pathways. The UPR
is triggered by several events, including protein unfolding/misfolding,
hypoxia, low adenosine triphosphate levels, ER calcium depletion, and
protein/sterol over-expression, causing dissociation of 78 kDa glucose-
regulated protein (GRP78) from the three UPR sensors, (A) inositol-requiring
enzyme 1α (IRE1α), (B) protein kinase RNA-like endoplasmic reticulum
kinase (PERK), and (C) activating transcription factor-6 (ATF6). Activated
IRE1α undergoes dimerization and autophosphorylation to generate
endogenous RNase activity; in turn, this is responsible for splice truncation of
X-box binding protein 1 (XBP1S) mRNA. Additionally, IRE1α may also
activate the extrinsic apoptosis pathway, in which tumor necrosis factor
(TNF) receptor-associated factor 2 (TRAF2)-dependent downstream
activation of c-Jun N-terminal kinase (JNK) and caspase-12 takes place.
Once activated, PERK undergoes homodimerisation and autophosphorylation
to activate eukaryotic translation initiation factor 2 (eIF2α). In turn, this
induces ATF4 expression. Separately, dissociation of GRP78, allows ATF6
processing by the Golgi complex, where proteases S1P and S2P cleave an
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active 50 kDa (p50) ATF6 domain that is free to translocate to the nucleus.
Xbp1s, ATF4 and ATF6, as well as other unlisted factors, are responsible for
three dominant cell responses to UPR. The folding pathway induces
increased expression of molecular chaperones, including GRP78, assisting in
compensatory ER protein folding. Alternatively, the cell may respond by
increasing ER-associated protein degradation (ERAD) pathway, whereby
gene products target and degrade unfolded proteins in the ER. Prolonged
UPR results in the activation of the intrinsic apoptosis pathway; this ATF6
and ATF4-dependent process induces C/EBP-homologous protein (CHOP)
expression. In turn, CHOP inhibits B-cell lymphoma 2 and induces apoptosis.
1.6.6. Nuclear Receptors
Nuclear receptors are ligand-dependent transcription factors that regulate
glucose and lipid metabolism in the liver. Nuclear receptors are divided into
seven subfamilies named as NR0-NR6 [54] and NR1 subfamily is of
particular importance in NAFLD. Peroxisome proliferator-activated receptors
(PPAR) α, β, γ belongs to this subfamily. PPARα regulates β-oxidation and
cholesterol removal during the fasting state or when metabolism increases in
adipose and/or muscle tissues [54]. Hepatic PPARα expression decreases in
NAFLD leading to steatosis, but is enhanced following diet and exercise [55].
In animal models of steatosis and steatohepatitis, the use of PPARα
activators improves the disease.
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1.7. Epidemiology and Prevalence of NASH
With obesity reaching epidemic proportions in the 21st century, NAFLD is on
the rise. NAFLD is the most common etiology of chronic liver disease in the
United States and other developed countries. The annual incidence of
NAFLD has been estimated to be as high as 10% with the development of
NAFLD associated most directly with the metabolic syndrome and preceding
weight gain. Worldwide, the prevalence of NAFLD in the general population
ranges from 9%-37%. In the United States, recent estimates suggest that
NAFLD affects 30% of the general population and as high as 90% of the
morbidly obese [13].
There is no reliable data on the prevalence of NASH in the general
population. A number of studies undertaken in obese individuals undergoing
bariatric surgery. In these series the frequency of NASH varies between14%
to 56%. In hospital series of patients undergoing liver biopsy the frequency
ranges from 1 to 32% [56]. These large variations on the prevalence of NASH
can partly be attributed to different definitions of NASH and which
histopathological findings are required for the diagnosis to be set.
In an autopsy series of 351 apparently non-alcoholic patients the frequency of
NASH was 6.3%. NASH was defined as ballooning of hepatocytes with
clearing of the hepatocellular cytoplasm accompanied by large-droplets
steatosis. NASH was found in 18.5% of obese and in 2.7% of lean patients [7].
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Whereas, in a study considering 61868 patients over the period 2002-2012
found that NASH-related HCC increases from 8.3% to 13.5%, an increase of
near 63%. Furthermore, they found that the number of NASH-HCC patients
undergoing liver transplantation increased nearly 4-fold during this period
[57].
1.8. Treatment for NASH
The standard of care for patients with NAFLD/NASH is life style modification with
weight loss as the mainstay therapy. As overall weight loss is difficult to achieve and
maintain for most patients, pharmacological therapy is often needed. Treatment of
NAFLD includes aggressive cardiovascular risk factor management, including
obesity, dyslipidemia, and diabetes. At present there are no FDA-approved agents
with an indication for the treatment of NASH. Multiple pharmacological
intervention have been attempted for NASH, including thiazolinediones,
Dipeptidyl peptidase-4 inhibitors, glucagon-like peptide-1 receptor agonist,
sodium/glucose cotransporter 2 inhibitors, antioxidants/supplements, lipid
lowering drug, nonselective phosphodiesterase inhibitors, farnesoid X receptor
agonist. There are no good large randomized controlled trials in humans for many of
these compounds, except vitamin E [58]. Vitamin (800 IU/day) is an option for
patients biopsy proven NASH without diabetes [59].
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Treatment strategies for NASH aim to improve insulin sensitivity, modify
underlying metabolic risk factors, or to protect the liver from further insult by
oxidative stress and inflammation.
1.9. CURCUMIN
Curcumin [1,7-bis (4-hydroxyl -3-methoxyphenyl)-1,6-heptadiene-3,5-dione] or
[diferuloylmethane] is a natural polyphenol compound found in the turmeric,
Curcuma longa plant & possesses potent anti-oxidant, anti-inflammatory
properties, modulate the activity of protein kinases, membrane ATPases, and
transcription factors.
To date several clinical trials have been completed; most of these trials have
suggested that curcumin is safe and effective in a number of human diseases. The
most promising effects of curcumin have been observed with cancer, inflammatory
conditions, skin, liver and neurological disorders and pain.
Diferuloylmethane
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Accumulating evidences suggest that curcumin has a diverse range of
molecular targets, including complete inhibition of the activity of protein
kinase C. It can enhance the pancreatic β cell function by inhibiting
phosphodiesterase activity and regulated insulin secretion under glucose
stimulated condition. Curcumin treatment can protect the liver from drug –
induced toxicity.
Curcumin protects liver injury against tetra chloromethane, aflatoxin B1,
alcohol and paracetamol toxicity. A previous study has pointed to the
Aggarwal BB, et al., Int. J Biochem Cell Biol, 2009; 41: 40-59
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protective effect of curcumin on acute liver injury by inhibiting NF-κB and
oxidative stress. It is also reported that curcumin possesses the activity
against ER stress. Several studies suggested that curcumin is safe and well
tolerated treatment [60].
1.10. Le Carbone
Le Carbone (LC) is a charcoal supplement, enriched with dietary fibers.
Today, beyond use in hospitals as an antidote for drugs and poisons,
activated charcoal is a global remedy for general detoxification, digestion
issues, gas bloating, heart health, and anti- aging [61]. Toxicology studies
show activated charcoal to be harmless to human health.
In the early 20th century, the charcoal activation process development
sparked many medical journal to publish research revealing activated
charcoal as an antidote for poisons and way to improve intestinal disorder. In
patients with high cholesterol, activated charcoal able to reduce the total
cholesterol levels. Some study proved that co-treatment of activated charcoal
with anti-cancer drug increase the survival rate after stomach cancer surgery
[62]. Experiment evidence suggest that LC has the adenosine
monophosphate-activated protein kinase (AMPK) enhancing and anti-
inflammatory activities[63]. By enhancing AMPK it may contribute in lipid
metabolism. Several reports suggested that that activated charcoal also has
the anti-atherosclerosis and lipid lowering activity [64].
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Based on the above reports and for the strong biological activities against
different diseases along with inflammation, oxidative stress and lipid
lowering activity, for my study I selected the curcumin and LC as treatment
option for my experiment.
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SCOPE OF STUDY
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The Present studies were aimed:
1. To study the effect of type 1 DM on liver and its treatment option.
2. To investigate the relationship between NASH and HCC under
diabetic condition and their mechanisms.
3. To investigate the treatment and prevention options of NASH-HCC
under diabetic background.
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CHAPTER ONE
Curcumin Ameliorates Streptozotocin Induced Liver
Damage through Modulation of Endoplasmic Reticulum
Stress Mediated Apoptosis in Diabetic Rats
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1. Introduction
DM is one of the most common endocrine metabolic disorders. Studies have
shown that hepatobiliary disorders, such as the inflammation and necrosis or
fibrosis of non-alcoholic fatty liver disease can follow diabetes [65], [66]. It
has been reported that the standardized mortality rate from end-stage liver
disease (i.e. cirrhosis) in diabetic patients is higher than those with
cardiovascular disease [67]. Several mechanisms can cause pancreatic β-cell
dysfunction, including chronic inflammation, oxidative stress, excessive
hyperglycemia and nutrient levels, lipotoxicity, ER stress (ERS), etc. [68],
[69], [70]. A previous study has shown that hepatic fat accumulation and
oxidative stress play a critical role in the development of diabetic liver injury
[71] and a number of reports have shown that antioxidants could attenuate
the complications of diabetes, including fatty changes in patients and in
experimental models [72], [73]. Nowadays, ERS has attracted significant
attention and has been proposed to play a crucial role in the development of
insulin resistance [69]. It is also reported that insulin resistance, a common
underlying reason for the β cell failure that occurs in type 2 diabetes, is
associated with higher levels of ERS in β cells in animal models of disease
[74], [75] and also in humans [76], [77]. It is proposed that oxidative damage
that is caused by ERS may be the fundamental in the etiology of the β cell
failure associated with both type 1 and 2 diabetes [78]. Liver, as the major
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target organ of insulin, plays important roles in the development of insulin
resistance and diabetes mellitus.
The ER is a complex intracellular membranous network that regulates
protein synthesis and folding. Alterations in ER homeostasis due to increased
protein synthesis, accumulation of misfolded proteins or alterations in the
calcium or the redox balance of the ER, lead to a condition called ERS [79]. To
overcome the deleterious effects of ERS induction, cells have evolved various
protective strategies, which are known as the UPR [80]. Furthermore, the
results from these reports have suggested that induction of ERS is closely
associated with the energy metabolism, especially the lipid metabolism with
the involvement of UPR signaling pathways. Rutkowski and Cols examined
the contribution of each arm of the UPR pathway to the regulation of
metabolic genes and development of hepatic dyslipidemia and concluded that
all three arms of UPR signaling pathway double stranded RNA depended
protein kinase – like ER kinase (PERK), inositol requiring enzyme 1α
(IRE1α) and activating transcription factor 6 (ATF6) are activated, leading to
metabolic dysregulation [81].
When ERS is prolonged in the presence of hyperglycemia condition, the ER
triggers the apoptotic pathway by activating the CCAAT/ enhancer – binding
protein homologous protein (CHOP) [82], the IRE1-TRAF2-ASK1-MAP
kinase pathway [83], and/or the ER-localized cysteine protease caspase-12
[84]. Moreover, cell death signaling cascades including p38 mitogen activated
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protein kinase (p38MAPK) and apoptosis signal regulating kinase 1 (ASK1)
are also activated by pro-inflammatory cytokine tumor necrosis factor α
(TNFα), IL-1β, TLR4 and by the activation of the UPR signaling marker
IRE1α in diabetic liver. In diabetes involving both inflammation and ERS
lead to hepatic apoptosis and liver damage.
Curcumin is the active ingredient of the traditional herbal remedy and
dietary spice turmeric (Curcuma longa) and is the subject of clinical trials for
various diseases such as cancer, Alzheimer’s disease, and ulcerative colitis
[85]. The polyphenol curcumin (diferuloylmethane) comprises 2-8% of most
turmeric preparations and is generally regarded as its most active
component, having potent antioxidant, anti-inflammatory and anti-
carcinogenic properties. Curcumin has been shown to modulate the activity of
protein kinases [86], membrane ATPases [87], and transcription factors [88],
[60]. It is also reported that curcumin plays important role to diminish
myocardial ERS signaling proteins and to decrease the key regulator or
inducer of apoptosis in experimental autoimmune myocarditis rats [89]. A
previous study has pointed to the protective effect of curcumin on acute liver
injury by inhibiting NF-κB and oxidative stress [90]. However, to the best of
my knowledge, studies have not been revealed the effect of curcumin on ERS
in diabetic liver. Although many aspects of curcumin-induced cytoprotection
are studied, the molecular mechanism by which curcumin protects liver
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tissues against streptozotocin (STZ) - induced liver injury is not clear. The
present study was designed to shed light on this issue.
2. Materials and methods
2.1. Materials
Unless otherwise stated, all reagents were of analytical grade and purchased
from Sigma (Tokyo, Japan).
2.2. Animals and Experimental design
All animals were treated in accordance with the guidelines for animal
experimentation of our institute [91]. Male Sprague-Dawley rats (weight 250-
300 g) were obtained from Charles River Japan Inc. (Kanagawa, Japan).
Animals were housed in a temperature of 23 ± 2 °C and humidity of 55 ± 15%,
and were submitted to a 12 hour light/dark cycle, and allowed free access to
standard laboratory chow and tap water. Animals were allowed to fast for 4
hours and then induced diabetes by a single intraperitoneal (i.p.) injection of
freshly prepared solution of STZ (Sigma-Aldrich, Inc. Saint Louis, MO, USA)
at a dose of 55 mg/kg, diluted in citrate buffer 20 mM (pH 4.5). Forty eight
hours later, blood glucose was measured by tail-vein sampling using Medi-
safe chips (Terumo Inc., Tokyo, Japan). Diabetes was defined as a morning
blood glucose reading of ≥ 300 mg/dL. Thirty rats were randomly divided into
three groups (n = 10/group): nondiabetic normal control group (Normal),
diabetic rats treated with vehicle, 1% gum Arabic (Control) and diabetic rats
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
55 | P a g e
treated with curcumin 100 mg/kg/day [92] diluted in vehicle, 1% gum Arabic
(Curcumin) (curcumin was purchased from Sigma-Aldrich, Tokyo, Japan).
Curcumin treatment was started at 3 weeks after STZ injection and was
administered via oral gavage for 8 weeks. All rats were sacrificed at 11 weeks
after the induction of diabetes for analysis of liver tissues.
2.3. Biochemical analysis
Each week, rats were weighed and their blood glucose levels were measured.
Urine samples were collected over a 24 h period in individual metabolic cages
for the measurement of protein in urine at 1, 3, 7, and 11 weeks and were
determined by the Bradford method. At the end of experimentation,
heparinized whole blood was collected from anesthetized rats via heart
puncture. EDTA-blood was centrifuged at 3000 g, 15 min at 4°C for the
separation of plasma. The plasma was used for the estimation of triglyceride
(TG) and total cholesterol (TC), alanine aminotransferase (ALT).
2.4. Histopathological analysis
Formalin-fixed liver sections (4 μm) were stained with hematoxylin and eosin
(H&E) or periodic acid–Schiff (PAS). Morphological analysis was done by
computerized image analysis system on ten microscopic fields per section
examined in a 400-fold magnification (CIA-102; Olympus), with the observer
blind to the study group [93], [94].
2.5. Western blotting analysis
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
56 | P a g e
The liver tissue protein lysate was prepared using a method similar to that
described by Soetikno [95]. The total protein concentration in the samples
was measured by the bicinchoninic acid (BCA) method. For the determination
of protein levels, equal amounts of protein extracts (50 µg) were separated by
7.5–15% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis
(Bio-Rad, CA, USA) and transferred electrophoretically to nitrocellulose
membranes. Membranes were blocked with 5% non-fat dry milk or bovine
serum albumin (BSA) in Tris buffered saline Tween (20 mM Tris, pH 7.6, 137
mM NaCl, and 0.1% Tween 20). Primary antibodies against GRP78, IRE1α,
p-IRE1α, TRAF2, ATF6, PERK, p-PERK, CHOP/GADD153, TNFα, IL-1β,
TLR4 and β Tubulin were obtained from Santa Cruz Biotechnology, Santa
Cruz, CA, USA. Primary antibodies against bcl2, cleaved caspase-3, p-
p38MAPK and ASK1 were obtained from Cell Signaling Technology Inc.,
Beverly, MA, USA. And primary antibody cleaved caspase-12 was obtained
from Bio Vision Inc., Milpitas, CA, USA. All the antibodies were used at a
dilution of 1:1000. The membrane was incubated overnight at 40C with the
primary antibody, and the bound antibody was visualized using the
respective horseradish peroxidase (HRP) -conjugated secondary antibodies
(Santa Cruz Biotechnology Inc.) and chemiluminescence developing agents
(Amersham Biosciences, Buckinghamshire, UK). The levels of β Tubulin,
PERK (for p-PERK) and IRE1α (for p- IRE1α) were estimated in every
sample to check for equal loading of samples. Films were scanned, and band
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
57 | P a g e
densities were quantified by densitometric analysis using the Scion Image
program (Epson GT-X700, Tokyo, Japan).
2.6. Immunohistochemistry
Formalin-fixed, paraffin-embedded liver tissue sections were used for
immunohistochemical staining. After deparaffinization and hydration, the
slides were washed in Tris-buffered saline (TBS; 10 mM/L Tris HCl, 0.85%
NaCl, pH 7.2). Endogenous peroxidase activity was quenched by incubating
the slides in methanol and 0.3% H2O2 in methanol. After overnight
incubation with the primary antibody, that is, mouse monoclonal anti- ED1
antibody (diluted 1:50) (sc-59103; Santa Cruz Biotechnology Inc. CA, USA) at
4°C, the slides were washed in TBS and (HRP) -conjugated goat anti-mouse
secondary antibody was then added and the slides were further incubated at
room temperature for 1 h. The slides were washed in TBS and incubated with
diaminobenzidine tetra hydrochloride as the substrate, and counterstained
with hematoxylin. A negative control without primary antibody was included
in the experiment to verify the antibody specificity. ED1 positive hepatocytes
were counted in 100 hepatocytes/ group under 200-fold magnification and
expressed as cells/hepatocyte cross section [96].
2.7. Statistical analysis
All values are expressed as means ± standard error of mean (SEM) and were
analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s
methods for post hoc analysis or two-tailed t-test when appropriate. A value
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
58 | P a g e
of p<0.05 was considered statistically significant. For statistical analysis,
Graph Pad Prism 5 software (San Diego, CA, USA) was used.
3. Results
3.1. Biochemical parameters in experimental animals
For the confirmation of diabetic model and the effect of treatment periodically
the blood glucose level was checked, which is shown in Figure 1, before
treatment the blood glucose level was significantly higher than that of
normal group but during the treatment period from 6 weeks curcumin
treatment significantly decreased this blood glucose level. Moreover, it is also
reported that curcumin has the capability to improve the pancreatic islets
[97] and has been demonstrated in prevention of isolated β cells death and
dysfunction induced by STZ [97], [98].
During treatment
period
0
200
400
600
800
1000
Normal
3
(Before Treatment)
4 5 6
Curcumin
7 8 9
Control***
***
*** *** *** *** ***
######
### ###
###
******
** ***** **
**
10 11
***
###
**
Duration (Weeks)
Blo
od G
luco
se l
evel
(m
g/d
L)
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
59 | P a g e
Fig 1: Time-course changes in blood glucose. Blood glucose increased
progressively in the untreated diabetic rats following induction of diabetes.
Curcumin treatment significantly reduced blood glucose in the beginning of
treatment and these were maintained throughout the study period until
sacrifice. Values are means ± SEM. **p < 0.01, ***p < 0.001 vs Normal, ###p<
0.001 vs Control.
As shown in Table 1, body weight was significantly decreased in diabetic rats,
but curcumin treatment could not prevent this declined body weight. The
ratio of liver weight and body weight (g/kg) were significantly increased in
untreated diabetic rats compared to normal rats and curcumin treatment
reduced this ratio. Compared with the normal group, diabetic rats developed
elevated mean plasma glucose. Plasma TG, TC and ALT were also elevated in
the diabetic rats in comparison to the normal rats (p<0.01, p<0.001). All of
these abnormalities significantly attenuated by curcumin treatment (p< 0.05,
p< 0.001). To evaluate the effect of curcumin on preventing hyperfiltration
induced by STZ, we measured 24-h urine volume and urinary protein
excretion. Although the control group showed a marked elevation of 24-h
urine and urinary protein excretion, curcumin treatment could not reduce
this elevated urinary excretion but significantly reduced the urinary protein
excretion (p< 0.05).
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
60 | P a g e
Table 1: Changes in Biochemical Parameters after 8 Weeks of Treatment
with Curcumin in Diabetic Rats Induced by Streptozotocin
Normal
(n=10)
Control
(n=10)
Curcumin
(n=10)
Body weight (BW) (g)
539.5 ± 38.48
316.33 ± 15.7***
368.75 ± 24.62***
Liver weight (LW) /BW
(g/kg)
28.57 ± 1.47
43.23 ± 1.35***
37.92 ± 4.64# *
Plasma glucose (mg/dL)
116.25 ± 22.1 761.8 ± 50.8*** 287.66 ± 78.4### **
TG (mg/dL) 94.75 ± 5.72 431.25 ±
118.92***
109 ± 11.94###
TC (mg/dL) 58.50 ± 3.8 105.5 ± 10.9*** 81.25 ± 13.72#
ALT (IU/L) 39.75 ± 2.36 124 ± 28.82** 97 ± 30.26*
Urine Volume (mL/Kg/24h) 19 ± 1 618 ± 137*** 592 ± 109***
Protein in urine/24h (g) 13 ± 2 38 ± 15.63* 15 ± 10#
Normal, age matched normal rats; Control, untreated diabetic rats;
Curcumin, diabetic rats treated with curcumin 100 mg/kg/day. TG:
triglyceride; TC: total cholesterol, ALT: alanine aminotransferase. Values are
expressed as means ± SEM.
*p <0.05, **p <0.01, ***p <0.001 vs normal, #p< 0.05, ###p< 0.001 vs control.
3.2. Effect of curcumin on ERS marker protein
Activation of GRP78 protein expression was used to demonstrate the ERS
induction, whose expression is reported to be increased in the diabetic rats.
ERS leads to the activation of caspase-12 and other markers involved in the
hepatic changes associated with DM. My study with diabetic rats also
revealed the involvement of ERS as evidenced by a significant increase in the
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
61 | P a g e
hepatic GRP78 expression but cleaved caspase-12 expression was not so
significant. Prevention of ERS is one of the strategies involved in the
reduction of liver complications of diabetes. In the present study curcumin
treated animals had shown significant attenuation of GRP78 and cleaved
caspase-12 was slightly attenuated when compared with the control group
(Figure 2A and 2B).
Fig 2: Expression of hepatic protein involved in ERS. Representative western
blots (lower panel) show specific bands for GRP78 (A) and Caspase12 (B) and
the representative histograms (upper panel) show the band densities with
relative β Tubulin. The blots are representatives of five independent
experiments. Each bar represents mean ± SE. Normal, age matched normal
rats; Control, untreated diabetic rats; Curcumin, diabetic rats treated with
curcumin 100 mg/kg/day. **p<0.01 vs Normal, #p<0.05 vs Control based on
one way ANOVA followed by Tukey’s test.
Normal Control Curcumin0.0
0.5
1.0
1.5
2.0
2.5
**
#
GR
P78/
Tubulin
GRP78
β Tubulin
A
Normal Control Curcumin0
2
4
6
8
Cle
aved c
asp
ase
-12/
Tubulin
β Tubulin
Cleaved caspase-12
B
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
62 | P a g e
3.3. Effect of curcumin on expression of UPR signaling proteins in liver
The signals from activated UPR molecules to the relevant effector proteins
are conveyed by three different proteins, PERK, IRE1α, and ATF6α. As
expected, here I have found that the phosphorylation of PERK protein was
significantly increased in the diabetic rats compared with the normal SD rats
(Figure 3A). Diabetic rats have displayed a significant up-regulation in the
hepatocyte expression levels of p-IRE1α protein compared with the normal
SD rats (Figure 3B). And curcumin treatment significantly attenuated the
increased liver p-PERK and p-IRE1α levels. But interestingly, I did not find
any significant difference in the level of ATF6α protein expression in the
diabetic rats compared with the normal SD rats (Figure 3C).
A
Normal Control Curcumin0.0
0.1
0.2
0.3***
#*
p-I
RE
1
/t-
IRE
1
B
Normal Control Curcumin0.00
0.05
0.10
0.15
*#
p-P
ER
K/P
ER
K
p-IRE1α
t-IRE1α PERK
p-PERK
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
63 | P a g e
Fig 3: Expression of hepatic proteins involved in UPR signaling pathway.
Representative western blots (lower panel) show specific bands for p-PERK
(A), p-IRE1α (B) and ATF6α (C) and the representative histograms (upper
panel) show the band densities with relative β Tubulin, PERK (for p-PERK)
and IRE1α (p-IRE1a) . The blots are representatives of five independent
experiments. Each bar represents mean ± SE. Normal, age matched normal
rats; Control, untreated diabetic rats; Curcumin, diabetic rats treated with
curcumin 100 mg/kg/day. *p<0.05, ***p<0.001 vs Normal, #p<0.05 vs Control
based on one way ANOVA followed by Tukey’s test.
3.4. Effect of curcumin on hepatocyte protein expression levels of
CHOP/GADD153 in the diabetic rats
It has been proposed that enhancement of the hepatocyte protein expression
of CHOP/GADD153 gene could eventually induce apoptosis. Immunoblot
analysis revealed that the diabetic rats have demonstrated significant
C
Normal Control Curcumin0
1
2
3
4
AT
F6
/ T
ubulin
β Tubulin
ATF6α
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
64 | P a g e
elevation of CHOP/GADD153 protein expression compared with the normal
rats (Figure 4A) and curcumin treatment significantly decreased the
increased liver CHOP expression.
3.5. Effect of curcumin on liver expression of TRAF2, ASK1 and p-p38MAPK
The TRAF2 protein expression was significantly higher in the diabetic rats
than the normal rats, which was significantly attenuated by curcumin
treatment (Figure 4B). There was a significant increase in the expression of
p-p38MAPK and ASK1 in the diabetic control rats. This result suggests
activation of IRE1α recruit TRAF2, then ASK1 directly binds to TRAF2 and
activated. Curcumin treatment modified these changes in the liver of diabetic
rats. Protein expression of these markers were attenuated significantly in the
curcumin treated rats (Figure 4B, 4C and 4D).
Normal Control Curcumin0
1
2
3
###
***
TR
AF
2/
Tubulin
TRAF2
β Tubulin
B
Normal Control Curcumin0.0
0.5
1.0
1.5
2.0
2.5 ***
##
CH
OP
/ T
ubulin
CHOP
β Tubulin
A
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
65 | P a g e
Fig 4: Hepatic expressions of CHOP, TRAF2, ASK1 and p-p38MAPK.
Representative western blots (lower panel) show specific bands for
CHOP/GADD153 (A), TRAF2 (B), ASK1 (C) and p-p38MAPK (D) and the
representative histograms (upper panel) show the band densities with
relative β Tubulin. The blots are representatives of five independent
experiments. Each bar represents mean ± SE. Normal, age matched normal
rats; Control, untreated diabetic rats; Curcumin, diabetic rats treated with
curcumin 100 mg/kg/day. **p<0.01, ***p<0.001 vs Normal, ##p<0.01, ###p<0.001
vs Control.
3.6. Effect of curcumin on hepatocyte protein expression levels of cleaved
caspase-3 and bcl2 in diabetic rats
Immunoblot analysis revealed that the diabetic rats have displayed an
increase in the hepatocyte protein expression of cleaved caspase-3 compared
with the normal rats (Figure 5A) and decreased protein expression level of
Normal Control Curcumin0.0
0.1
0.2
0.3
***
###
p-p
38M
AP
K/
Tubulin
p-p38MAPK
β Tubulin
ASK1
β Tubulin
D C
Normal Control Curcumin0.00
0.05
0.10
0.15
**
##
AS
K1/
Tubulin
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
66 | P a g e
bcl2. This increase in liver of cleaved caspase-3 protein expression was
markedly suppressed and significantly increased the bcl2 protein expression
by curcumin treatment in the liver of diabetic rats. This clearly indicates that
curcumin attenuated the hepatocytes apoptosis.
Fig 5: Hepatic expressions of cleaved caspae-3 and bcl2. Representative
western blots (lower panel) show specific bands for cleaved caspase-3 (A) and
bcl2 (B) and the representative histograms (upper panel) show the band
densities with relative β Tubulin. The blots are representatives of five
independent experiments. Each bar represents mean ± SE. Normal, age
matched normal rats; Control, untreated diabetic rats; Curcumin, diabetic
rats treated with curcumin 100 mg/kg/day. *p< 0.05, **p< 0.01 vs Normal, #p<
0.05, ##p < 0.01 vs Control.
Normal Control Curcumin0.0
0.1
0.2
0.3
0.4
**
##
Cle
aved c
aspase-3
/ T
ubulin
Cleaved caspase-3
β Tubulin
B A
Normal Control Curcumin0.0
0.5
1.0
1.5
*
#
bcl2
/ T
ubulin
bcl2
β Tubulin
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
67 | P a g e
3.7. Effect of curcumin on inflammatory marker proteins in the diabetic liver
Inflammation is involved in the mechanism of metabolism related diseases
such as diabetes mellitus and obesity [99], [100]. Inflammatory cytokines
produced by liver infiltrated autoreactive immune cells are the major factors
causing cell death in type 1 diabetes [101]. It has been proposed that ERS
pathways play crucial roles in the inflammatory response. Therefore, I
performed western blot analysis to measure the hepatocyte protein
expression levels of TNFα, IL-1β and TLR4 in diabetic rats. The diabetic rats
displayed significantly upregulated protein expression levels of TNFα, IL-1β
and TLR4 compared with those in normal rats (p< 0.05 and p< 0.001), and
these increases were significantly attenuated by curcumin treatment. (Figure
6A, 6B and 6C).
Normal Control Curcumin0.0
0.5
1.0
1.5
*
#
TN
F
/ T
ubulin
TNFα
β Tubulin
B A
Normal Control Curcumin0.0
0.5
1.0
1.5
2.0
2.5
***
###
IL-1
/ T
ubulin
β Tubulin
IL-1β
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
68 | P a g e
Fig 6: Hepatic expressions of TNFα, IL-1β and TLR4. Representative western
blots (lower panel) show specific bands for TNFα (A), IL-1β (B) and TLR4 (C)
and the representative histograms (upper panel) show the band densities
with relative β Tubulin. The blots are representatives of five independent
experiments. Each bar represents mean ± SE. Normal, age matched normal
rats; Control, untreated diabetic rats; Curcumin, diabetic rats treated with
curcumin 100 mg/kg/day. *p<0.05, ***p<0.001 vs Normal, #p<0.05, ##p<0.01
and ###p<0.001 vs Control.
3.8. Histopathological findings
Normal histology was seen in the normal control rats (Figure 7). The normal
liver contains a large amount of glycogen. This therefore leads to the staining
of hepatocytes intensely pink with a PAS stain. In the PAS staining,
examined liver sections of normal control rats showed the normal pattern
distribution of glycogen granules, a liver section of diabetic rats showed
marked depletion in the glycogen granules, meanwhile this glycogen level
β Tubulin
TLR4
Normal Control Curcumin0
5
10
15
20 *
##
TL
R4/
Tubulin
C
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
69 | P a g e
became increased in curcumin treated animals (Figure 7A). Fatty liver was
shown by H&E staining as an unstained area in liver parenchymal cells
(Figure 7B). In the untreated diabetic rats, microvacular vacuolization, focal
necrosis and inflammation in the portal area were significantly apparent
compared with the normal rats and curcumin treated diabetic rats (Figure
7B) improved these findings.
3.9. Effect of curcumin on macrophage (kupffer cells) infiltration and
fibronectin accumulation
Macrophages are a heterogeneous population of myeloid –derived
mononuclear cells that are a critical component of innate immune response
[102], [103]. I investigated that livers from normal rats did not show any
significant macrophage infiltration (Figure 7C). On the other hand, diabetic
rats demonstrated prominent macrophage (ED1 positive cells) recruitment in
the liver (Figure 7C and 7C1), whereas diabetic rats treated with curcumin
showed marked reduction of macrophage activation (p < 0.01) (Figure 7C and
7C1). Fibronectin accumulation in diabetic rat liver was also significantly
higher compared to that of normal control group (Figure 7D). When curcumin
was given to STZ diabetic groups, the sections of liver showed reduced
fibronectin accumulation (Figure 7D).
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
70 | P a g e
Normal Control Curcumin
A
PAS
B
H&E
C
ED1
D
Fibronectin
Normal Control Curcumin0
10
20
30
40
**
##
Num
ber
of
ED
1 p
osi
tive c
ells/
100 f
ield
s
C1
20µm 20µm 20µm
20µm 20µm 20µm
100µm 100µm 100µm
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
71 | P a g e
Fig 7: Effect of curcumin on histopathological changes. Histological staining
with Periodic acid-Schiff (PAS) in liver (A) shows that glycogen contents of
rat liver decreased in diabetic animals when compared to normal control
animals but these levels increased to near normal after treatment with
curcumin. In hematoxylin and eosin (H&E), (B) light microscopic
photographs of livers of experimental animal showed the liver of normal
control group, lipid accumulation indicated by the unstained area in liver
tissues, microvascular fattening and focal necrosis, portal inflammation in
the untreated diabetic group, in curcumin treated diabetic group, the severity
of these changes was less than those in the untreated diabetic group. (C and
C1) Immunohistochemical staining for macrophage (ED1 positive cells) and
its quantification graph in each group. (D) Imunohistochemical staining for
fibronectin in liver section. Each bar represents mean ± SE. Normal, age
matched normal rats; Control, untreated diabetic rats; Curcumin, diabetic
rats treated with curcumin 100 mg/kg/day. **p<0.01 vs Normal, ##p<0.01 vs
Control.
4. Discussion
The ER is a membranous network that provides a specialized environment
for processing and folding newly synthesized proteins. The ER regulates
protein folding, calcium storage and the biosynthesis of macromolecules such
as steroids, lipids and carbohydrates. Disruption of ER homeostasis, often
termed ERS, has been observed in liver and adipose tissue of humans with
nonalcoholic fatty liver disease and/or obesity [65], [104], [105], [106], [107].
As the metabolic demands increase, which can perturb the protein folding in
the ER, and then does the work load of this protein factory collectively called
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
72 | P a g e
ERS [108]. Song and Scheuner indicated that nutrient fluctuations and
insulin resistance increase proinsulin synthesis in β cells beyond the capacity
for folding of nascent polypeptide within the ER lumen, thereby disrupting
ER homeostasis and triggering the UPR and chronic ERS promoted apoptosis
[78]. Since hepatocytes have a well- developed ER structure, ERS is involved
in liver- related disease [109]. As curcumin also has the ERS inhibitory effect
[89], in light of the important role of the liver in glucose homeostasis and the
pathogenesis of diabetes, I sought to examine the potential effect of curcumin
on the ERS response signal in hepatocytes of diabetic animals. So, I question
whether the beneficial function of curcumin on suppressing the ERS in the
liver.
The ER lumen provides a specialized environment for protein folding and
maturation and a unique complement of molecular chaperones and folding
enzymes [110]. Recent studies have suggested that ERS and UPR signaling
are tightly associated with hepatic lipid metabolism [111], [112]. In
mammalian cells, UPR activation involves three ER-localized proteins IRE1α,
PERK, and ATF6α [113]. It is currently thought that in unstressed cells all
three proteins are maintained in an inactive state via their association with
the ER protein chaperone GRP78. GRP78 acts as a master regulator of the
activation of UPR signaling pathways. Upon ERS, GRP78 is released and
sequestered on unfolded proteins, allowing activation of PERK, IRE1α and
ATF6α [114]. In the present study, I investigated ERS signaling in diabetic
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
73 | P a g e
liver and observed the elevated protein expression of GRP78, p-PERK and
IRE1α in the liver of diabetic rats, which were attenuated by curcumin
treatment (Figure 2A, 3A and 3B). But the expression of ATF6α was not
significant in the liver of diabetic rats compared with normal rats (Figure
3C).
Considering all these findings, the increase of GRP78 expression and
activation of the UPR is often used as indicators of ERS due to the complexity
of directly measuring the ER integrity or protein aggregates and I
hypothesize that the increase in the protein expression of GRP78 and UPR
signaling proteins might actually indicate the induction of ERS in the
diabetic rats. Curcumin treatment attenuated the ERS in the liver of diabetic
rats by reducing these marker proteins.
Prolonged or insufficient ERS may turn physiological mechanisms into
pathological consequences. When ERS inducing stresses is too severe or
prolonged to allow for recovery of ER function, the apoptosis pathway is
activated to remove damaged cells [115], [116], [117],[118], [119]. It is
proposed that at least three pathways are involved in the ER stress-
mediated apoptosis [115],[116], [117], [118],[119],[120]. The first is
transcriptional activation of the gene for CHOP. The second is activation of
the IRE1-TRAF2-ASK1-MAP kinase pathway. The third is activation of ER-
associated caspase-12.
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
74 | P a g e
CHOP is expressed at low levels under physiological conditions, but it is
strongly induced at the transcriptional level in response to ER stress [78],
[115], [116], [121], [122]. The transcription of the chop gene is activated by all
three ER stress sensors (IRE1α, ATF6 and PERK) signaling pathways. The
activation of PERK plays a dominant role in the induction of transcription
factor CHOP/GADD153 [123] over that of ATF6α and IRE1α signaling
pathways, although the presence of all three signaling pathways is required
to achieve the maximal induction of CHOP [116], [122]. Moreover, the failure
of the UPR to ameliorate ERS, can lead to cell death via several mechanisms
and CHOP is among the best characterized of the UPR-regulated
proapoptotic proteins [116].
Correlates with these results, the phosphorylation of hepatic PERK and
CHOP gene expression were shown to be significantly increased in the
diabetic rats when compared with normal rats. Treatment with curcumin
significantly attenuated the changes in the hepatic expression of p-PERK and
CHOP/GADD153 and provided evidence for the prevention of ERS, one of the
possible mechanisms of hepatic apoptosis in diabetic rats (Figure 3A and 4A).
Whereas, IRE1α also appears to mediate rapid degradation of specific
mRNAs, presumably in an effort to reduce production of proteins that require
folding in the ER lumen [124], [125]. Under ERS conditions, activated IRE1α,
one of the ERS sensors, recruits TRAF2; then ASK1 directly binds to TRAF2
and is activated. It is thought that the mitochondria pathway is involved in
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
75 | P a g e
ASK1 mediated apoptosis. In this study, the liver of diabetic rats has
displayed a significant increase of TRAF2 and ASK1 protein expression
(Figure 4B and 4C). Treatment with curcumin significantly down regulated
the protein expression of TRAF2 and ASK1 in the liver of diabetic rats.
IRE1α activation has also been linked to the activation of p38MAPK and JNK
[126], [127], [128]. The p38MAPK and JNK are subfamily of the MAPK
superfamily and are classified as a stress response. In the present study, the
hepatic levels of phosphorylated forms of p38MAPK are significantly lesser in
the curcumin treated rats when compared with the control group (Figure 4D)
suggesting that curcumin treatment avoided the activation of MAPK
signaling cascade with the prevention of stress in the liver of diabetic rats
through which it has prevented the liver injury involved in STZ- induced
diabetic rats. Interestingly, in this experiment I did not observe any
significant difference in the phosphorylation of JNK among the three groups
(data is not shown here).
Pro-caspase-12 is localized to the cytosolic side of ER membrane and is
activated by ERS but the mechanism has not been confirmed. It is reported
that caspase-12 knockout cells are partially resistant to ERS induced
apoptosis [117]. Interestingly, in this study, I did not observe any significant
difference in the expression of cleaved caspase-12 among the normal, control
and treatment group (Figure 2B). For further confirmation of apoptosis in the
liver of diabetic rats and the effect of curcumin treatment on diabetic liver, I
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
76 | P a g e
checked the apoptotic marker protein cleaved caspase-3 and anti-apoptotic
protein bcl2. In this study, I found that the expression levels of activated
caspase-3 were significantly increased and the anti-apoptotic proteins bcl2
were reduced in the diabetic liver. Curcumin treatment prevented all of these
alterations (Figure 5A and 5B).
Furthermore, it is reported that the ERS pathway plays crucial roles in the
inflammatory response [127], [129], [130]. IRE1α.TRAF2 complex also
involved in the transcriptional induction of inflammation related genes. Nanji
et al. (2003) have demonstrated the protective effect of curcumin in rat liver
injury induced by alcohol, where curcumin administration prevented ALT
increases, blocked the activation of NF-κB, the expression of
proinflammatory cytokines (TNFα) and iNOS [131] . In this experiment, I
found that activation of TLR4 as well as proinflammatory cytokine
expression; TNFα and IL-1β were increased in diabetic rats compared with
the levels in normal rats. Curcumin prevented all of these alterations (Figure
6A, 6B and 6C). I showed that the significant elevation level of ALT in
diabetic rats was reduced slightly by curcumin treatment.
Stimuli that injure the liver can activate multiple intracellular stress
responses, such as the inflammatory response. Macrophage or kupffer cells
have been implicated in the pathogenesis of various liver diseases [132]. In
the present study, using the accumulation of ED1 as a marker of macrophage
activation, I have demonstrated increased activation of macrophage/ kupffer
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
77 | P a g e
cells in the hepatic tissue of diabetic animals (Figure 7C and 7C1) and
increased fibronectin accumulation in diabetic liver (Figure 7D). In this
study, I demonstrated that macrophage recruitment in diabetic liver is
ameliorated by the administration of curcumin. Curcumin is a representative
polyphenolic compound found in the dietary spice turmeric. From the
morphological study, I found that, long-term curcumin treatment improved
many pathological changes, degenerated hepatocytes with polymorphic
nuclei, dilated sinusoids and mononuclear cell infiltrate extending through
hepatic tissue in diabetic rats. In the present study, curcumin significantly
reduced the blood glucose level. It is also reported that it has the capability to
improve the activity of pancreas thus curcumin able to increase the insulin
secretion [97]. Pancreatic islet cell death is the cause of deficient insulin
production in diabetes. Approaches towards prevention of cell death are of
prophylactic importance in control and management of hyperglycemia.
Meghana et al investigated and reported that islet viability and insulin
secretion in curcumin pretreated islets are significantly higher than islets
exposed to STZ alone [98]. Recently it is reported that curcumin enhances
pancreatic β cell function by inhibiting phosphodiesterase activity and
regulated insulin secretion under glucose stimulated condition [133]. From
this experiment, I can conclude that curcumin may indirectly reduce the ERS
by reducing hyperglycemia or it may directly reduce the ERS in diabetic liver
and protect the liver damage (Figure 8).
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Figure 8: Schematic summary of the experiment. Diabetes mellitus (Type 1
diabetes) causes hyperglycemia and increases advanced glycation end products
(AGE). Hyperglycemia enhances ER malfunction, which causes dissociation of
GRP78 and activation of UPR proteins (IRE1a, PERK) and caspase pathway. Then
leads to hepatic apoptosis, which may cause cell death. Ultimately caused the
liver damage. Curcumin treatment significantly reduced the hyperglycemia
in diabetic rats and protected ER function and reduced liver damage.
Hyperglycemia and
Advanced
Glycation End
Products
ER malfunction
p-IRE-1α p-PERK
Induction of
ER stress
(GRP78)
Recruitment of
TRAF2 CHOP/GADD153
gene
Activation of
Caspase
pathway
Cleaved caspase -3
and 12 ASK1 Metabolic
dysfunctio
n
Diabetic
Hepatopathy
Diabetes
Mellitus (Type
1 Diabetes)
GRP78
IRE-1α ATF-6
PERK
p-p38MAPK
Hepatic
apoptosis
Decreased
bcl2
Curcumin
Normal state
ER
Activation of
inflammatory
marker
TNFα, IL-1β, TLR4
Cell death Liver
Damage
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79 | P a g e
5. Conclusion
The ERS related inflammation and apoptosis are involved in the
pathogenesis of various diseases, including hepatopathy. Therefore, the ERS
pathway can be a new target for the treatment of those diseases. The result
presented here show that the administration of curcumin inhibits ERS, ERS
related apoptosis and inflammation in the liver of STZ-induced diabetic rats.
All the above results suggest that the beneficial effect of curcumin occurs, at
least in part through modulation of the UPR signaling pathway. Given these
promising preclinical findings, I believe that the curcumin, might be
considered as a potential adjuvant entity for preventing diabetic liver
damage.
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CHAPTER TWO
Curcumin ameliorates liver damage and progression of
NASH in NASH-HCC mouse model possibly by
modulating HMGB1-NF-κB translocation
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1. Introduction:
HCC is the fifth most common form of cancer and the third common cause of
cancer-related deaths [134]. Recently, NAFLD is the well-known reason for
persistent liver disease in well-off countries. There is a range of liver
diseases, extending from hepatic steatosis (simple intrahepatic accumulation
of lipid droplets) and steatosis with inflammation and fibrosis (NASH) to
cirrhosis and HCC [135]. Since NASH is considered as a hepatic
manifestation of the metabolic disorder and exceedingly connected with
diabetes, it is speculated that the relationship amongst diabetes and HCC is
identified with the progression of NASH [136]. Fujii et al., established a novel
NASH-HCC model in mice under diabetic background [11]. Inflammation
promotes the progression of NASH [137] and has also been a critical factor in
the progression of HCC [138]. Understanding the molecular mechanisms
linking hepatocytes necrosis to inflammation and progressive liver damage is,
therefore, critical to the development of novel therapeutic strategies for
NASH-HCC.
HMGB1, a DNA-binding nuclear protein is a leader-less pro-inflammatory
cytokine and is an important member of inflammatory cascade [139]. HMGB1
can be either actively regulated from activated immune cells or passively
from injured cells [140] and signals tissue damage [141]. More importantly,
the role of HMGB1 as a DAMP has led to its implication in a variety of
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
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disease states mediated by inflammatory stimuli, including, sepsis, arthritis,
liver ischemia-reperfusion injury, liver transplantation, cancer, HCC
[142,143] and atopic dermatitis [144]. Extracellular HMGB1 can bind to
different cell surface receptors, including TLR2, TLR4 and the RAGE act on
target cells and promotes inflammation [145]. Activation of these receptors
results in the activation of NF-κB, extracellular signal-regulated kinase
(ERK) signaling, thereby triggering a variety of inflammatory and pro-
inflammatory cytokines. Furthermore, the elevated levels of these cytokines
can induce NF-κB activation and thereby worsen the disease condition. The
role of pro-inflammatory transcription factor like NF-κB [146], and HMGB1
[147] in the development and progression of HCC is also well established.
Curcumin (diferuloylmethane) is a polyphenol comprising 2-8% of most
turmeric preparations. It acts as an active ingredient and exerts potent lipid
regulatory, anti-inflammatory, anti-oxidative and anti-carcinogenic
properties that are important for protecting against NASH. One of the main
properties of curcumin is its hepatoprotective activity. It has been reported
that curcumin protects several drugs induced liver injury [148,149]. In our
previous study, we reported that curcumin protects the liver damage of
streptozotocin (STZ) induced diabetic rats by inhibiting ERS and ERS-related
apoptosis and inflammation [150]. It has also been reported that curcumin
inhibits NF-κB, an oncogenic signaling molecule in liver cancer [151].
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Although some studies reported that curcumin reduces Propionibacterium
acnes [152], carbon tetra chloride [153] and concanavalin A [154] induced
acute liver injury through inhibiting the HMGB1 release, to the best of our
knowledge, no other experimental studies revealed the effect of either
curcumin or other treatments on this novel NASH-HCC model. Thus, in the
present study, I explored the possible protective mechanisms of curcumin on
the liver damage and progression of NASH in NASH-HCC mouse model.
2. Materials and methods
2.1. Animals and experimental designs
All animals were treated in accordance with the guidelines for animal
experimentation of our institute (Approve No. H270310) [150]. C57BL/6J
mice were bred in my laboratory. Animals were housed in a temperature of
23 ± 2°C and humidity of 55 ± 15% and were submitted to a light/dark cycle,
allowed free access to standard laboratory chow and tap water. NASH-HCC
was induced in male mice by a single subcutaneous injection of 200 µg STZ
(Sigma, MO, USA) at 2 days after birth. They were being started feeding with
high-fat diet (HFD) 32 (CELA Japan) ad libitum at the age of 4 weeks, and
continued up to 14 weeks age of mice. The normal diet provided 404 Kcal/100
g (water 10%, protein 25.0%, fat 4.5%, ash 6.7%, carbohydrate 49.3% and
fiber 4.5%), whereas, the HFD 32 provided 507.6 Kcal/100 g (water 6.2%,
protein 25.5%, fat 32.0%, ash 4.0%, carbohydrate 29.4%, and fiber 2.9%).
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
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Mice were randomly selected into three groups (n=6/group): the normal group
(Normal) were normal mice subjected to the normal diet; the NASH group
(NASH) were STZ injected mice subjected to the HFD32 treated with 1% gum
Arabic (the vehicle of curcumin); the NASH and Curcumin group (NASH +
Curcumin) were STZ injected mice subjected to the HFD32 (NASH mice)
treated with curcumin (100 mg/kg/day, Sigma-Aldrich, Tokyo, Japan)
suspended in 1% gum Arabic. Curcumin treatment was started at the age of
10 weeks and administered via oral gavage continued up to 14 weeks of age
along with HFD. All mice were sacrificed at the age of 14 weeks, and serum
was separated from blood. Liver tissue was isolated for histological,
biochemical, and molecular biological analysis [11].
2.2. Biochemical analysis
Fasting blood glucose was measured using the G-checker (Sanko Junyaku,
Tokyo, Japan). Serum alanine aminotransferase (ALT), aspartate
aminotransferase (AST), alkaline phosphatase (ALP), triglyceride (TG) and
total cholesterol (TC) were measured by FUJI DRI-CHEM 7000 (Fujifilm,
Tokyo, Japan) as previously described [150].
2.3. Histological examination
Liver sections (4 µm) of different groups of mice were immediately fixed in
10% formaldehyde solution, embedded in paraffin, cut into several
transversal sections and mounted on glass slides. Then the liver tissue
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
85 | P a g e
sections were deparaffinized and stained with hematoxylin and eosin (H&E).
Morphological analysis was done by computerized image analysis system on
ten microscopic fields per section examined in a 40-fold magnification
(AmScope, USA), with the observer blind to the study group [150]. Here,
macrovesicular steatosis, microvesicular steatosis and hepatocellular
hypertrophy were separately scored and the severity was graded, based on
the percentage of the total area affected, into the following categories: 0
(<5%), 1 (5-33%), 2 (34-66%) and 3 (>66%). Inflammation was evaluated by
counting the number of inflammatory foci per field, a focus has defined a
cluster of ≥5 inflammatory cells. Different fields were counted and scored as
per field: 0 (<0.5), 1 (0.5-1.0), 2 (1.0-2.0) and 3 (>2). By adding the score of the
above four parameters were used to calculate the NAFLD activity score,
resulting in a total clinical score ranging from 0 (healthy) to 12 (maximal
severity of NASH) [14]. NAFLD score ≥ 5 was identified as definite NASH.
2.4. Determination of liver collagen content
Formalin-fixed, paraffin-embedded, liver sections (4 µm) were deparaffinized
and stained with Masson trichrome (MT) [155]. MT staining was performed
following the manufacturer’s instructions (Accustain HT15, Sigma-Aldrich,
St. Louis, MO).
2.5. Liver homogenates and nuclear extract preparation
Briefly, 100 mg of liver tissue was homogenized in 0.5 mL of buffer A
containing 10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM MgCl2 , 1 mM DTT, 0.1
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
86 | P a g e
mM EDTA, 0.1 mM PMSF, 1 µM pepstatin, and 1 mM p-amino benzamidine
using a tissue homogenizer for 20 s. Homogenates were kept on ice for 15
min, and then 125 µL of a 10% Triton X-100 solution was added and mixed
for 15 s and the mixture was centrifuged for 2 min at 12 000 rpm. The
supernatant containing cytosolic proteins was collected. The pelleted nuclei
were washed once with 200 µL of buffer A plus 25 µL of 10% Triton X-100,
centrifuged, then suspended in 50 µL of buffer B containing 50 mM HEPES,
pH 7.8, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM
PMSF, and 10% v/v glycerol, mixed for 20 min, and centrifuged for 5 min at
12 000 rpm. The supernatant containing nuclear proteins was stored at –
80°C [156].
2.6. Western blot analysis
The frozen liver tissues were weighed and homogenized in an ice-cold buffer
(50 mM Tris-HCl, pH 7.4, 200mM NaCl, 20 mM NaF, 1 mM Na3VO4, 1 mM 2-
mercaptoethanol, 0.01 mg/mL leupeptin, 0.01 mg/mL aprotinin).
Homogenates were then centrifuged (3000 × g, 10 min, 4°C) and the
supernatants were collected and stored at -80°C. The total protein
concentration in samples was measured by the BCA method [150]. Samples
with equal amounts of protein were separated in polyacrylamide gel,
transferred and identified on nitrocellulose membrane with specific
antibodies followed by HRP-labelled secondary antibodies. The antibodies
applied: connective tissue growth factor (CTGF), interferon gamma (IFNγ)-
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
87 | P a g e
inducible protein (IP) 10, IL-1β, IFN γ, TLR4, p-NF-κB, nuclear factor of
kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) α, nuclear
factor-erythroid 2-related factor (Nrf)-2, HMGB1, cluster of differentiation
(CD) 68, CYP2E1, p67phox, vascular endothelial growth factor (VEGF),
SREBP-1c, peroxisome proliferator-activated receptor (PPAR) α, PPARγ,
oxidized low-density lipoprotein receptor (Ox-LDL R) 1, adipose
differentiation related protein (ADRP), CCAAT/enhancer binding protein
(C/EBP)β, glypican-3, β tubulin, and LaminA (Santa Cruz Biotechnology,
Santa Cruz, CA, USA), NF-κB, p-ERK1/2, ERK1/2 and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) (Cell Signaling Technology Inc., Beverly,
MA, USA), prothrombin (Abcam, Cambridge, UK). The levels of GAPDH,
NF-κB (for p-NF-κB), ERK1/2 (for p-ERK1/2), LaminA (for HMGB1 and
C/EBPβ) and β tubulin (for TLR4, PPARγ and Nrf2) were estimated in every
sample to check for equal loading of samples.
2.7. Statistical analysis
Data are shown as means ± SEM and were analyzed using ANOVA followed
by Tukey’s method or Kruskal-Wallis test followed by Dunn’s multiple
comparison when appropriate. A value of p< 0.05 was considered statistically
significant. GraphPad Prism 5 software (San Diego, CA, USA) was used.
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
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3. Results
3.1. Effect of curcumin on clinicopathology and biochemical parameters in
NASH-HCC mice
There was no huge difference in energy consumption and body weight among
the three groups (Table 1). The relative liver weight (%) was significantly
increased in NASH group compared to normal, but in the curcumin treated
group this increased proportion was significantly less. Fasting blood glucose
level and serum TG, TC, ALT, AST and ALP levels were also significantly
increased in NASH group compared to the normal group. These biomarkers
were of lower concentration in the NASH+ Curcumin group compared to
NASH group (Table 1). The liver of the NASH group also showed pale yellow
color, swelling and granular surface with tumor protruding whereas the liver
of the NASH + Curcumin group showed moderate fatty liver with a smooth
surface (Fig. 1A). H&E staining showed extreme steatosis, enlarged
hepatocytes, scattered lobular inflammatory cells in NASH mice. However,
these changes were lesser in NASH + Curcumin group than the NASH group
(Fig. 1B, Table 2).
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
89 | P a g e
Table 1: Changes in biochemical parameters after 4 weeks of treatment with
curcumin in NASH-HCC mice
Normal
group
(n=6)
NASH group
(n=6)
NASH + Curcumin
group
(n=6)
Food intake /day/mouse (g) 4.0 ± 0.6 3.27 ±0.055 3.5 ±0.7
Energy
consumption/day/mouse (Kcal)
16.16 ± 2.5 16.59 ± 0.28 17.76 ± 3.2
Body weight (BW) (g) 24.8 ± 2.7 20.7 ± 5.9 24.6 ± 2.2
% of Relative liver weight
(LW) /BW
4.01 ± 0.21 8.91 ± 1.5***
7.2 ± 0.43#***
Blood glucose (mg/dL) 101.3 ± 23.4 488.0 ± 19.9*** 363.0 ± 23.9###
***
Serum TG (mg/dL) 17.5 ± 1.3 46.3 ± 12.1* 40.3 ± 11.7*
Serum TC (mg/dL) 81.8 ± 3.8 253.8 ± 18.9*** 189.5 ± 10.6###
***
Serum ALT (IU/L) 38.0 ± 9.0 304.3 ± 218.7* 61.3 ± 15.9#
Serum AST (IU/L) 72.4 ± 5.4 270.5 ± 84.4*** 108.3 ± 25.0##
Serum ALP (IU/L) 322.8 ± 49.3 653.8 ± 141.9*** 408.4 ± 47.2##
Normal, age matched normal mice; NASH, untreated NASH-HCC mice;
NASH + Curcumin; NASH-HCC mice treated with curcumin 100 mg/kg/day.
TG: triglyceride; TC: total cholesterol, ALT: alanine aminotransferase, ALP:
alkaline phosphatase, AST: aspartate aminotransferase, NAFLD: non-
alcoholic fatty liver disease. Values are expressed as means ± SEM.
Statistical analysis was carried out using One way ANOVA followed by
Tukey’s method where, *p <0.05, ***p <0.001 vs normal, #p< 0.05, ##p <0.01,
###p< 0.001 vs NASH.
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
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Fig. 1. Curcumin attenuates the clinicopathology in NASH-HCC mice. (A),
Representative macroscopic appearance of livers (Black arrow: liver tumors).
(B), H&E staining (Black arrow: macrovesicular steatosis, red arrow:
microvesicular steatosis, yellow arrow: hypertrophy, circles: inflammatory
cells). (C) Fibrosis deposition by Masson trichrome staining (blue area). (D),
the hepatic protein level of CTGF. Data are mean ± SE, (n= 6/ group).
Normal NASH NASH + Curcumin0.000
0.005
0.010
0.015
0.020
*
##
CT
GF
/GA
PD
H
CTGF
GAPDH
D
Normal M
acr
osc
op
ic
Ap
pea
ra
nce
H &
E s
tain
ing
M
ass
on
tric
hrom
e
sta
inin
g
A
B
C
NASH NASH + Curcumin
20 µm 20µm
20 µm 20 µm 20 µm
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
91 | P a g e
Normal, age matched normal mice; NASH, untreated NASH-HCC mice;
NASH + Curcumin; NASH-HCC mice treated with curcumin 100 mg/kg/day.
Statistical analysis was carried out using One way ANOVA followed by
Tukey’s method where, *p<0.05 vs Normal, ##p<0.01 vs NASH.
3.2. Effect of curcumin on hepatic fibrosis in NASH-HCC mice
To examine the effect of curcumin in hepatic nutritional fibrosis, I performed
MT staining of hepatic tissues of three groups. MT staining demonstrated
pericellular fibrosis around central veins, and its positive area (%) was
(median (interquartile range)): in normal group (0.26 (0.075-0.5)); in NASH
group (1.755 (1.34-2.375)); while in NASH + Curcumin group (0.6075 (0.131-
1.470)) (p=0.0291), (Fig. 1C and Table 2). Consistent with the reduced
fibrosis, the hepatic protein expression of fibrosis promoter, CTGF was
significantly lower in NASH + Curcumin mice (p<0.01 vs. NASH) (Fig. 1D).
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
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Table 2: Histopathological analysis scores after 4 weeks of treatment with
curcumin in NASH-HCC mice.
Item Definition Score Normal
group
(n=6)
NASH
group
(n=6)
NASH +
Curcumin
group
(n=6)
Kruskal-
Wallis
P value
A. Macrovesicular
steatosis
<5%
5-33%
33-66%
>66%
0
1
2
3
0 (0-0) 2 (2-2)** 1 (1-1) 0.0183
B. Microvesicular
steatosis
<5%
5-33%
33-66%
>66%
0
1
2
3
0 (0-0) 2 (1-2)** 1 (1-1) 0.0289
C. Hepatocellular
hypertrophy
<5%
5-33%
33-66%
>66%
0
1
2
3
0 (0-0) 2 (2-2)** 1 (1-1)
0.0183
D. Inflammation:
Number of
inflammatory
foci
/field
<0.5
0.5-1.0
1.0-2.0
>2.0
0
1
2
3
0 (0-0) 2 (2-3)*
2 (1-2) 0.0344
NAFLD activity
score
Score of A+ B
+ C + D
0-12 0 (0-0) 8 (7-9)* 5 (4-5) 0.0234
MT score
(Fibrosis Score)
(Blue stained
areas / non-
stained
areas )×100
0-4 0.26
(0.075-0.5)
1.755
(1.34-
2.375)*
0.6075
(0.131-
1.470)
0.0291
Normal, age matched normal mice; NASH, untreated NASH-HCC mice;
NASH + Curcumin; NASH-HCC mice treated with curcumin 100 mg/Kg/day.
NAFLD: non-alcoholic fatty liver disease, MT: Masson trichrome. Values are
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expressed as medians (interquartile ranges). Statistical analysis was carried
out using Kruskal-Wallis test followed by Dunn’s multiple comparison where,
*p <0.05, **p <0.01 vs normal.
3.3. Effect of curcumin on the cytoplasmic HMGB1 level in liver
To find the altered distribution of HMGB1 in NASH liver of mice, Western
blot was performed to investigate the HMGB1 expression in the cytoplasm
and nuclear fraction from the liver of NASH and normal mice. The cytosolic
expression of HMGB1 was higher than nuclear expression in NASH group. In
contrast, the translocation of HMGB1 from the nucleus to cytoplasm was
significantly lower in the NASH + Curcumin group compared to NASH group
(Fig. 2A).
Normal NASH NASH + Curcumin0 .0 0 0
0 .0 0 1
0 .0 0 2
0 .0 0 3
0 .0 0 4*
#
HM
GB
1 c
yts
ol/
nu
cle
us
Lamin A (Nucleus)
HMGB1 (Cytosol)
HMGB1 (Nucleus)
GAPDH (Cytosol)
A
β tubulin
TLR4
Normal NASH NASH + Curcumin0
2
4
6***
##
TL
R 4
/ tubulin
B
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Fig. 2. Curcumin attenuates hepatic inflammatory cytokines and macrophage
infiltration in NASH-HCC mice.
Representative Western blots show specific bands for nuclear HMGB1 (for
LaminA) and cytosolic HMGB1 (A), TLR4 (for β tubulin) (B), IP10 (C), IFNγ
(D), IL-1β (E), and CD68 (F) and the representative histograms show the
band densities with relative to that GAPDH. The ratio of HMGB1 expression
in the cytoplasmic fraction to nuclear fraction was measured by densitometric
quantification (A). Each bar represents mean ± SE. Normal, age matched
normal mice; NASH, untreated NASH-HCC mice; NASH + Curcumin; NASH-
HCC mice treated with curcumin 100 mg/kg/day. Statistical analysis was
Normal NASH NASH + Curcumin0.00
0.01
0.02
0.03
0.04**
###
IP10/G
AP
DH
IP10
GAPDH
C
GAPDH
IFNγ
D
Normal NASH NASH + Curcumin0.00
0.02
0.04
0.06*
#
IL-1
/GA
PD
H
GAPDH
IL-1β
E
Normal NASH NASH + Curcumin0.0
0.5
1.0
1.5*
#
IFN/
GA
PD
H
Normal NASH NASH + Curcumin0.00
0.02
0.04
0.06*
###**
CD
68/G
AP
DH
GAPDH
CD68
F
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95 | P a g e
carried out using One way ANOVA followed by Tukey’s method
where,*p<0.05, **p<0.01, ***p<0.001 vs Normal, #p<0.05, ##p<0.01,
###p<0.001 vs NASH.
3.4. Effect of curcumin on hepatic chemokines, cytokines, and other pro-
inflammatory molecules
The hepatic IP10, IFNγ and IL-1β protein expression were significantly
increased in NASH mice. I observed that the liver protein level of TLR4 and
the expression of KCs, CD68, a marker of macrophages [157], were
significantly increased in NASH group compared to the normal group.
Curcumin treatment essentially suppressed these protein levels in the liver
of the NASH + Curcumin group compared to NASH group (Fig. 2B-F).
3.5. Effect of curcumin on NF-κB pathway
I noticed that the increased hepatic nuclear translocation of NF-κB subunits
p65 and diminished cytosolic IκBα protein level (Fig. 3A & B) in NASH group
compared to the normal group. The expression of Ox-LDL-R1 was also
significantly increased in NASH liver, which activates the NF-κB signal
transduction pathway [158] (Fig. 3C). But curcumin treatment markedly
prevented all of these alterations as shown in the NASH + Curcumin group
compared to NASH group.
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Fig. 3. Curcumin attenuates NF-κB pathway in NASH-HCC mice
Representative Western blots show specific bands for nuclear and cytosolic p-
NF-κB (for NF-κB) (A), IκBα (B) and Ox-LDL-R1 (C), and the representative
histograms show the band densities with relative to that GAPDH. The ratio
of p-NF-κB expression in the nuclear fraction to cytosolic fraction was
measured by densitometric quantification (A). Each bar represents mean ±
SE. Normal, age matched normal mice; NASH, untreated NASH-HCC mice;
NASH + Curcumin; NASH-HCC mice treated with curcumin 100 mg/kg/day.
Statistical analysis was carried out using One way ANOVA followed by
Tukey’s method where, *p<0.05, **p<0.01 vs Normal, #p<0.05, ##p<0.01 vs
NASH.
Normal NASH NASH + Curcumin0.0
0.5
1.0
1.5
*
#
I B
/GA
PD
H
GAPDH
IκBα
A B
Normal NASH NASH + Curcumin0.00
0.02
0.04
0.06
**
##
Ox-L
DL
-R1/G
AP
DH
GAPDH
Ox-LDL-R1
C
Normal NASH NASH + Curcumin0
2
4
6
8
**
#
p-N
F-
B n
ucl
eus/
cyto
sol
p-NFκB (Nucleus)
NFκB (Nucleus)
p-NFκB (Cytosol)
NFκB (Cytosol)
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3.6. Effect of curcumin on hepatic lipogenesis
The hepatic protein expression of lipogenic controllers that include SREBP-
1c, ADRP and genes which regulate adipogenesis, such as PPARγ were
significantly increased in NASH group compared to normal mice. On the
other hand, these increased hepatic protein expressions were significantly
lesser in the NASH + Curcumin group compared to NASH group (Fig. 4A-C).
The protein expression of PPARα was reduced in the NASH group while it
was increased in the liver of NASH + Curcumin group but non-significantly
(Fig. 4D).
Normal NASH NASH + Curcumin0.00
0.05
0.10
0.15
0.20
0.25**
###
SR
EB
P1c/G
AP
DH
Normal NASH NASH + Curcumin0.0
0.1
0.2
0.3
0.4
**
##
AD
RP
/GA
PD
H
Normal NASH NASH + Curcumin0
2
4
6
8
**
###*
PP
AR/
tubulin
PPARγ
C
Normal NASH NASH + Curcumin0.00
0.01
0.02
0.03
0.04
PP
AR
/GA
PD
H
D
PPARα
β tubulin GAPDH
GAPDH
SREBP1c
GAPDH
ADRP
A B
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
98 | P a g e
Fig. 4. Curcumin attenuates hepatic lipogenesis in NASH-HCC mice.
Western blots show specific bands for hepatic SREBP1c (A), ADPR (B),
PPARγ (for β tubulin) (C) and PPARα (D) and the representative histograms
show the band densities with relative to that GAPDH. Each bar represents
mean ± SE. Normal, age matched normal mice; NASH, untreated NASH-
HCC mice; NASH + Curcumin; NASH-HCC mice treated with curcumin 100
mg/kg/day. Statistical analysis was carried out using One way ANOVA
followed by Tukey’s method where, *p<0.05, **p<0.01 vs Normal, ##p<0.01,
###p<0.001 vs NASH.
3.6. Curcumin attenuated oxidative stress in NASH-HCC liver
I evaluated the hepatic levels of C/EBPβ and CYP2E1 using Western blot
analysis. Both nuclear C/EBPβ and cytosolic CYP2E1 protein expression were
significantly increased in the NASH group compared to the normal group,
whereas, in the NASH + Curcumin group these were significantly less
corresponding to NASH mice (Fig. 5A and B). The expression of cytosolic
NADPH oxidase component p67phox and p-ERK1/2 were markedly increased
but the expression of Nrf2 was significantly decreased in the liver of NASH-
HCC mice compared to normal mice. However, curcumin treatment markedly
reversed all of these expressions in the liver of the NASH + Curcumin group
(Fig. 5C-E).
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
99 | P a g e
Normal NASH NASH + Curcumin0.000
0.005
0.010
0.015
0.020
0.025
**
#
C/E
BP
/Lam
in A
C/EBPβ
Lamin A
Normal NASH NASH + Curcumin
0.00
0.05
0.10
0.15
0.20
**
###
CY
P2E
1/G
AP
DH
GAPDH
CYP2E1
A B
Normal NASH NASH + Curcumin0.0
0.1
0.2
0.3
0.4
**
###
p-E
RK
1/2
/ER
K1/2
ERK1/2
p-ERK1/2
C
Normal NASH NASH + Curcumin0.00
0.01
0.02
0.03
0.04
**
#
p67phox/G
AP
DH
GAPDH
p67phox
D
Normal NASH NASH + Curcumin0.0
0.5
1.0
1.5
*
##
Nrf
2/
tubulin
β tubulin
Nrf2
E
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
100 | P a g e
Fig. 5. Curcumin attenuates hepatic oxidative stress in NASH-HCC mice.
Western blots show specific bands for hepatic C/EBPβ (for-Lamin A) (A),
CYP2E1 (B), p-ERK1/2 (for ERK1/2) (C) and p67phox (D) and Nrf2 (for β
tubulin) (E) and the representative histograms show the band densities with
relative to that GAPDH. Each bar represents mean ± SE. Normal, age
matched normal mice; NASH, untreated NASH-HCC mice; NASH +
Curcumin; NASH-HCC mice treated with curcumin 100 mg/kg/day.
Statistical analysis was carried out using One way ANOVA followed by
Tukey’s method where, p<0.05, **p<0.01 vs Normal, #p<0.05, ##p<0.01,
###p<0.001 vs NASH.
3.7. Effect of curcumin on hepatic VEGF, glypican-3 and prothrombin
expression
I investigated the hepatic protein expression level of VEGF, glypican-3, and
prothrombin in this NASH-HCC mouse model, I found that these were
significantly increased in NASH group compared to the normal group, but in
curcumin treated group the expression of these proteins were significantly
less when compared to NASH group (Fig. 6A-C).
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
101 | P a g e
Fig. 6. Curcumin attenuates hepatic VEGF, Glypican-3 and prothrombin
expression in NASH-HCC mice. Western blots show specific bands for hepatic
VEGF (A), Glypican-3 (B) and prothrombin (C) and the representative
histograms show the band densities with relative to that GAPDH. Each bar
represents mean ± SE. Normal, age matched normal mice; NASH, untreated
NASH-HCC mice; NASH + Curcumin; NASH-HCC mice treated with
curcumin 100 mg/kg/day. Statistical analysis was carried out using One way
ANOVA followed by Tukey’s method where, *p<0.05, **p<0.01 vs Normal,
#p<0.05, ##p<0.01 vs NASH.
Normal NASH NASH + Curcumin0.00
0.05
0.10
0.15
0.20
**
#
VE
GF
/GA
PD
H
GAPDH VEGF
Normal NASH NASH + Curcumin0.0
0.1
0.2
0.3
**
#
Gly
pic
an-3
/GA
PD
H
Normal NASH NASH + Curcumin0
2
4
6
*
##
Pro
thro
mbin
/GA
PD
H
GAPDH
Glypican-3
GAPDH
Prothrombin
A B
C
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4. Discussion
Animal models are key to translate human diseases and testing the
effectiveness of various available drugs for treatment. There are several
models of NASH and HCC, but they neither show high reproducibility nor
meet the clinicopathological process from fatty liver, NASH, fibrosis to HCC
under diabetic background [159]. Although, diabetes is a known danger
element for HCC, it is also reported that neither diabetes nor obesity alone
causes of HCC [160]. In this experiment, I considered a novel NASH-HCC
model, which was developed by injecting a low-dose STZ and feeding a HFD
both in mice. This model shows 100% reproducibility for HCC, with quick
results for the study of the disease progression and maintains a sequence
from fatty liver, NASH, fibrosis to HCC under diabetic background [11]. To
date, there is no effective treatment to manage NASH. In this study, I
investigated the possible hepatoprotective mechanism of curcumin at a
dosage that translates to a curcumin intake of around of 6 g/day in adult
humans [161].
The replication of human NASH histopathology in animal model remains the
only mean of accurately assessing NASH [159]. Briefly, the two key features
of NASH, steatosis, and inflammation; categorized by Liang et al. as
micro/macrosteatosis, hepatocellular hypertrophy and inflammatory cell
aggregation [14]. In our experiment, based on this scoring the mice in NASH-
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103 | P a g e
HCC group reached a higher NAFLD activity score along with fibrosis (Table
2) and increased serum aminotransferases and blood glucose levels (Table 1).
Increase in aminotransferase levels leads to focal inflammation, hepatic
necrosis and fibrosis without developing resistance to insulin [137]. The
CTGF also plays a key role in the progression of liver fibrosis in mice. High
level of CTGF protein expression was found in NASH liver, with pericellular
fibrosis (Fig. 1D); which represents human NASH “chicken wire fibrosis”.
Interestingly, these biomarkers were of lower concentration in the NASH +
Curcumin group compared with the NASH group along with reduced NAFLD
score and fibrosis marker.
It is well established the importance of liver inflammation in the initiation
and progression of cancer. Several study groups worked on characterizing the
role of DAMPs, including HMGB1, in liver inflammation [142]. It is proved
that the release of nuclear HMGB1 to the extracellular, not only mediates
acute phase of liver injury in ischemia reperfusion, but also promotes HCC
invasion and provides a link between inflammations to HCC [147]. The
HMGB1 release can be correlated with its ability to signal through receptors
TLR2, TLR4 and RAGE [145]. As it is reported that the TLR4-deficient mice
exhibited reduced hepatic ischemia-reperfusion injury, partially protects from
diet-induced steatohepatitis and downregulated the inflammatory cytokines
[162], brought an attention to TLR4 in NASH. In line with other
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104 | P a g e
inflammation-associated HCC, in this study, I identified the significantly
increased levels of cytosolic translocation of HMGB1 in the liver of NASH
mice with upregulation of TLR4 protein levels. Interestingly, both the protein
expression levels of TLR4 and cytosolic translocation of HMGB1 were
significantly less in the liver of curcumin treated mice compared to NASH
group (Fig. 2A-B). In similarity, glycyrrhizin, a direct HMGB1 inhibitor [163],
has been used clinically [164] and experimentally [165] to prevent liver
cirrhosis and HCC for decades in Japan. Interaction of HMGB1 with TLR4
induces nuclear translocation of activated NF-κB, and activates ERK
signaling and ultimately resulting the release of pro-inflammatory cytokines
[144]. The secreted stimuli like IFNγ [166], and IL-1β [167] can also
stimulate the release of HMGB1 from macrophages. Accordingly, I found the
elevated hepatic protein expression levels of pro-inflammatory cytokines
IP10, IFNγ, IL-1β and macrophage infiltration (CD68) in NASH group,
whereas, in the NASH + Curcumin group these levels were significantly
lesser (Fig. 2C-F). It is already proved that NF-κB is a key regulator of early
hepatic inflammatory recruitment and liver injury in NASH [146] and HCC
[168]. While KCs CD68 display powerful NF-κB activation in NASH liver,
resulting in the production and secretion of pro-inflammatory cytokines that
strongly implicated as a promoter of fibrosis and HCC [169]. Therefore,
curcumin treatment also protects the nuclear translocation of NF-κB
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
105 | P a g e
pathway and its upstream and downstream inflammatory effectors, which
induce liver injury and progression of steatohepatitis (Fig.3).
My data also suggest that curcumin treatment protects the STZ-HFD-
induced liver from steatosis and liver injury. Where, steatosis sensitizes the
liver to injury by cytokines, endo-toxin, adipokines, mitochondrial
dysfunction and oxidative stress, which result in de novo lipogenesis and
increased release of free fatty acids and abnormal lipid deposition in the
liver. The protein expression levels of SREBP-1c and ADRP were
significantly lower in the curcumin treated group when compared with the
NASH group (Fig. 4A and B), which are involved in de novo fatty acid
synthesis. Hepatic expression of PPARγ is augmented in various models of
fatty liver disease in which liver regeneration has been reported to be
impaired [170]. In contrast, Gazit et al. suggested that increased hepatic
PPARγ activity might improve regeneration of fatty liver [171], but, in my
study, I found that the elevated level of PPARγ in NASH liver was
significantly downregulated by curcumin treatment (Fig. 4C). Curcumin
treatment increased the protein expression of fatty acid oxidation regulatory
gene PPARα in NASH liver (Fig. 4D).
Although, HMGB1 does not contain a leader sequence, the mechanisms for
regulating HMGB1 release and activity vary depending on context. Many
experimental studies reveal that oxidative stress is likely a common
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
106 | P a g e
mechanism regulating HMGB1 translocation, release and activity [172]. In
another study, Wang et al., reported that high blood glucose level induced
HMGB1 translocation through NADPH oxidase and PKC dependent pathway
[173]. Consistent with these reports, I found that the hepatic protein
expression of CYP2E1 and its upstream C/EBPβ, downstream p-ERK1/2 and
NADPH oxidase subunit p67phox were significantly lesser in curcumin
treated group along with reduced blood glucose level compared to NASH
group (Fig. 5A-D, Table 1). Activation of CYP2E1, is involved in the
metabolism of fatty acid and produce ROS directly or indirectly. Redox
systems, including antioxidants and phase ІІ detoxifying enzymes provide
protection against ROS actuated tissue damage. Nrf2 is a vital cytoprotective
transcriptional variable that induces the expression of several antioxidants
and phase ІІ detoxifying enzymes [156] and acts as a defense system in the
development of NASH [174]. Actually, besides Nrf2’s part in directing cellular
antioxidant guard, it likewise has anti-inflammatory capacities [156]. Here, I
found that the significantly higher expression of Nrf2 in the liver of the
NASH + Curcumin group when compared to NASH group (Fig. 5E).
Oxidative stress and inflammatory pathways lead to the transformation of a
normal cell to a tumor cell, its survival, and proliferation [175]. In my
experiment, I noticed the tumor protruding in the liver of NASH group, in
contrast, it was absent in the NASH + Curcumin group (Fig.1A). It is
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
107 | P a g e
reported that VEGF may act as a major regulator of hepatic tumorigenesis in
a mouse model of experimental liver metastasis [176]. This tumor protruding
may be malignant or progress to HCC, and, as it has been reported that the
hepatic expression of glypican-3 [177] and DCP or prothrombin [178] are
significantly increased in most HCCs compared with benign liver lesions and
normal liver, they act as a useful prognostic tumor marker for HCC. In my
study, I observed that VEGF, glypican-3 and prothrombin, markers for HCC
were significantly higher in the NASH group compared to the normal group
(Fig. 6A-C). But, these expressions were significantly lower in the curcumin
treated group compared to NASH group.
In summary, my data indicate that curcumin has a beneficial effect in
NASH, it might be partly by reducing the cytosolic and nuclear translocation
of HMGB1 and NF-κB, either directly or indirectly by reducing oxidative
stress and blood glucose level in NASH liver. Furthermore, by inhibiting the
progression of NASH to HCC curcumin might be useful in the management of
NASH and may constitute a powerful approach for the novel NASH-HCC
model.
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CHAPTER THREE
Le Carbone modulates liver damage in non-alcoholic
steatohepatitis-hepatocellular carcinoma mouse model
through activation of AMPKα-SIRT1 signaling
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109 | P a g e
1. Introduction
NAFLD has become increasingly emergent due to changes in lifestyle and
resultant over-nutrition [179]. NASH is an extended form of NAFLD,
characterized by necro-inflammation, lipid accumulation and fibrosis, act as a
significant risk factor for the development of cirrhosis and HCC [180].
Although still obscure, the transition from benign steatosis to steatohepatitis
in NASH is regarded as the increased levels of toxic lipids, induced by
dysregulated lipid metabolism and triggered by the oxidative stress and pro-
inflammatory cytokines [181,182].
Several animal model of NAFLD is associated with the impairment of the
hepatic SIRT1 -AMPK axis, a central signaling system for controlling the
lipid metabolism pathway [183]. SIRTs belong to the silent information
regulator-2 family. SIRT1 deacetylation has been recognized as a regulatory
mechanism for several proteins involved in NAFLD pathogenesis [184] and
low SIRT1 expression has been noticed in different NAFLD models [185].
Similarly, the AMPK signaling system plays a vital role in cellular and
organismal survival during stress by maintaining metabolic homeostasis
[186].
The activation of SIRT1-AMPK signaling in several metabolic tissues,
including liver has been proposed to increase the rate of fatty acid oxidation
and restrain the lipogenesis mostly by modulating activity of PPARγ
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
110 | P a g e
coactivator-α (PGC-1α) /PPARα or SREBP-1 through deacetylation and
phosphorylation, respectively [187], [188].
PPARγ is a transcription factor, thought to be the master regulator of fat
storage and energy homeostasis [189], and although the function of PPARγ is
controversial, the increased expression of PPARγ is found in obese mice and
which is associated to increase the lipogenesis. PPARα acts as a regulator of
β-oxidation and removes cholesterol at the time of necessity. It is also found
that hepatic PPARα expression is low in NAFLD due to steatosis, but
enhanced following diet and exercise [190].
Dietary supplementation along with vitamin E found to improve the NAFLD
activity score in non-diabetics with biopsy proven NASH patients [191]. LC, a
charcoal supplement, comprises with a large amount of dietary fibers. In my
previous study, I found that LC supplementation able to enhance the AMPK-
SIRT1 expression in colon tissue of experimental colitis in a mouse model and
reduced the inflammation [63]. It is also suggested that, activated charcoal
possesses wound healing, anti-aging properties [61]. Some study proved that
chemotherapy with mitomycin C adsorbed onto activated charcoal (MMC-CH)
may increase the survival rates after stomach cancer surgery, when
anticancer drugs fail to reduce the secondary cancer development [62].
Furthermore, in patients with trimethylaminuria (a metabolic disorder),
activated charcoal supplement improve the life of individual suffering [192].
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
111 | P a g e
Activated charcoal also used to prevent abnormal hardening (sclerosis) in the
heart and coronary blood vessels by improving lipid profile [64]. For the
above-mentioned beneficial effects of charcoal supplement, in this experiment
I hypothesized that LC may prevent the liver damage in a novel mouse model
of NASH-HCC by activating AMPK-SIRT1 expression.
2. Materials and Methods
2.1. Drugs and chemicals
HFD32 was purchased from CLEA, Japan. LC was obtained from mcprot.,
Biotechnology- EJC, Japan. All other chemicals used were purchased from
Sigma, Japan unless mentioned otherwise.
2.2. Composition of normal diet, HFD32 and LC
The normal diet provided 404 Kcal/100 g (water 10%, protein 25.0%, fat 4.5%,
ash 6.7%, carbohydrate 49.3% and fiber 4.5%), whereas, the HFD 32 provided
507.6 Kcal/100 g (water 6.2%, protein 25.5%, fat 32.0%, ash 4.0%,
carbohydrate 29.4%, and fiber 2.9%). LC was formulated as capsule; entirely
with the activated Carbone and contained a minor quantity of minerals (in
100 g of LC) such as sodium 2.2 mg, potassium 77.6 mg and manganese 0.4
mg. Where other minerals like non-phosphorous, iron, calcium, magnesium,
copper, zinc and heavy metals (cadmium, lead, arsenic and total mercury)
were not present. According to the Corporation Vision Bio-test results, the
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
112 | P a g e
nutritional value of LC (in 100 g): energy 4 Kcal, moisture 0.6 g, protein
minimum, lipid minimum, carbohydrate 1.1 g, ash 0.2 g, water soluble
dietary fiber 0.6 g, insoluble dietary fiber 97.5 g and the dietary fiber total
amount 98.1 g.
2.3. Animals and experimental designs
All animals were treated in accordance with the guidelines for animal
experimentation of our institute (Approve No. H270313) [150]. Animals were
housed in a temperature of 23 ± 2°C and humidity of 55 ± 15% and were
submitted to a light/dark cycle, allowed free access to standard laboratory
chow and tap water. C57BL/6J mice were bred in my laboratory. NASH-HCC
was induced in male mice by a single subcutaneous injection of 200 µg STZ
(Sigma, MO, USA) at 2 days after birth and after then they were being
started feeding with HFD32 ad libitum at the age of 4 weeks, and continued
up to 16 weeks age of mice. Mice were randomly selected into three groups
(n=6/group): group 1 (Normal) were normal mice subjected to the normal diet;
group 2 (NASH) were STZ injected mice subjected to the HFD32 treated with
1% methyl cellulose (MC) (the vehicle of LC); group 3 (LC) were STZ injected
mice subjected to HFD32 treated with LC suspension at (5 mg/mouse/day,
suspended in 1% MC). LC treatment was started at the age of 6 weeks and
administered via oral gavage continued up to 16 weeks of age along with
HFD. All mice were sacrificed at the age of 16 weeks, and serum was
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113 | P a g e
separated from blood. Liver tissue was isolated for histological, biochemical,
and molecular biological analysis [11].
2.4. Biochemical analysis
Fasting blood glucose level was measured using the G-checker (Sanko
Junyaku, Tokyo, Japan). Serum ALT, AST, ALP, TG and TC were measured
by FUJI DRI-CHEM 7000 (Fujifilm, Tokyo, Japan) as previously described
[150].
2.5. Histological examination
Formalin-fixed liver sections (4 μm) were stained with H&E. Morphological
analysis was done by computerized image analysis system on ten microscopic
fields per section examined in a 20-fold magnification (CIA-102; Olympus,
Tokyo, Japan), with the observer blind to the study group [150]. Here,
macrovesicular steatosis, microvesicular steatosis and hepatocellular
hypertrophy were separately scored and the severity was graded, based on
the percentage of the total area affected, into the following categories: 0
(<5%), 1 (5-33%), 2 (34-66%) and 3 (>66%). Inflammation was evaluated by
counting the number of inflammatory foci per field, a focus has defined a
cluster of ≥5 inflammatory cells. Different fields were counted and scored as
per field: 0 (<0.5), 1 (0.5-1.0), 2 (1.0-2.0) and 3 (>2). By adding the scores of
the above four parameters the NAFLD activity score was calculated, which
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114 | P a g e
result in a total clinical score ranging from 0 (healthy) to 12 (maximal
severity of NASH) [14]. NAFLD score ≥ 5 was identified as definite NASH.
2.6. Determination of liver fibrosis content
Formalin-fixed, paraffin-embedded, liver sections (4 µm) were stained with
MT [155]. MT staining was performed following the manufacturer’s
instructions (Accustain HT15, Sigma-Aldrich, St. Louis, MO).
2.7. Western blot analysis
The liver tissue protein lysate was prepared using a method similar to that
described previously [193]. The total protein concentration in the samples
was measured by the BCA method. For the determination of protein levels,
equal amounts of protein extracts (50 µg) were separated by 7.5–15% SDS
polyacrylamide gel electrophoresis (Bio-Rad, CA, USA) and transferred
electrophoretically to nitrocellulose membranes. Membranes were blocked
with 5% non-fat dry milk or BSA in Tris buffered saline Tween (20 mM Tris,
pH 7.6, 137 mM NaCl, and 0.1% Tween 20). Primary antibodies against
SIRT1, Heme-oxygenase-1 (HO-1), p47phox, nuclear factor-erythroid 2-
related factor (Nrf)-2, glucose transporter type (GLUT) 4,
phosphoenolpyruvate carboxykinase (PEPCK)-C, PPARα, PPARγ, ADRP,
adiponectin receptor (AdipoR)1, IL-10, IL-1β, IL-6, tissue inhibitor of
metalloproteinases (Timp) 4, matrix metalloproteinase (MMP)-9, p53, p-p53,
and glypican-3 were obtained from Santa Cruz Biotechnology, Santa Cruz,
CA, USA. Phospho-AMPKα, AMPKα, PGC1α, JNK, p-JNK, ERK1/2, p-
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
115 | P a g e
ERK1/2, β-actin and GAPDH antibodies were obtained from Cell Signaling
Technology Inc., Beverly, MA, USA. Collagen IV and prothrombin were
obtained from Abcam, Cambridge, UK. The levels of AMPKα (for p-AMPKα),
JNK (for p-JNK), ERK1/2 (for p-ERK1/2), p53 (for p-p53) and GAPDH or β-
actin were estimated in every sample to check for equal loading of samples.
2.8. Immunohistochemistry
Formalin-fixed, paraffin-embedded liver tissue sections were used for
immunohistochemical staining. After deparaffinization and hydration, the
slides were washed in Tris-buffered saline (TBS; 10 mM/L Tris HCl, 0.85%
NaCl, pH 7.2). Endogenous peroxidase activity was quenched by incubating
the slides in methanol and 0.3% H2O2 in methanol. After overnight
incubation with the primary antibody, that is, mouse monoclonal anti-cluster
of differentiation (CD)36 antibody (diluted 1:100) (sc-59103; Santa Cruz
Biotechnology Inc. CA, USA) at 4°C, the slides were washed in TBS and
(HRP) -conjugated goat anti-rabbit secondary antibody was then added and
the slides were further incubated at room temperature for 1 h. The slides
were washed in TBS and incubated with diaminobenzidine tetra
hydrochloride as the substrate, and counterstained with hematoxylin. A
negative control without primary antibody was included in the experiment to
verify the antibody specificity. CD36 positive hepatocytes were counted in
100 fields/ group under 20 fold magnification and expressed as cells/field [63].
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2.9. Statistical analysis
Data are shown as means ± SEM and were analyzed using ANOVA followed
by Tukey’s method or Kruskal-Wallis test followed by Dunn’s multiple
comparison when appropriate. A value of p< 0.05 was considered statistically
significant. GraphPad Prism 5 software (San Diego, CA, USA) was used.
3. Results
3.1. Effect of LC on clinicopathology and biochemical parameters in
NASH-HCC mice
There was no huge difference in energy consumption and body weight among
the three groups during the experimental period (Table 1). The ratio of liver
weight and body weight (g/Kg) was significantly increased in NASH group
compared to normal (p<0.01), but in LC treated group this increased
proportion were significantly lesser than the NASH group (p<0.05) (Fig. 1B).
Fasting blood glucose level and serum TG, TC, ALT, AST and ALP levels
were also significantly elevated in NASH group compared to the normal
group. The serum ALT and AST levels, indicators of liver damage, were
significantly prevented in LC treated group (p<0.05), and other lipid profile
markers were also noticeably lesser in LC group when compared to NASH
group (Table 1). But the administration of LC did not show any effect on the
increased blood glucose level (Table 1). Macroscopically, the liver of the
NASH showed the abnormal shape and swelling with tumor protruding
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
117 | P a g e
whereas the liver of LC group showed moderate fatty liver with a smooth
surface (Fig. 1A). H&E staining showed extreme steatosis, enlarged
hepatocytes, and scattered lobular, inflammatory cells with an increased
NAFLD activity score in NASH mice. However, these changes were lesser in
LC group than the NASH group (Fig. 1C-D).
Table 1: Changes in biochemical parameters after treatment with Le Carbone
in NASH-HCC mice
Normal group
(n=6)
NASH group
(n=6)
LC
(n=6)
Food intake /day/mouse (g) 3.133 ± 0.151 2.575 ±0.053 2.505 ± 0.154
Energy
consumption/day/mouse
(Kcal)
12.66 ± 0.611 13.1 ± 0.27 12.72 ± 0.783
Body weight (BW) (g) 24.23 ± 0.75 22.70 ± 4.66 22.33 ± 1.62
Liver weight (LW) /BW
(g/Kg)
53.48 ± 2.61 86.85 ± 14.32**
63.33 ± 11.29#
Blood glucose (mg/dL) 110.75 ± 13.67 469.60 ± 65.00*** 490.50 ± 14.18***
Serum TG (mg/dL) 47.33 ± 13.8 59.00 ± 18.33 30.50 ± 13.53
Serum TC (mg/dL) 90.00 ± 7.81 202.00 ± 29.02*** 148.50 ± 19.94#*
Serum ALT (IU/L) 62.00 ± 3.46 367.00 ± 173.53* 65.75 ± 27.29#
Serum AST (IU/L) 145.67 ± 46.29 799.25 ± 353.26* 186.00 ± 66.39#
Serum ALP (IU/L) 358.33 ± 35.84 646.25 ± 215.13 334.25 ± 89.20#
Normal, age matched normal mice; NASH, untreated NASH-HCC mice; LC,
NASH-HCC mice treated with Le Carbone suspension at 5 mg/mouse/day.
TG: triglyceride; TC: total cholesterol, ALT: alanine aminotransferase, ALP:
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
118 | P a g e
alkaline phosphatase, AST: aspartate aminotransferase. Values are
expressed as means ± SEM. *p <0.05, **p<0.01, ***p <0.001 vs normal, #p< 0.05
vs NASH.
Fig. 1. Le Carbone attenuates the clinicopathology in NASH-HCC mice. (A),
Representative macroscopic appearance of livers (circles: liver tumors). (B),
Histogram for the ratio of liver weight (LW) and body weight (BW) in gm/kg.
B
Normal NASH LC0
2
4
6
8
*
NA
FL
D a
ctiv
ity s
core
/fie
ld
Normal NASH LC0.0
0.5
1.0
1.5
*
% o
f fi
bro
sis/
field
D F
Normal NASH LC0
20
40
60
80
100**
#
LV
/BW
(g/K
g)
LC NASH Normal
H&
E
Sta
inin
g
Ma
crosc
op
ic
Ap
pea
ran
ce
A
C
E
MT
Sta
inin
g
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
119 | P a g e
(C), H&E staining (yellow arrow: macrovesicular steatosis, black arrow:
microvesicular steatosis, red arrow: hypertrophy, circles: inflammatory cells).
(D), Histogram of non-alcoholic fatty liver disease (NAFLD) activity score.
(E), Fibrosis deposition by Masson trichrome staining (blue area). (F),
percentage of fibrosis in each group. Data are mean ± SE, (n= 6/ group).
Normal, age matched normal mice; NASH, untreated NASH-HCC mice; LC,
NASH-HCC mice treated with Le Carbone suspension at 5 mg/mouse/day.
Statistical analysis was carried out using One way ANOVA followed by
Tukey’s test where **p<0.01 vs Normal, and #p<0.05 vs NASH, except
*p<0.05 vs Normal analyzed by Kruskal-Wallis test.
3.2. Effect of LC on hepatic fibrosis in NASH-HCC mice
To examine the effect of LC in hepatic fibrosis, I performed MT staining of
hepatic tissues of three groups. MT staining demonstrated pericellular
fibrosis around central veins, and its percentage of positive area was [median
(interquartile range)]: in normal group [0.03 (0.023-0.0456)]; in NASH group
[0.95 (0.6131-1.23)]; whilst in LC group [0.135 (0.065-0.228)] (p=0.0273), (Fig.
1E-F).
3.3. LC administration stimulated hepatic p-AMPKα and SIRT1
expression in NASH-HCC mice
Since AMPK and SIRT1 play an important role in liver lipid metabolism I
investigated the protein expression levels in NASH livers using Western blot.
The protein expression of p-AMPKα (p<0.01) and SIRT1 (p<0.05) were
significantly reduced in the NASH group compared to that of normal group.
The phosphorylation of AMPKα was increased in the liver of LC treated
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
120 | P a g e
group by 1.5-fold, compared to the NASH group but did not reach a
significant level (Fig. 2A). The hepatic protein expression of SIRT1 was
significantly increased in LC group by 1.7-fold when compared to NASH
group (p<0.01) (Fig. 2B).
Fig. 2. Effect of Le Carbone on hepatic AMPKα, SIRT1, GLUT4, PGC1α and
PEPCK-C expression in NASH-HCC mice.
Normal NASH LC0.0
0.2
0.4
0.6
0.8
1.0
**
p-A
MP
K
/AM
PK
Normal NASH LC0 .0 0
0 .0 5
0 .1 0
0 .1 5
0 .2 0
*
##
SIR
T1
/ -a
cti
np-AMPKα
AMPKα β-actin
SIRT1
A B
Normal NASH LC0.0
0.1
0.2
0.3
0.4
0.5
GL
UT
4/G
AP
DH
GLUT4
GAPDH
C
Normal NASH LC0.0
0.1
0.2
0.3
**
*
PG
C-1
/GA
PD
H
PGC1α
GAPDH
D
Normal NASH LC0.00
0.05
0.10
0.15
0.20
0.25
PE
PC
K-C
/ G
AP
DH
PEPCK-C
GAPDH
E
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
121 | P a g e
Representative Western blots show specific bands for hepatic (A), p-AMPKα
(for AMPKα); (B), SIRT1 (for β-actin); (C), GLUT4; (D), PGC1α; and (E),
PEPCK-C and the representative histograms show the band densities with
relative GAPDH. Each bar represents mean ± SE. Normal, age matched
normal mice; NASH, untreated NASH-HCC mice; LC, NASH-HCC mice
treated with Le Carbone suspension at 5 mg/mouse/day. Statistical analysis
was carried out using One way ANOVA followed by Tukey’s test where,
*p<0.05, **p<0.01 vs Normal, and ##p<0.01 vs NASH.
3.4. Effect of LC on hepatic GLUT4, PGC1α, and PEPCK-C expression in
NASH-HCC mice
The upregulation of AMPK and SIRT1, reduce the hepatic lipid accumulation
[183], [194]. To check this I detected the downstream proteins of AMPK using
Western blot, including GLUT4, PGC1α and PEPCK-C, which are all
associated with lipid metabolism. The hepatic protein expression of GLUT4
and PEPCK-C were trended to lesser in the NASH group by 0.84-fold and
0.612-fold respectively, while, PGC1α (p<0.01) expression was significantly
lesser by 0.42-fold when compared to the normal mice. But, in LC group,
hepatic expression of GLUT4 were trended to increase by 1.1-fold compared
to NASH mice (Fig. 2C). The protein levels of PGC1α and PEPCK-C were
about 1.6-fold higher in LC treated liver than the NASH liver (Fig. 2D-E).
3.5. Effect of LC on hepatic expression of PPARα, PPARγ, ADRP, and
AdipoR1 protein levels in NASH-HCC mice
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
122 | P a g e
I examined the effect of LC on the central regulator of lipid metabolism,
PPARα, and steatosis-related protein expression such as PPARγ, ADRP and
AdipoR1 in the liver of NASH mice. I observed that the hepatic expression of
PPARα was modestly lesser by 0.72-fold in NASH mice than the normal,
whereas, in the mouse of LC treated group, the reduced PPARα level was
significantly increased by 2.1-fold than the NASH mice (p<0.01).
Interestingly, I found that the hepatic expression of PPARα in LC group was
also significantly higher than the normal mice by 1.4-fold (p<0.05) (Fig. 3A).
The expression of PPARγ and ADRP (p<0.01) protein were increased in the
NASH liver by 1.9-fold and 3.2-fold respectively than the normal mice. In LC
treated mice, the increased levels of PPARγ was significantly prevented by
(p<0.01) and the level of ADRP was 0.77-fold lesser but did not reach a
significant level when compared to the NASH mice (Fig. 3B-C). On the other
hand, the expression of AdipoR1 was modestly decreased in the NASH group
by 0.72-fold than the normal liver, but in the mice of LC group it was slightly
higher (by 1.1-fold) than the NASH mice (Fig. 3D).
PPARα
β-actin
A
Normal NASH LC0.0
0.2
0.4
0.6
0.8
##
PP
AR/
GA
PD
H
PPARγ
GAPDH
B
Normal NASH LC0 .0 0
0 .0 2
0 .0 4
0 .0 6
0 .0 8
0 .1 0 *##
PP
AR
/ -a
cti
n
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
123 | P a g e
Fig. 3. Le Carbone attenuates hepatic lipogenesis in NASH-HCC mice.
Western blots show specific bands for hepatic (A), PPARα (for β-actin); (B),
PPARγ; (C), ADRP; and (D), AdipoR1 and the representative histograms
show the band densities with relative to GAPDH. Each bar represents mean
± SE. Normal, age matched normal mice; NASH, untreated NASH-HCC mice;
LC, NASH-HCC mice treated with Le Carbone suspension at 5
mg/mouse/day. Statistical analysis was carried out using One way ANOVA
followed by Tukey’s test where, *p<0.05, **p<0.01 vs Normal, ##p<0.01 vs
NASH.
3.6. Effect of LC on hepatic oxidative and anti-oxidative marker expression
in NASH-HCC mice
Since, oxidative stress plays important role in progression of NASH and
AMPK-SIRT1 signaling can repress ROS production [195], I checked some
protein expression levels by Western blot including anti-oxidative marker
proteins HO-1, Nrf2 and NAD(P)H oxidative subunit p47phox in all groups.
The hepatic protein expression of HO-1 (p<0.05) and Nrf2 were about 0.70-
Normal NASH LC0.00
0.05
0.10
0.15
0.20
**
*A
DR
P/G
AP
DH
ADRP
GAPDH
C
Normal NASH LC0 .0 0 0
0 .0 0 5
0 .0 1 0
0 .0 1 5
0 .0 2 0
Ad
ipo
R1
/GA
PD
H
AdipoR1
GAPDH
D
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
124 | P a g e
fold lower and p47phox was 1.9-fold higher (p<0.01) in NASH mice compared
to normal mice. But the protein expression level of HO-1 and Nrf2 were
significantly higher in LC group by 1.4-fold and 1.85-fold respectively than
the NASH group (p<0.05) (Fig. 4A & C). Interestingly, the increased p47phox
level was significantly prevented in LC group (p<0.01) than the NASH liver
(Fig. 4B).
Normal NASH LC0.00
0.01
0.02
0.03
0.04
*
#
HO
-1/G
AP
DH
HO-1
GAPDH
Normal NASH LC0.0
0.2
0.4
0.6
**
##
p47phox/G
AP
DH
GAPDH
p47phox
B
GAPDH
Nrf2
C
A
Normal NASH LC0.0
0.5
1.0
1.5
Nrf
2/G
AP
DH
#
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
125 | P a g e
Fig. 4. Effect of Le Carbone on hepatic HO-1, p47phox, Nrf2, p-JNK and p-
ERK1/2 expression in NASH-HCC mice
Representative Western blots show specific bands for (A), HO-1; (B), p47phox;
(C), Nrf2; (D), p-JNK (for JNK); (E), p-ERK1/2 (ERK1/2) and the
representative histograms show the band densities with relative to GAPDH.
Each bar represents mean ± SE. Normal, age matched normal mice; NASH,
untreated NASH-HCC mice; LC, NASH-HCC mice treated with Le Carbone
suspension at 5 mg/mouse/day. Statistical analysis was carried out using One
way ANOVA followed by Tukey’s test where, *p<0.05, **p<0.01 vs Normal,
#p<0.05, ##p<0.01 and ###p<0.001 vs NASH.
3.7. Effect of LC on the protein expression levels of p-JNK and p-ERK1/2 in
NASH-HCC liver
The phosphorylation of JNK, and ERK1/2 were markedly elevated in the liver
of NASH group by 1.5-fold, and 1.4-fold (p<0.01, p<0.05) respectively than the
Normal NASH LC0.000
0.005
0.010
0.015
0.020
**
#p-J
NK
/JN
K
JNK
p-JNK
D
Normal NASH LC0.00
0.02
0.04
0.06
0.08
0.10
###
*
p-E
RK
1/2
/ER
K1/2
ERK1/2
p-ERK1/2
E
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
126 | P a g e
normal group. While both of these protein expressions were markedly lesser
in LC group than that of the NASH group (p<0.05, p<0.001) (Fig. 4D-E).
Normal NASH LC0.00
0.02
0.04
0.06
0.08
0.10
*IL-1
0/G
AP
DH
GAPDH
IL-10
A
Normal NASH LC0.00
0.05
0.10
0.15
0.20
#
IL-1
/GA
PD
H
GAPDH
IL-1β
B
Normal NASH LC0.00
0.01
0.02
0.03
0.04
IL-6
/GA
PD
H
GAPDH
IL-6
C
Normal NASH C-20.00
0.02
0.04
0.06
0.08
*
#
Tim
p 4
/GA
PD
H
GAPDH
Timp4
D
Normal NASH LC0.0
0.1
0.2
0.3
0.4
Collag
en I
V/G
AP
DH
GAPDH
Collagen IV
E
Normal NASH LC0.000
0.002
0.004
0.006
0.008
#
MM
P-9
/GA
PD
H
GAPDH
MMP-9
F
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
127 | P a g e
Fig. 5. Effect of LC on the expression of inflammatory and fibrogenic proteins
in NASH-HCC liver
Western blots show specific bands for hepatic (A), IL-10; (B), IL-1β; (C), IL-6;
(D), Timp4; (E), collagen IV; and (F), MMP-9 and the representative
histograms show the band densities with relative GAPDH. Each bar
represents mean ± SE. Normal, age matched normal mice; NASH, untreated
NASH-HCC mice; LC, NASH-HCC mice treated with Le Carbone suspension
at 5 mg/mouse/day. Statistical analysis was carried out using One way
ANOVA followed by Tukey’s test where, *p<0.05 vs Normal, #p<0.05 vs
NASH.
3.8. Effect of LC on the expression of inflammatory and fibrogenic proteins
in NASH-HCC liver
I next investigated the protein expression of IL-10, IL-1β, IL-6, Timp4,
collagen IV and MMP-9 in NASH liver. The hepatic expression of IL-10 was
significantly lower where Timp4 was significantly and IL-1β, IL-6, collagen
IV, and MMP-9 were trended to increase in NASH group than the normal
group. Interestingly, the expression of IL-10, IL-6 and collagen IV were
trended to reverse and the protein expression of IL-1β, Timp4 and MMP-9
were significantly reduced in LC treated group (Fig. 5A-F). Along with these
data immunohistochemistry data showed that the CD36 positive cells were
significantly lower in the hepatic tissue of NASH group than the normal
group (p<0.01), but in LC treated group CD36 positive cells were trended to
higher than in the NASH hepatic tissue (Fig. 6A&B).
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
128 | P a g e
Fig. 6. Effect of LC on the expression of CD36 positive cells in NASH-HCC
liver
(A-B), Immunohistochemical staining of CD36 positive cells and their
quantitative data. Data are mean ± SE, (n= 6/ group). Normal, age matched
normal mice; NASH, untreated NASH-HCC mice; LC, NASH-HCC mice
treated with Le Carbone suspension at 5 mg/mouse/day. Statistical analysis
was carried out using One way ANOVA followed by Tukey’s test where,
**p<0.01 vs Normal.
CD
36
A
NASH Normal
Normal NASH LC0
10
20
30
40
50
**
Avera
ge n
um
ber
of
CD
36
posi
tive c
ells/
field
B
LC
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
129 | P a g e
3.9. Effect of LC on p-p53, p53, glypican-3 and prothrombin in NASH-HCC
liver
The hepatic protein expression of p53, glypican-3 and prothrombin were
elevated in NASH group (Fig. 7B-D) but the phosphorylation of p53 was
trended to decrease in NASH liver (Fig. 7A) when compared to that of normal
liver. The protein expression of p53, glypican-3 and prothrombin were
prevented in LC treated group, whereas the phosphorylation of p53 was
trended to increase in this group when compared to that of NASH group (Fig.
7A-D).
Normal NASH LC0.0
0.2
0.4
0.6
p-p
53/p
53
p53
p-p53
A
Normal NASH LC0.00
0.02
0.04
0.06
0.08
0.10
p53/G
AP
DH
GAPDH
p53
B
Normal NASH LC0.00
0.05
0.10
0.15
0.20
0.25
Gly
pic
an-3
/GA
PD
H
#*
Glypican-3
C
Normal NASH LC0.00
0.05
0.10
0.15
0.20
0.25
***
Pro
thro
mb
in /
-act
in
β-actin
Prothrombin
D
GAPDH
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
130 | P a g e
Fig. 7. Effect of LC on p-p53, p53, glypican-3 and prothrombin in NASH-HCC
liver
Western blots show specific bands for hepatic (A), p-p53 (for p53); (B), p53;
(C), Glypican-3; and (D), prothrombin (for β-actin); (E), and the
representative histograms show the band densities with relative GAPDH.
Each bar represents mean ± SE. Normal, age matched normal mice; NASH,
untreated NASH-HCC mice; LC, NASH-HCC mice treated with Le Carbone
suspension at 5 mg/mouse/day. Statistical analysis was carried out using One
way ANOVA followed by Tukey’s test where, *p<0.05, **p<0.01 vs Normal,
#p<0.05 vs NASH.
4. Discussion
In the present study, I have demonstrated that LC supplement plays an
important role to prevent or protect the liver damage by attenuating
steatohepatitis in NASH-HCC mice. Accordingly, in the liver of NASH mice,
LC suspension stimulated the AMPK-SIRT1 signaling system, activated
PPARα signaling, suppressed PPARγ expression, increased the rate of fatty
acid oxidation, reduced lipid synthesis, and attenuated liver fat deposition.
Furthermore, LC treatment reduced the oxidative stress, inflammation,
fibrosis, and the progression of NASH to HCC.
To gain insight into the pathogenesis and evaluate the treatment options,
mouse models of NASH-HCC are of utmost importance. There is a high
phenotypical variety in the available mouse models, however models that
truly display the full spectrum of histopathological and metabolic features
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
131 | P a g e
associated with human NASH are rare. Since NASH is considered as a
hepatic manifestation of the metabolic disorder and exceedingly connected
with diabetes, it is also suspected that there is a relationship amongst
diabetes and HCC with the progression of NASH [196]. In the present study,
I considered a novel NASH-HCC mouse model, which was developed by
injecting a low-dose STZ and feeding a HFD in mice, meet the
clinicopathological process from fatty liver, NASH, fibrosis to HCC under
diabetic background [11]. Some reports suggested that charcoal has the
ability to detoxify the fatty liver in obese and alcoholic subjects and can also
lower the concentration of total lipids, cholesterol and triglycerides in the
blood serum, liver, heart and brain [197], [64]. In this study, I investigated
the possible hepatoprotective mechanism of LC, a charcoal supplement in
this NASH-HCC mouse model.
It is well established that AMPK is the master regulator of energy
homeostasis, plays a concerning role in diabetes, metabolic syndrome,
cardiovascular diseases and cancers [198], [199]. Reduced AMPK activity
contributes to lipogenesis, thereby influencing steatosis and possibly
lipotoxicity, as well as hepatocellular injury and prolongation of liver
inflammation in steatohepatitis, thoroughly in the NASH-HCC mouse model.
AMPK and SIRT1 are evolutionary conserved partners, which have similar
functions in metabolism and cellular survival [200]. As energy metabolism is
critical in cancer development, AMPK/SIRT1 might be a promising target for
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
132 | P a g e
HCC treatment. It is also reported that, in clinical samples, the
phosphorylation of AMPK is lower in HCC tissues than the normal tissues
[201]. Along with these data, in my experiment, phosphorylation of AMPK
and the expression of SIRT1 were significantly lower in NASH liver than the
normal liver. LC treatment enhanced the p-AMPK and SIRT1 expressions in
LC group (Fig. 2A-B). Emerging evidence indicating that activation of the
AMPK pathway reduces the intracellular ROS to prevent cellular oxidative
stress damage triggered by different insult; hyperglycemia, fatty acids [195],
[202]. ROS also downregulate SIRT1 [203]. Many target proteins of AMPK
are so called- longevity factors such as, SIRT1, PGC1α, HO-1, and FOXOs,
which not only increase the stress resistance but also inhibit inflammatory
response [204]. The nutrient sensor SIRT1, also exerts the critical control of
gluconeogenic gene expression via deacetylation of PGC-1α [205]. It is shown
that the activation of PGC-1α in liver diminished triglyceride production and
secretion [206] and the rates of fatty acid oxidation is also diminished in
hepatocytes isolated from PGC-1α deficient mice [207]. Similarly, in my
study, I found that the protein expression of PGC-1α was significantly
diminished in NASH liver, but the LC treatment enhanced this expression
(Fig. 2D). In addition, the expression GLUT4 and PEPCK-C were trended to
reduce in this NASH group where trended to restore in LC treated group and
play a role in glucose utilization (Fig. 2C&E).
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
133 | P a g e
Moreover repression of AMPK induces the lipid accumulation in the
hepatocytes, decreases the ability of mitochondria to oxidize free fatty acids,
and subsequently increases the production of ROS ultimately results in the
progression of NAFLD to NASH [208]. Here, I found that the LC treatment
markedly enhanced the expression of PPARα in NASH liver, which is a
powerful fatty acid oxidation inducer involved in lipid breakdown (Fig. 3A).
Activation of PPARγ is associated with hepatic lipid accumulation in both
alcoholic and nonalcoholic fatty liver [209], [210]. There are some reports that
heterozygous PPARγ-deficient mice are protected from HFD or aging induced
adipocyte hypertrophy, obesity and insulin resistance [211], [212].
Interestingly, my present study showed that the hepatic protein expression of
PPARγ and ADRP were elevated in NASH mice, but significantly reduced in
the liver of LC treated mice (Fig. 3B-C). The expression of AdipoR1 was
reduced in NASH liver, but LC treatment did not show any effective role to
restore this expression (Fig. 3D). These data, might suggest that LC protects
the liver from steatosis other than non-adiponectin –mediated mechanisms.
These findings were corroborated by improved liver histology through
reduced macro-and micro-steatosis, and serum ALT, AST levels in LC treated
group (Fig. 1C, Table 1).
A large body of evidence has proved an undeniable contribution of oxidative
stress and proinflammatory cytokines to the advancement of steatosis to
steatohepatitis. In my study, I found the expression of NAD(P)H oxidase
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
134 | P a g e
subunit p47phox was significantly lesser in LC treated mice (Fig. 4B). Redox
systems, including antioxidants and phase ІІ detoxifying enzymes provide
protection against ROS actuated tissue damage. Nrf2 is a vital cytoprotective
transcriptional variable that induces the expression of several antioxidants
and phase ІІ detoxifying enzymes [156] and acts as a defense system in the
development of NASH [174]. Actually, besides Nrf2’s part in directing cellular
antioxidant guard, it likewise has anti-inflammatory capacities [156].
Moreover, HO-1 acts as an anti-oxidant marker and can also inhibit the
inflammatory response; some study reported that the expression of HO-1 is
upregulated in NASH [213]. In contrast, it is reported that the over
expression of HO-1 suppresses HCC progression by inhibiting the
proliferation and metastasis of HepG2 [214]. Here, I found that the markedly
higher expression of HO-1 and Nrf2 in the liver of the LC treated mice than
the liver of NASH-HCC mice (Fig. 4A, C). Oxidative stress and JNK-
activation progress, ERK1/2 activation become evident and enhance
inflammatory cytokine accumulation [215]. It is reported that the JNK, ERK
also act as oncogenic mediators in HCC [216]. Phosphorylation of JNK, ERK
were enhanced by HFD feeding in HCC progenitor cells derived tumors [216].
Consistent with these reports, in my study, I found the tumors protrusion in
NASH liver (Fig. 1A) and the expression of p-JNK, and p-ERK1/2 were also
elevated in NASH groups (Fig. 4D-E). Where the expressions of these
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
135 | P a g e
increased protein levels were markedly lower in LC treated group and no
tumor protrusion was found.
Inflammation seems to play a leading role in NASH progression as
recruitment of inflammatory macrophages occurs to create inflammatory foci
and to phagocyte lipid droplets [217]. Phagocytosis of macrophage decreased
later as seen in human NASH [218], indicating the altered function of
macrophages at a later phase of inflammation. Macrophages residing by
activated fibroblast and both participate in fibrotic foci in liver. Increased
hepatic IL-6 is suggested to contribute to accelerate macrophage fibroblast
interaction within Disse’s space in the liver [219]. Furthermore, in a pilot
study, increased levels of plasma IL-6 were found in NASH patients and it is
also reported that circulating IL-6 is strongly correlated with adverse
prognosis in HCC [220]. On macrophages CD36 involves in phagocytosis.
Studies showed that deletion of CD36 in mice increased MCP-1 in
hepatocytes, promotes macrophage migration to liver and aggravates hepatic
inflammatory response and fibrosis [221]. Where, IL-10 is the protective
cytokine against diet-induced liver inflammation. In my study, I observed
that liver inflammation and fibrosis were substantially increased in NASH
group. Here, I found that administration of LC in NASH-HCC mice displayed
attenuation of inflammation and fibrosis in liver by H&E and MT staining
(Fig. 1C-F), and enhanced the anti-inflammatory cytokine IL-10 (Fig. 5A) and
CD36 positive cells in the hepatic tissue (Fig. 6A-B) and suppressed the
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
136 | P a g e
hepatic protein expression of IL-1β, IL-6, Timp4, collagen IV and MMP-9
(Fig. 5B-F).
Oxidative stress and inflammatory pathways lead to the transformation of a
normal cell to a tumor cell, its survival, and proliferation. The p53 tumor
suppressor is a crucial regulator in response to various stress signals, such as
DNA damage, hypoxia and abnormal oncogenic events. Phosphorylation of
p53 is a key mechanism responsible for the activation of its tumor suppressor
functions in response to DNA damage [222], and p-p53 could trigger the
AMPK-dependent cell cycle arrest [223]. The tumor protruding may be
malignant or progress to HCC, and, as it has been reported that the hepatic
expression of glypican-3 [177] and DCP or prothrombin [178] are significantly
increased in most HCCs compared with benign liver lesions and normal liver,
they act as a useful prognostic tumor marker for HCC. In my study, I
observed that p53, glypican-3 and prothrombin, markers for HCC were
higher, but the phosphorylation of tumor suppressor p53 lower in the NASH
group compared to the normal group (Fig. 7A-D). But, all of these expressions
were altered in the LC treated group compared to NASH group.
In summary, my present study demonstrates that the preventive
action of LC against NASH-HCC in mice is mediated, at least in part,
through stimulating an important hepatic signaling system, the AMPK-
SIRT1 axis and consequently by reducing lipogenesis, oxidative stress and
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
137 | P a g e
inflammation in NASH liver. Furthermore, by inhibiting the progression of
NASH to HCC, LC might be useful in the management of NASH and may
constitute a powerful approach for the novel NASH-HCC model.
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CHAPTER FOUR
Comparison between diabetes and NASH
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
139 | P a g e
1. Introduction
NAFLD is a global health problem, affecting 6-45% of the general population,
rising up to 70% with type 2 DM and 90% in morbidly obese patients [224].
NASH is part of the spectrum of NAFLD that leads to progressive liver
disease, which can progress to cirrhosis and its associated complications,
including hepatic failure and HCC. In NASH patients, advanced fibrosis is
the major predictor of morbidity and liver-related mortality. High incidence
of perisinusoidal hepatic fibrosis is also reported in experimental type 1 DM,
while in humans perisinusoidal fibrosis often parallels with diabetic
microangiopathy [33]. Recent studies suggest that NAFLD may be more
common in type 1 DM and may serve as an independent risk marker [27],
although the attention given to NAFLD in type 2 DM [225]. It is also clearly
established that diabetes acts as an independent risk factor for HCC. Since
NASH is considered as a hepatic issue of metabolic syndrome and highly
associated with diabetes [226]. NASH and DM frequently coexist as they
share the pathogenic abnormalities within the liver, including hepatocyte
apoptosis, ER stress, lipotoxic mediators, oxidative stress and inflammations
are key contributors to hepatocellular injury. However, there is no direct
evidence that sequentially depicts the liver pathogenesis from type 1 diabetes
to NASH. To investigate the causal involvement of diabetes in NASH, I
considered a novel progressive NASH model in mice, which established under
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
140 | P a g e
diabetic condition [227]. In this study, I have shown the pathogenic
correlation between the experimental type 1 diabetic rat liver and the NASH
mouse liver.
2. Materials and methods
2.1. Materials
Unless otherwise stated, all reagents were of analytical grade and purchased
from Sigma (Tokyo, Japan).
2.2. Animals and Experimental design
All animals were treated in accordance with the guidelines for animal
experimentation of our institute [91]. Male Sprague-Dawley rats (weight 250-
300 g) were obtained from Charles River Japan Inc. (Kanagawa, Japan).
C57BL/6J mice were bred in my laboratory. Animals were housed in a
temperature of 23 ± 2 °C and humidity of 55 ± 15%, and were submitted to a
12 hour light/dark cycle, and allowed free access to standard laboratory chow
and tap water. Rats were allowed to fast for 4 hours and then induced
diabetes by a single intraperitoneal (i.p.) injection of freshly prepared
solution of STZ (Sigma-Aldrich, Inc. Saint Louis, MO, USA) at a dose of 55
mg/Kg, diluted in citrate buffer 20 mM (pH 4.5). Forty eight hours later,
blood glucose was measured by tail-vein sampling using Medi-safe chips
(Terumo Inc., Tokyo, Japan). Diabetes was defined as a morning blood
glucose reading of ≥ 300 mg/dL. Twelve rats were randomly divided into two
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
141 | P a g e
groups (n = 6/group): nondiabetic normal control group (Normal), and diabetic
rats (DM). NASH was induced in male mice by a single subcutaneous
injection of 200 µg STZ (Sigma, MO, USA) at 2 days after birth. They were
being started feeding with HFD32 (CELA Japan) ad libitum at the age of 4
weeks, and continued up to 14 weeks age of mice. Mice were randomly
selected into two groups (n=6/group): the normal group (Normal) were normal
mice subjected to the normal diet; and the NASH group (NASH) were STZ
injected mice subjected to the HFD32. All rats and mice were sacrificed at 11
and 14 weeks, respectively after the induction of STZ for analysis of liver
tissues.
2.3. Biochemical analysis
Fasting blood glucose was measured using G-checker (Sanko Junyaku,
Tokyo, Japan). Serum ALT, AST, ALP, TG and TC were measured by FUJI
DRI-CHEM 7000 (Fujifilm, Tokyo, Japan) as previously described [150].
2.4. Histological examination
Liver sections (4 µm) of different groups of animals were immediately fixed in
10% formaldehyde solution, embedded in paraffin, cut into several
transversal sections and mounted on glass slides. Then the liver tissue
sections were deparaffinized and stained with hematoxylin and eosin (H&E).
Morphological analysis was done by computerized image analysis system on
ten microscopic fields per section examined in a 40-fold magnification
(AmScope, USA), with the observer blind to the study group [150].
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
142 | P a g e
2.5. Western blot analysis
The frozen liver tissues were weighed and homogenized in an ice-cold buffer
(50 mM Tris-HCl, pH 7.4, 200mM NaCl, 20 mM NaF, 1 mM Na3VO4, 1 mM 2-
mercaptoethanol, 0.01 mg/mL leupeptin, 0.01 mg/mL aprotinin).
Homogenates were then centrifuged (3000 × g, 10 min, 4°C) and the
supernatants were collected and stored at -80°C. The total protein
concentration in samples was measured by the BCA method [150]. Samples
with equal amounts of protein were separated in polyacrylamide gel,
transferred and identified on nitrocellulose membrane with specific
antibodies followed by HRP-labelled secondary antibodies. The antibodies
applied: SREBP1c, PPARα, p-PERK, PERK, p-IRE1α, IRE1α, CHOP,
p47phox, CYP2E1, IL-1β, TLR4, VEGF-B, glypican-3, and β tubulin (Santa
Cruz Biotechnology, Santa Cruz, CA, USA), p-AMPKα, AMPKα, cl.caspase-
12, cl.caspase-3 and GAPDH (Cell Signaling Technology Inc., Beverly, MA,
USA), prothrombin (Abcam, Cambridge, UK). The levels of AMPKα (for p-
AMPKα), PERK (for p-PERK), IRE1α (for p-IRE1α), and GAPDH or β-tubulin
were estimated in every sample to check for equal loading of samples.
2.6. Statistical analysis
Data are shown as means ± SEM and were analyzed using unpaired t test. A
value of p< 0.05 was considered statistically significant. GraphPad Prism 5
software (San Diego, CA, USA) was used.
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
143 | P a g e
3. Results
3.1. Biochemical parameters in experimental animals
There was no huge difference in energy consumption and body weight
between the NASH mice and its respective normal mice. In case of DM rats
the energy consumption was significantly increased and body weight was
significantly decreased when compared to the normal rats (Table 1). The ratio
of liver weight to body weight (g/Kg) was significantly increased both in DM
and NASH groups when compared to their respective normal group (Table 1).
Fasting blood glucose level, serum TG, TC, ALT, ALP were also markedly
elevated in both groups DM and NASH group than their respective normal
group (Table 1). And serum AST level was trended to increase in DM group
but significantly increased in NASH group comparing to their normal group
(Table 1).
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
144 | P a g e
Table 1: Changes of biochemical parameters in DM rats and NASH mice.
SD Rats C57BL/6J mice
Normal group
(n=6)
DM group
(n=6)
Normal group
(n=6)
NASH group
(n=6)
Food intake /day (g) 22.71 ± 0.53 36.36 ± 0.89*** 4.0 ± 0.6 3.27 ±0.055
Energy consumption/day
(Kcal)
91.75 ± 2.14 146.89 ± 3.6*** 16.16 ± 2.5 16.59 ± 0.28
Body weight (BW) (g) 539.5 ± 38.48 316.33 ± 15.7*** 24.8 ± 2.7 20.7 ± 5.9
Liver weight (LW) /BW
(g/Kg)
28.57 ± 1.47 43.23 ± 1.35*** 40.1 ± 0.21 89.1 ± 1.5***
Blood glucose (mg/dL) 116.25 ± 22.1 761.8 ± 50.8*** 101.3 ± 23.4 488.0 ± 19.9***
Serum TG (mg/dL) 94.75 ± 5.72 431.25 ± 118.92** 17.5 ± 1.3 46.3 ± 12.1**
Serum TC (mg/dL) 58.50 ± 3.8 105.5 ± 10.9*** 81.8 ± 3.8 253.8 ± 18.9***
Serum ALT (IU/L) 39.75 ± 2.36 124 ± 28.82* 38.0 ± 9.0 304.3 ± 218.7*
Serum AST (IU/L) 141.33 ± 16.74 187.8 ± 99.28 72.4 ± 5.4 270.5 ± 84.4**
Serum ALP (IU/L) 180 ± 0.0 554.25 ± 351 322.8 ± 49.3 653.8 ± 141.9***
Normal, age matched normal rat or mice; DM, untreated type 1 diabetic rats; NASH,
untreated NASH-HCC mice; TG: triglyceride; TC: total cholesterol, ALT: alanine
aminotransferase, ALP: alkaline phosphatase, AST: aspartate aminotransferase, NAFLD:
non-alcoholic fatty liver disease. Values are expressed as means ± SEM. Statistical
analysis was carried out using unpaired two t -test where, *p <0.05, **p<0.01, ***p <0.001
vs respective normal group.
3.2. Histopathological findings
Normal histology was found in the normal rats and mice liver (Figure 1).
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
145 | P a g e
H&E staining showed extreme steatosis, enlarged hepatocytes, scattered
lobular inflammatory cells in NASH mice. Microvesicular vacuolization, focal
necrosis and inflammation in the portal area were also found in the liver of
DM rat but in lesser extent than the NASH mice (Figure 1).
Figure 1. Histopathological changes. Histological staining with H&E in liver
(A) shows light microscopic photograph of normal rat and DM rat; (B) normal
mice and NASH mice.
3.3. AMPKα expression in the liver of experimental animals
The phosphorylation of AMPKα in the diabetic liver was trended to reduce
while in NASH liver it was significantly reduced (Figure 2A-B).
A
B
20 µm
Normal DM
H&E Staining
20 µm
20 µm 20 µm
SD
Rat
C5
7B
L/6
J
mou
se
Normal NASH
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
146 | P a g e
Figure 2: Hepatic expression of p-AMPKα in DM and NASH liver. Western
blots show specific band for hepatic (A) p-AMPKα for SD rats and (B) p-
AMPKα for mice; and the representative histograms show the band densities
with relative to that AMPKα. Each bar represents mean ± SE. Normal, age
matched normal rat or mice; DM, STZ injected type 1 diabetic rats; NASH,
STZ injected mice subjected to HFD. Statistical analysis was carried out
using unpaired t test where, *p<0.05 vs respective normal group.
3.4. Hepatic lipogenesis in experimental animals
The hepatic protein expression of lipogenic controllers including SREBP1c
was significantly increased both in DM and NASH group than their
respective normal group (Figure 3A & B). But expression of PPARα was
Normal DM0.0
0.1
0.2
0.3
0.4
SD rats
p-A
MP
K
/AM
PK
p-AMPKα
AMPKα
A
Normal NASH0.0
0.2
0.4
0.6
0.8
1.0
*
p-A
MP
K
/AM
PK
C57BL/6J mice
p-AMPKα
AMPKα
B
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
147 | P a g e
significantly reduced in DM liver while trended to reduce in NASH liver
(Figure 3C &D).
Figure 3: Hepatic lipogenesis in DM and NASH liver. Western blots show
specific band for hepatic (A) SREBP1c (for β-Tubulin) for SD rats and (B)
SREBP1 in mice; (C) PPARα for SD rats c and (D) PPARα for mice and the
representative histograms show the band densities with relative to that
GAPDH. Each bar represents mean ± SE. Normal, age matched normal rat or
mice; DM, STZ injected type 1 diabetic rats; NASH, STZ injected mice
subjected to HFD. Statistical analysis was carried out using unpaired t test
where, *p<0.05, **p<0.01 vs respective normal group.
Normal NASH 0.00
0.05
0.10
0.15
0.20
0.25**C57BL/6J mice
SR
EB
P1c/G
AP
DH
SREBP1c
GAPDH
Normal DM 0
1
2
3
4
5**
SR
EB
P 1
c/
-Tubulin
SD Rats
SREBP 1c
β-Tubulin
A B
Normal DM0.0
0.2
0.4
0.6
0.8
*
SD Rats
PP
AR
/GA
PD
H
PPARα
GAPDH
Normal NASH 0.00
0.01
0.02
0.03
0.04 C57BL/6J mice
PP
AR
/GA
PD
H
PPARα
GAPDH
D C
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
148 | P a g e
3.5. Expression of UPR signaling proteins
Diabetic rats and NASH mice both have displayed a significant upregulation
in the hepatocytes expression levels of p-PERK (p<0.01) compared with their
respective normal liver (Figure 4A-B). The phosphorylation IRE1α (p<0.05)
was also significantly increased in DM liver (p<0.001) and NASH liver
(p<0.05) when compared to their respective normal group (Figure 4C-D).
Figure 4: Hepatic UPR signaling protein expression in DM and NASH liver.
Western blots show specific band for hepatic (A), p-PERK (PERK) for SD rats;
Normal DM0.0
0.1
0.2
0.3***SD Rats
p-I
RE
1
/IR
E1
PERK
p-PERK
Normal DM0.00
0.05
0.10
0.15
**
SD Rats
p-P
ER
K/P
ER
K
p-IRE1α
IRE1α
A B
Normal NASH 0.0
0.5
1.0
1.5
2.0
2.5
*
p-I
RE
1
/IR
E1
C57BL/6J mice
IRE1α
p-IRE1α
Normal NASH 0.00
0.05
0.10
0.15
**
C57BL/6J mice
p-P
ER
K/P
ER
K
p-PERK
PERK
C D
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
149 | P a g e
(B) p-PERK (PERK) for mice; (C) p-IRE1α (for IRE1α) for SD rats and (D) p-
IRE1α (for IRE1α) for mice and the representative histograms show the band
densities. Each bar represents mean ± SE. Normal, age matched normal rat
or mice; DM, STZ injected type 1 diabetic rats; NASH, STZ injected mice
subjected to HFD. Statistical analysis was carried out using unpaired t test
where, *p<0.05, **p<0.01, ***p<0.001 vs respective normal group.
3.6. Expression of apoptotic markers in experimental animals
Hepatic protein expression of cleaved caspase-12, cleaved caspase-3 and
CHOP were significantly increased in the liver of diabetic rats (non-
significant, p<0.01, p<0.001) and the NASH mice (p<0.01, p<0.01, p<0.05)
when compared to their representative normal groups (Figure 5A-F).
β-Tubulin
Cl.caspase-12
Normal DM0.0
0.1
0.2
0.3
0.4
**
Cl. c
aspa
se
-3/
-Tubulin SD Rats
Cl. caspase-3
β-Tubulin
A B
C
Normal NASH 0.00
0.05
0.10
0.15
##
**C57BL/6J mice
Cl.C
asp
ase
12/G
AP
DH
GAPDH
Cl. Caspase 12
D
GAPDH
Cl. Caspase 3
Normal NASH 0.00
0.02
0.04
0.06
0.08
**
C57BL/6J mice
Cl. C
asp
ase
3/G
AP
DH
Normal DM0
2
4
6
8SD Rats
Cle
aved
cas
pase
-12/
-Tub
ulin
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
150 | P a g e
Figure 5: Hepatic apoptotic protein expression in DM and NASH liver.
Western blots show specific band for hepatic (A-B), cl.caspase-12 for SD rats
and mice; (C-D) cl.caspase-3 for SD rats and mice; and (E-F) CHOP in SD
rats and mice (for Lamin A) and the representative histograms show the
band densities with relative to that β-Tubulin or GAPDH. Each bar
represents mean ± SE. Normal, age matched normal rat or mice; DM, STZ
injected type 1 diabetic rats; NASH, STZ injected mice subjected to HFD.
Statistical analysis was carried out using unpaired t test where, *p<0.05,
**p<0.01, ***p<0.001 vs respective normal group.
3.7. Oxidative stress in experimental animals
The hepatic expression of NAD(P)H oxidative subunits p47phox and CYP2E1
were significantly increased both in DM (p<0.05) and NASH (p<0.05, p<0.01)
group when compare to their respective normal group (Figure 6A-D).
CHOP
β-Tubulin
CHOP
Lamin A
E F
Normal NASH 0.000
0.005
0.010
0.015
0.020
0.025
*C57BL/6J mice
CH
OP
/Lam
in A
Normal DM0.0
0.5
1.0
1.5
2.0
2.5 ***SD Rats
CH
OP
/ -T
ubulin
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
151 | P a g e
Figure 6: Hepatic oxidative stress in DM and NASH liver. Western blots
show specific band for hepatic (A-B) p47phox for SD rats and mice; (C-D)
CYP2E1 for SD rats and mice and the representative histograms show the
band densities with relative to that GAPDH. Each bar represents mean ± SE.
Normal, age matched normal rat or mice; DM, STZ injected type 1 diabetic
rats; NASH, STZ injected mice subjected to HFD. Statistical analysis was
carried out using unpaired t test where, *p<0.05, **p<0.01 vs respective
normal group.
3.8. Hepatic inflammation in experimental animals
Inflammation is involved in metabolism related diseases including, diabetes
and NASH. Here, I found the protein expression of pro-inflammatory
Normal DM0.0
0.5
1.0
1.5
2.0
2.5
*
SD Rats
p47phox/G
AP
DH
p47phox
GAPDH
Normal NASH0.0
0.2
0.4
0.6
*
p47phox/G
AP
DH
C57BL/6J
GAPDH
p47phox
C
A
Normal DM0.0
0.5
1.0
1.5
*
SD Rats
CY
P2E
1/G
AP
DH
CYP2E1
GAPDH
Normal NASH0.00
0.05
0.10
0.15
0.20
**C57BL/6J mice
CY
P2E
1/G
AP
DH
GAPDH
CYP2E1
D
B
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
152 | P a g e
cytokine IL-1β, and inflammatory cascade TLR4 were significantly increased
both in DM and NASH liver when compared with their respective normal
liver (Figure 7A-D).
Figure 7: Hepatic inflammation in DM and NASH liver. Western blots
show specific band for hepatic (A-B), IL-1β for SD rats and mice and (C-D),
TLR4 for SD rats and mice; and the representative histograms show the
band densities with relative to that β-Tubulin or GAPDH. Each bar
represents mean ± SE. Normal, age matched normal rat or mice; DM, STZ
injected type 1 diabetic rats; NASH, STZ injected mice subjected to HFD.
Normal DM0.0
0.5
1.0
1.5
2.0
2.5***
IL-1
/ -T
ubulin
SD Rats
β-Tubulin
IL-1β
Normal NASH 0.00
0.02
0.04
0.06 *
IL-1
/GA
PD
H
C57BL/6J mice
GAPDH
IL-1β
β-Tubulin
TLR4
β-Tubulin
TLR4
A B
C D
Normal DM0
5
10
15
20**SD Rats
TL
R4/
-Tubulin
Normal NASH 0
2
4
6**C57BL/6J mice
TL
R 4
/ -T
ubulin
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
153 | P a g e
Statistical analysis was carried out using unpaired t test where, *p<0.05,
**p<0.01, ***p<0.001 vs respective normal group.
3.9. Hepatic protein expression of VEGF-B, glypican-3 and prothrombin
in experimental animals
I investigated the hepatic protein expression level of VEGF-B, glypican-3,
and prothrombin in all groups, I found that these expression were
significantly increased both in DM and NASH groups than their respective
group (Figure 8A-B, D-F), except the glypican-3, was trended to increase in
DM group (Figure 8C).
Normal DM0
1
2
3
4
5
**SD Rats
VE
GF
-B/G
AP
DH
VEGF-B
GAPDH
SD Rats
Normal DM0.0
0.2
0.4
0.6
0.8
1.0
Gly
pica
n-3/
GA
PD
H
Glypican-3
GAPDH
A B
C
Normal NASH 0.00
0.05
0.10
0.15
0.20
**
C57BL/6J mice
VE
GF
-B/G
AP
DH
GAPDH
VEGF-B
D
Normal NASH0.0
0.1
0.2
0.3
**C57BL/6J mice
Gly
pic
a-3/G
AP
DH
GAPDH
Glypican-3
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
154 | P a g e
Figure 8: Hepatic VEGF-B, glypican-3, and prothrombin expression in DM
and NASH liver. Western blots show specific band for hepatic (A-B),
VEGF-B for SD rats and mice; (C-D), glypican-3 for SD rats and mice ,
and (E-F), prothrombin for SD rats and mice and the representative
histograms show the band densities with relative to that GAPDH. Each
bar represents mean ± SE. Normal, age matched normal rat or mice; DM,
STZ injected type 1 diabetic rats; NASH, STZ injected mice subjected to
HFD. Statistical analysis was carried out using unpaired t test where,
*p<0.05, **p<0.01 vs respective normal group.
4. Discussion
In the present study, I have demonstrated that the correlative effect of
DM and NASH on liver. Since diabetes and NASH both act as the
critical risk factor for HCC; the third common cause of cancer related
death. Although, NASH is a cause of serious health issue, there is no
effective treatment as well as diagnostic marker at present [228]. To
Normal DM0
5
10
15
**SD Rats
Pro
thro
mbin
/GA
PC
DH
Prothrombin
GAPDH
Normal NASH 0
2
4
6
*
Pro
thro
mbin
/GA
PD
H
C57BL/6J mice
GAPDH
Prothrombin
E F
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
155 | P a g e
investigate the pathogenesis and finding the treatment, suitable
animal model is the most important.
In this study, I found the similar pathogenic abnormalities in the liver of both
DM and NASH animals. In NASH, first chemical intervention induced mild
inflammation, a key pathogenesis of diabetes and metabolic disorders [229].
As β-cell replication and neogenesis are actively found early after birth [230],
low-dose of STZ at this time causing of both islets islet injury and
regenerative responses, leading to diabetic conditions. Continuation of
dietary intervention enhances fat deposition in the liver with increased
lipogenesis and fatty acid oxidation, leading to the hepatocellular injury.
Accordingly, in H&E staining, I found the severe fat deposition in NASH liver
when compared to the DM liver and both livers showed the inflammation and
cellular hypertrophy (Figure 1). Fat accumulation is the prerequisite for the
development of NASH. AMPK is well established for the lipid metabolism
and found the decreased levels of p-AMPKα both in DM and NASH liver
(Figure 2A-B). The extent of lipogenesis marker expression including,
SREBP1c and serum lipid profile were significantly elevated in both DM and
NASH group compared to their respective normal liver (Figure 3A-B, Table
1). While the expression fatty acid regulatory gene significantly reduced in
DM liver and slightly in NASH liver (Figure 3C-D). The serum ALT and AST
level indicator of liver damage markedly elevated in NASH group but in DM
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
156 | P a g e
group AST level was trended to increase but non-significantly while ALT was
increased significantly when compared to its respective normal group (Table
1). Transition of steatosis to steatohepatitis is the crucial event in NASH
pathophysiology, although it is still unclear. Several studies suggested that
lipotoxicity play important role in this action, which associated with ER
stress or prolonged UPR stress, oxidative stress and inflammation [53]. In
this study, it is found that the significant elevation of phosphorylated PERK
and IRE1α in both DM and NASH liver (Figure 4). Prolonged UPR stress
leads to hepatocyte apoptosis, and generation of apoptotic bodies has been
recognized as a potent inflammatory and fibrogenic stimulus [231], direct
link between apoptosis and NASH has been demonstrated by a number of
experimental studies. Several pathways lead to lipotoxic apoptosis, here
significant elevation of hepatic cleaved caspase 12, cleaved caspase 3 and ER
stress induced apoptotic protein CHOP were found in both groups DM and
NASH (Figure 5), while in diabetic rats the elevation of cleaved caspase 12
did not reach a significant level (Figure 5A). Oxidative stress has been
considered one of the one of the responsible pathway for the progression of
steatosis to steatohepatitis [232]. Oxidative subunit p47phox and oxidative
marker CYP2E1 were significantly increased both in DM and NASH liver
(Figure 6). CYP2E1 promotes oxidative stress and inflammation, resulting in
hepatocyte injury and progression to NASH [40]. Inflammation represents a
crucial aspect in NASH pathogenesis. Excess of toxic lipids causes cellular
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
157 | P a g e
stress which triggers the hepatocyte apoptosis, the prevailing mechanism of
cell death in NASH, associated with the degree of liver inflammation [41].
The protein expression pro-inflammatory cytokine IL-1β and inflammatory
marker protein TLR4 were significantly elevated in the liver of DM rat and
NASH when compared with their respective normal group (Figure 7).
Oxidative stress and inflammation pathways lead to the switch of a normal
cell to a tumor cell, its survival and proliferation [175]. Since diabetes and
NASH both are risk factor for the HCC, here I checked the protein expression
of VEGF-B, glypican-3 and prothrombin, while VEGF may help to regulate
hepatic tumorigenesis [176], glypican-3 [177] and prothrombin [178] are
elevated in most HCC liver. The expression of VEGF-B and prothrombin
were significantly elevated in both groups, DM and NASH with their
respective normal group (Figure 8A-B, E-F) while, the gypican-3 was
significantly elevated in NASH group but in DM it was very slightly
increased than its respective normal group (Figure 8C-D). From the other
studies and my data, it is confirmed that glypican-3 is a potential liver cancer
therapeutic target as it is over-expressed in HCC but not expressed or
expressed at a low levels in normal and DM liver tissue.
In conclusion, my data indicate that diabetes is closely linked with the NASH
and its pathophysiology. Thus, type 1 diabetic liver and the STZ-induced
NASH liver are showing the similar pathogenic abnormalities, this novel
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
158 | P a g e
NASH model is comprised under diabetic background. This an open a new
resource to understand the mechanism and novel therapeutic strategies
against NASH.
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
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SUMMMARY
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
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SUMMARY
From my study and others it has been speculated that NASH will overtake
HCV/HBV as the most common associated risk factor for HCC in the next
decade due to the prevalence of diabetes and NAFLD. Because an increasing
number of patients are being diagnosed with NASH, a more comprehensive
understanding of this disease mechanism is critical to developing new clinical
strategies for early detection, treatment, and even prevention.
Hyperglycemia, hyperlipidemia, and inflammation are the main metabolic
abnormalities in diabetes, which are able to stimulate generation of ROS. All
of these metabolic abnormalities along with ER stress are closely associated
with the building of steatosis in liver and lipotoxicity in steatohepatitis.
Oxidative stress and pro-inflammatory cytokines progress the NASH to HCC
in some cases. Inflammatory cascade NF-κB-HMGB1 signaling are
attractively involved in the pathogenesis of NASH and its progression to
HCC. Where, AMPK is well established for the metabolic activity and
inhibition of the phosphorylation AMPK also associated with the
carcinogenesis.
Curcumin regulates blood glucose and antioxidant. Curcumin treatment
protects the liver against ER stress and apoptosis in experimental type 1
diabetes. It ameliorates liver damage and reduce the progression of NASH to
HCC through modulating HMGB1-NF-κB translocation in experimental
NASH-HCC under diabetic condition. Where, Le Carbone improves the
metabolic abnormalities and through activating AMPK and SIRT1 in the
NASH-HCC liver. Le Carbone attenuates oxidative stress and inflammation
along with preventing the lipogenesis in the experimental NASH-HCC liver.
In conclusion, curcumin and Le Carbone might be useful in the management
of NASH may constitute a powerful approach in treatment and prevention.
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List of Publications as first author
1. Mst. Rejina Afrin, Somasundaram Arumugam, Md. Azizur Rahman,
Mir Imam Ibne Wahed, Vengadeshprabhu Karuppagounder, Meilei
Harima, Hiroshi Suzuki,, Shizuka Miyashita, Kenji Suzuki, Hiroyuki
Yoneyama, Kazuyuki Ueno, Kenichi Watanabe. Curcumin ameliorates
liver damage and progression of NASH in NASH-HCC mouse model
possibly by modulating HMGB1-NF-κB translocation. International
Immunopharmacology, 2017; 44:174-182.
2. Mst. Rejina Afrin, Somasundaram Arumugam, Md. Azizur Rahman,
Vengadeshprabhu Karuppagounder, Remya Sreedhar, Meilei Harima,
Hiroshi Suzuki, Takashi Nakamura, Shizuka Miyashita, Kenji Suzuki,
Kazuyuki Ueno and Kenichi Watanabe. Le Carbone, a charcoal
supplement, modulates DSS-induced acute colitis in mice through
activation of AMPKα and downregulation of STAT3 and caspase 3
dependent apoptotic pathways. International Immunopharmacology,
2017;43:70-78.
3. Mst. Rejina Afrin, Somasundaram Arumugam, Mir Imam Ibne
Wahed, Vigneshwaran Pitchaimani, Vengadeshprabhu
Karuppagounder, Remya Sreedhar, Meilei Harima, Hiroshi
Suzuki, Shizuka Miyashita, Takashi Nakamura, Kenji
Suzuki, Masahiko Nakamura, Kazuyuki Ueno, and Kenichi Watanabe.
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
193 | P a g e
Attenuation of Endoplasmic Reticulum Stress-Mediated Liver Damage
by Mulberry Leaf Diet in Streptozotocin-Induced Diabetic Rats. The
American Journal of Chinese Medicine, January 2016; 44(1):87-101.
4. Mst. Rejina Afrin, Somasundaram Arumugam, Vivian Soetikno,
Rajarajan A Thandavarayan , Vigneshwaran Pitchaimani,
Vengadeshprabhu Karuppagounder, Remya Sreedhar, Meilei Harima,
Hiroshi Suzuki , Shizuka Miyashita, Mayumi Nomoto, Kenji Suzuki,
Kenichi Watanabe. Curcumin ameliorates streptozotocin induced liver
damage through modulation of endoplasmic reticulum stress mediated
apoptosis in diabetic rats. Free Radical Research 2015; 49(3):279-89.
List of publications as co-author
1. Vigneshwaran Pitchaimani , Somasundaram Arumugam, Rajarajan A
Thandavarayan, Vengadeshprabhu Karuppagounder, Mst. Rejina
Afrin , Remya Sreedhar, Meilei Harima, Hiroshi Suzuki, Shizuka
Miyashita, Takashi Nakamura, Kenji Suzuki, Masahiko Nakamura,
Kazuyuki Ueno, and Kenichi Watanabe. Fasting time duration
modulates the onset of insulin-induced hypoglycemic seizures in mice.
Epilepsy Research, September 2016; 125:47-51.
2. Vengadeshprabhu Karuppagounder, Somasundaram Arumugam ,
Rajarajan A Thandavarayan, Remya Sreedhar, Vijayasree V
Giridharan, Rejina Afrin , Meilei Harima, Miyashita Suzuki, Hara M,
Kenji Suzuki, Masahiko Nakamura, Kazuyuki Ueno, Kenichi
Watanabe. Curcumin alleviates renal dysfunction and suppresses
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
194 | P a g e
inflammation by shifting from M1 to M2 macrophage polarization in
daunorubicin induced nephrotoxicity in rats. Cytokine, August 2016;
84: 1-9.
3. Vigneshwaran Pitchaimani, Somasundaram Arumugam, Rajarajan A
Thandavarayan, Vengadeshprabhu Karuppagounder, Mst. Rejina
Afrin , Remya Sreedhar, Meilei Harima, Hiroshi Suzuki, Shizuka
Miyashita, Kenji Suzuki, Masahiko Nakamura, Kazuyuki Ueno, and
Kenichi Watanabe. Hypothalamic glucagon signaling in fasting
hypoglycemia. Life Sciences, May 2016; 153:118-23.
4. Remya Sreedhar, Somasundaram Arumugam, Vengadeshprabhu
Karuppagounder, Rajarajan A Thandavarayan, Vijayasree V
Giridharan, Vigneshwaran Pitchaimani, Mst. Rejina Afrin, Meilei
Harima, Takashi Nakamura, Masahiko Nakamura, Kenji Suzuki ,
Kenichi Watanabe. Jumihaidokuto effectively inhibits colon
inflammation and apoptosis in mice with acute colitis. International
Immunopharmacology 2015; 29(2):957-63.
5. Remya Sreedhar, Somasundaram Arumugam, Rajarajan A
Thandavarayan, Vijayasree V Giridharan, Vengadeshprabhu
Karuppagounder, Vigneshwaran Pitchaimani, Rejina Afrin, Meilei
Harima, Takashi Nakamura, Kazuyuki Ueno, Masahiko Nakamura,
Kenji Suzuki , Kenichi Watanabe. Toki-shakuyaku-san, a Japanese
kampo medicine, reduces colon inflammation in a mouse model of
acute colitis. International Immunopharmacology 2015; 29(2):869-75.
6. Remya Sreedhar, Somasundaram Arumugam , Rajarajan A
Thandavarayan, Vijayasree V Giridharan, Vengadeshprabhu
Karuppagounder, Vigneshwaran Pitchaimani, Rejina Afrin, Meilei
Harima, Masahiko Nakamura, Kenji Suzuki, Narasimman
Gurusamy, Prasanna Krishnamurthy, Kenichi Watanabe. Depletion of
cardiac 14-3-3η protein adversely influences pathologic cardiac
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
195 | P a g e
remodeling during myocardial infraction after coronary artery ligation
in mice. International Journal of Cardiology 2015; 202:146-53.
7. Vengadeshprabhu Karuppagounder, Somasundaram Arumugam,
Rajarajan A Thandavarayan, Vigneshwaran Pitchaimani, Remya
Sreedhar, Rejina Afrin, Meilei Harima, Hiroshi Suzuki, Kenji Suzuki,
Masahiko Nakamura, Kazuyuki Ueno, Kenichi Watanabe. Naringinin
ameliorates daunorubicin induced nephrotoxicity by mitigating AT1R,
ERK1/2-NFκB p65 mediated inflammation. International
Immunopharmacology 2015; 28(1):154-9.
8. Vengadeshprabhu Karuppagounder, Somasundaram Arumugam,
Rajarajan A Thandavarayan, Vigneshwaran Pitchaimani, Remya
Sreedhar, Rejina Afrin, Meilei Harima, Hiroshi Suzuki, Mayumi
Nomoto, Shizuka Miyashita, Kenji Suzuki, Masahiko Nakamura,
Kazuyuki Ueno, Kenichi Watanabe. Tannic acid modulates NFκB
signaling pathway and skin inflammation in NC/Nga mice through
PPARγ expression. Cytokine 2015; 76(2): 206-13.
9. Kenichi Watanabe, Vengadeshprabhu Karuppagounder,
Somasundaram Arumugam, Rajarajan A Thandavarayan,
Vigneshwaran Pitchaimani, Remya Sreedhar, Rejina Afrin, Meilei
Harima, Hiroshi Suzuki, Kenji Suzuki, Takashi Nakamura, Mayumi
Nomoto, Shizuka Miyashita, Kyoko Fukumoto, Kazuyuki Ueno. Purni
cortex ameliorates skin inflammation possibly through HMGB1-NFκB
pathway in house dust mite induced atopic dermatitis NC/Nga
transgenic mice. Journal of Clinical Biochemistry and Nutrition 2015;
5(3):186-94.
10. Meilei Harima, Somasundaram Arumugam, Juan Wen, Vigneshwaran
Pitchaimani, Vengadeshprabhu Karuppagounder, Remya Sreedhar,
Mst. Rejina Afrin, Shizuka Miyashita, Mayumi Nomoto, Kazuyuki
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
196 | P a g e
Ueno, Masahiko Nakamura, Kenichi Watanabe. Effect of carvedilol
against myocardial injury due to ischemia-reperfusion of the brain in
rats. Experimental and Molecular Pathology 2015; 98(3):558-62.
11. Somasundaram Arumugam, Remya Sreedhar, Rajarajan A
Thandavarayan, Vijayasree V Giridharan, Vengadeshprabhu
Karuppagounder, Vigneshwaran Pitchaimani, Mst. Rejina Afrin,
Shizuka Miyashita, Mayumi Nomoto, Meilei Harima , Takashi
Nakamura, Masahiko Nakamura, Kenji Suzuki , Kenichi Watanabe.
Telmisartan treatment targets inflammatory cytokines to suppress the
pathogenesis of acute colitis induced by dextran sulphate sodium.
Cytokine 2015; 74(2):305-12.
12. Vengadeshprabhu Karuppagounder, Somasundaram Arumugam,
Rajarajan A Thandavarayan, Vigneshwaran Pitchaimani, Remya
Sreedhar, Rejina Afrin, Meilei Harima, Hiroshi Suzuki, Mayumi
Nomoto, Shizuka Miyashita, Kenji Suzuki, Masahiko Nakamura,
Kenichi Watanabe. Modulation of HMGB1 translocation and
RAGE/NFκB cascade by quercetin treatment mitigates atopic
dermatitis in NC/Nga transgenic mice. Experimental Dermatology
2015; 24(6):418-23.
13. Remya Sreedhar, Somasundaram Arumugam , Rajarajan A
Thandavarayan , Vijayasree V Giridharan, Vengadeshprabhu
Karuppagounder, Vigneshwaran Pitchaimani, Rejina Afrin , Shizuka
Miyashita, Mayumi Nomoto, Meilei Harima , Narasimman Gurusamy,
Kenji Suzuki , Kenichi Watanabe. Myocardial 14-3-3η protein protects
against mitochondria mediated apoptosis. Cellular Signalling 2015;
27(4):770-6.
14. Vengadeshprabhu Karuppagounder, Somasundaram Arumugam,
Rajarajan A Thandavarayan, Vigneshwaran Pitchaimani, Remya
Sreedhar, Rejina Afrin, Meilei Harima, Hiroshi Suzuki, Mayumi
Non-alcoholic steatohepatitis onset of mechanisms under diabetic background and treatment strategies
197 | P a g e
Nomoto, Shizuka Miyashita, Kenji Suzuki, Kenichi Watanabe.
Resveratrol attenuates HMGB1 signaling and inflammation in house
dust mite-induced atopic dermatitis in mice. International
Immunopharmacology 2014; 23(2): 617-623.
15. Somasundaram Arumugam, Remya Sreedhar, Vengadeshprabhu
Karuppagounder, Rajarajan A Thandavarayan, Vijayasree V.
Giridharan, Vigneshwaran Pitchaimani, Rejina Afrin, Meilei Harima,
Kenji Suzuki, Kenichi Watanabe. Comparative evaluation of
torasemide and furosemide on rats with streptozotocin-induced
diabetic nephropathy. Experimental and Molecular Pathology 2014;
97(1).
16. Vigneshwaran Pitchaimani, Somasundaram Arumugam, Rajarajan A
Thandavarayan, Vengadeshprabhu Karuppagounder, Remya
Sreedhar, Rejina Afrin, Meilei Harima, Hiroshi Suzuki, Shizuka
Miyashita, Mayumi Nomoto, Hirohito Sone, Kenji Suzuki , Kenichi
Watanabe. Fasting mediated increase in p-BAD (ser155) and p-AKT
(ser473) in the prefrontal cortex of mice. Neuroscience Letters 2014;
579.
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198 | P a g e
Award
Awarded Japan Government Scholarship (Monbukagakusho) for the entire
course of Doctor of Philosophy (PhD), from October 2013 to March 2018.