non-alcoholic steatohepatitis onset of mechanisms under … · 2018-02-02 · non-alcoholic fatty...

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

2 | P a g e

DEDICATED TO

My beloved parents, brother, husband, respectable

teachers and specially to my supervisor


3 | P a g e

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


4 | P a g e

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


5 | P a g e

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


6 | P a g e

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.


7 | P a g e

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;


8 | P a g e

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;


9 | P a g e

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;


10 | P a g e

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;


11 | P a g e

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


12 | P a g e

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


13 | P a g e

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


14 | P a g e

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.


15 | P a g e

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


16 | P a g e

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


17 | P a g e

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.


18 | P a g e

INTRODUCTION


19 | P a g e

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].


20 | P a g e

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


21 | P a g e

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].


22 | P a g e

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


23 | P a g e

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.


24 | P a g e

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


25 | P a g e

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.


26 | P a g e

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


27 | P a g e

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


28 | P a g e

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


29 | P a g e

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


30 | P a g e

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].


31 | P a g e

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


32 | P a g e

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).


33 | P a g e

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.


34 | P a g e

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


35 | P a g e

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.


36 | P a g e

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


37 | P a g e

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


38 | P a g e

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


39 | P a g e

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.


40 | P a g e

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


41 | P a g e

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.


42 | P a g e

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].


43 | P a g e

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].


44 | P a g e

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


45 | P a g e

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


46 | P a g e

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].


47 | P a g e

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.


48 | P a g e

SCOPE OF STUDY


49 | P a g e

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.


50 | P a g e

CHAPTER ONE

Curcumin Ameliorates Streptozotocin Induced Liver

Damage through Modulation of Endoplasmic Reticulum

Stress Mediated Apoptosis in Diabetic Rats


51 | P a g e

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


52 | P a g e

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


53 | P a g e

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


54 | P a g e

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


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


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


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


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)


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).


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


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


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


0.1

0.2

0.3***

#*

p-I

RE

1

/t-

IRE

1

B


0.05

0.10

0.15

*#

p-P

ER

K/P

ER

K

p-IRE1α

t-IRE1α PERK

p-PERK


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



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


1

2

3

4

AT

F6

/ T

ubulin

β Tubulin

ATF6α


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).


1

2

3

###

***

TR

AF

2/

Tubulin

TRAF2

β Tubulin

B


0.5

1.0

1.5

2.0

2.5 ***

##

CH

OP

/ T

ubulin

CHOP

β Tubulin

A


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



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


0.1

0.2

0.3

***

###

p-p

38M

AP

K/

Tubulin

p-p38MAPK

β Tubulin

ASK1

β Tubulin

D C


0.05

0.10

0.15

**

##

AS

K1/

Tubulin


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.


0.1

0.2

0.3

0.4

**

##

Cle

aved c

aspase-3

/ T

ubulin

Cleaved caspase-3

β Tubulin

B A


0.5

1.0

1.5

*

#

bcl2

/ T

ubulin

bcl2

β Tubulin


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).


0.5

1.0

1.5

*

#

TN

F

/ T

ubulin

TNFα

β Tubulin

B A


0.5

1.0

1.5

2.0

2.5

***

###

IL-1

/ T

ubulin

β Tubulin

IL-1β


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



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


5

10

15

20 *

##

TL

R4/

Tubulin

C


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).


70 | P a g e

Normal Control Curcumin

A

PAS

B

H&E

C

ED1

D

Fibronectin


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


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


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


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.


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


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


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


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).


78 | P a g e

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


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.


80 | P a g e

CHAPTER TWO

Curcumin ameliorates liver damage and progression of

NASH in NASH-HCC mouse model possibly by

modulating HMGB1-NF-κB translocation


81 | P a g e

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


82 | P a g e

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].


83 | P a g e

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.


2.1. Animals and experimental designs


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%).


84 | P a g e

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].


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


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


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)-


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.


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.


88 | P a g e

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).


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.


90 | P a g e

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


91 | P a g e




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).


92 | P a g e

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


NASH + Curcumin; NASH-HCC mice treated with curcumin 100 mg/Kg/day.

NAFLD: non-alcoholic fatty liver disease, MT: Masson trichrome. Values are


93 | P a g e

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


94 | P a g e

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


0.01

0.02

0.03

0.04**

###

IP10/G

AP

DH

IP10

GAPDH

C

GAPDH

IFNγ

D


0.02

0.04

0.06*

#

IL-1

/GA

PD

H

GAPDH

IL-1β

E


0.5

1.0

1.5*

#

IFN/

GA

PD

H


0.02

0.04

0.06*

###**

CD

68/G

AP

DH

GAPDH

CD68

F


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.


96 | P a g e

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;



Tukey’s method where, *p<0.05, **p<0.01 vs Normal, #p<0.05, ##p<0.01 vs

NASH.


0.5

1.0

1.5

*

#

I B

/GA

PD

H

GAPDH

IκBα

A B


0.02

0.04

0.06

**

##

Ox-L

DL

-R1/G

AP

DH

GAPDH

Oｘ-LDL-R1

C


2

4

6

8

**

#

p-N

F-

B n

ucl

eus/

cyto

sol

p-NFκB (Nucleus)

NFκB (Nucleus)

p-NFκB (Cytosol)

NFκB (Cytosol)


97 | P a g e

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).


0.05

0.10

0.15

0.20

0.25**

###

SR

EB

P1c/G

AP

DH


0.1

0.2

0.3

0.4

**

##

AD

RP

/GA

PD

H


2

4

6

8

**

###*

PP

AR/

tubulin

PPARγ

C


0.01

0.02

0.03

0.04

PP

AR

/GA

PD

H

D

PPARα

β tubulin GAPDH

GAPDH

SREBP1c

GAPDH

ADRP

A B


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).


99 | P a g e


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


0.1

0.2

0.3

0.4

**

###

p-E

RK

1/2

/ER

K1/2

ERK1/2

p-ERK1/2

C


0.01

0.02

0.03

0.04

**

#

p67phox/G

AP

DH

GAPDH

p67phox

D


0.5

1.0

1.5

*

##

Nrf

2/

tubulin

β tubulin

Nrf2

E


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.


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).


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.


0.05

0.10

0.15

0.20

**

#

VE

GF

/GA

PD

H

GAPDH VEGF


0.1

0.2

0.3

**

#

Gly

pic

an-3

/GA

PD

H


2

4

6

*

##

Pro

thro

mbin

/GA

PD

H

GAPDH

Glypican-3

GAPDH

Prothrombin

A B

C


102 | P a g e

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-


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


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


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


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


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.


108 | P a g e

CHAPTER THREE

Le Carbone modulates liver damage in non-alcoholic

steatohepatitis-hepatocellular carcinoma mouse model

through activation of AMPKα-SIRT1 signaling


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


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].


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


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


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


113 | P a g e

separated from blood. Liver tissue was isolated for histological, biochemical,

and molecular biological analysis [11].


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].


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


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).


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-


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].


116 | P a g e


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


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


(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:


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


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.


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


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


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


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


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


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

#


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


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


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).


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


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


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


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


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).


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


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


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


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


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.


138 | P a g e

CHAPTER FOUR

Comparison between diabetes and NASH


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


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.


2.1. Materials

Unless otherwise stated, all reagents were of analytical grade and purchased

from Sigma (Tokyo, Japan).

2.2. Animals and Experimental design


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


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.


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].


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].


142 | P a g e


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.


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.


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).


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


(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).


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


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


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


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


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


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


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


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


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


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


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


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


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


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.


159 | P a g e

SUMMMARY


160 | P a g e

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.


161 | P a g e

REFERENCES


162 | P a g e

References

[1] R. Vuppalanchi, N. Chalasani, Nonalcoholic fatty liver disease and

nonalcoholic steatohepatitis: Selected practical issues in their evaluation and

management, Hepatology. 49 (2009) 306–317. doi:10.1002/hep.22603.

[2] J. Ludwig, T.R. Viggiano, D.B. McGill, B.J. Oh, Nonalcoholic steatohepatitis:

Mayo Clinic experiences with a hitherto unnamed disease., Mayo Clin. Proc.

55 (1980) 434–8. http://www.ncbi.nlm.nih.gov/pubmed/7382552.

[3] J.O. Westwater, D. Fainer, Liver impairment in the obese., Gastroenterology.


[4] H. THALER, [The fatty liver and its pathogenetic relation to liver cirrhosis].,

Virchows Arch. Pathol. Anat. Physiol. Klin. Med. 335 (1962) 180–210.

http://www.ncbi.nlm.nih.gov/pubmed/13920484.

[5] P.A. Batman, P.J. Scheuer, Diabetic hepatitis preceding the onset of glucose

intolerance., Histopathology. 9 (1985) 237–43.


[6] A.M. Diehl, Z. Goodman, K.G. Ishak, Alcohollike liver disease in

nonalcoholics. A clinical and histologic comparison with alcohol-induced liver

injury., Gastroenterology. 95 (1988) 1056–62.


[7] I.R. Wanless, J.S. Lentz, Fatty liver hepatitis (steatohepatitis) and obesity: an

autopsy study with analysis of risk factors., Hepatology. 12 (1990) 1106–10.


[8] A. Lonardo, M. Bellini, E. Tondelli, M. Frazzoni, A. Grisendi, M. Pulvirenti, et

al., Nonalcoholic steatohepatitis and the "bright liver syndrome":

should a recently expanded clinical entity be further expanded?, Am. J.

Gastroenterol. 90 (1995) 2072–4.



163 | P a g e

[9] Y. Falck-Ytter, Z.M. Younossi, G. Marchesini, A.J. McCullough, Clinical

features and natural history of nonalcoholic steatosis syndromes., Semin.

Liver Dis. 21 (2001) 17–26. http://www.ncbi.nlm.nih.gov/pubmed/11296693.

[10] G. Marchesini, E. Bugianesi, G. Forlani, F. Cerrelli, M. Lenzi, R. Manini, et

al., Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome.,

Hepatology. 37 (2003) 917–23. doi:10.1053/jhep.2003.50161.

[11] M. Fujii, Y. Shibazaki, K. Wakamatsu, Y. Honda, Y. Kawauchi, K. Suzuki, et

al., A murine model for non-alcoholic steatohepatitis showing evidence of

association between diabetes and hepatocellular carcinoma., Med. Mol.

Morphol. 46 (2013) 141–52. doi:10.1007/s00795-013-0016-1.

[12] B. Charrez, L. Qiao, L. Hebbard, Hepatocellular carcinoma and non-alcoholic

steatohepatitis: The state of play, World J. Gastroenterol. 22 (2016) 2494.

doi:10.3748/wjg.v22.i8.2494.

[13] B.Q. Starley, C.J. Calcagno, S.A. Harrison, Nonalcoholic fatty liver disease

and hepatocellular carcinoma: A weighty connection, Hepatology. 51 (2010)

1820–1832. doi:10.1002/hep.23594.

[14] W. Liang, A.L. Menke, A. Driessen, G.H. Koek, J.H. Lindeman, R. Stoop, et

al., Establishment of a general NAFLD scoring system for rodent models and

comparison to human liver pathology., PLoS One. 9 (2014) e115922.

doi:10.1371/journal.pone.0115922.

[15] R.G. Lee, Nonalcoholic steatohepatitis: tightening the morphological screws on

a hepatic rambler., Hepatology. 21 (1995) 1742–3.


[16] E.M. Brunt, C.G. Janney, A.M. Di Bisceglie, B.A. Neuschwander-Tetri, B.R.

Bacon, Nonalcoholic steatohepatitis: a proposal for grading and staging the

histological lesions., Am. J. Gastroenterol. 94 (1999) 2467–74.

doi:10.1111/j.1572-0241.1999.01377.x.

[17] C.P. Day, Genetic and environmental susceptibility to non-alcoholic fatty liver


164 | P a g e

disease., Dig. Dis. 28 (2010) 255–60. doi:10.1159/000282098.

[18] D.F. Wallace, V.N. Subramaniam, Co-factors in liver disease: the role of HFE-

related hereditary hemochromatosis and iron., Biochim. Biophys. Acta. 1790

(2009) 663–70. doi:10.1016/j.bbagen.2008.09.002.

[19] L. Valenti, A. Al-Serri, A.K. Daly, E. Galmozzi, R. Rametta, P. Dongiovanni, et

al., Homozygosity for the patatin-like phospholipase-3/adiponutrin I148M

polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver

disease., Hepatology. 51 (2010) 1209–17. doi:10.1002/hep.23622.

[20] C. Sun, J.-G. Fan, L. Qiao, Potential epigenetic mechanism in non-alcoholic

Fatty liver disease., Int. J. Mol. Sci. 16 (2015) 5161–79.

doi:10.3390/ijms16035161.

[21] L. Wang, H. Zhang, J. Zhou, Y. Liu, Y. Yang, X. Chen, et al., Betaine

attenuates hepatic steatosis by reducing methylation of the MTTP promoter

and elevating genomic methylation in mice fed a high-fat diet., J. Nutr.

Biochem. 25 (2014) 329–36. doi:10.1016/j.jnutbio.2013.11.007.

[22] K. Promrat, D.E. Kleiner, H.M. Niemeier, E. Jackvony, M. Kearns, J.R.

Wands, et al., Randomized controlled trial testing the effects of weight loss on

nonalcoholic steatohepatitis., Hepatology. 51 (2010) 121–9.

doi:10.1002/hep.23276.

[23] V.W.-S. Wong, R.S.-M. Chan, G.L.-H. Wong, B.H.-K. Cheung, W.C.-W. Chu,

D.K.-W. Yeung, et al., Community-based lifestyle modification programme for

non-alcoholic fatty liver disease: A randomized controlled trial, J. Hepatol. 59

(2013) 536–542. doi:10.1016/j.jhep.2013.04.013.

[24] M. Charlton, A. Krishnan, K. Viker, S. Sanderson, S. Cazanave, A. McConico,

et al., Fast food diet mouse: novel small animal model of NASH with

ballooning, progressive fibrosis, and high physiological fidelity to the human

condition., Am. J. Physiol. Gastrointest. Liver Physiol. 301 (2011) G825-34.

doi:10.1152/ajpgi.00145.2011.


165 | P a g e

[25] J.R. Garbow, J.M. Doherty, R.C. Schugar, S. Travers, M.L. Weber, A.E.

Wentz, et al., Hepatic steatosis, inflammation, and ER stress in mice

maintained long term on a very low-carbohydrate ketogenic diet., Am. J.

Physiol. Gastrointest. Liver Physiol. 300 (2011) G956-67.

doi:10.1152/ajpgi.00539.2010.

[26] P. Handa, V. Morgan-Stevenson, B.D. Maliken, J.E. Nelson, S. Washington,

M. Westerman, et al., Iron overload results in hepatic oxidative stress,

immune cell activation, and hepatocellular ballooning injury, leading to

nonalcoholic steatohepatitis in genetically obese mice, Am. J. Physiol. -

Gastrointest. Liver Physiol. 310 (2016) G117–G127.

doi:10.1152/ajpgi.00246.2015.

[27] S.E. Regnell, Å. Lernmark, Hepatic steatosis in type 1 diabetes., Rev. Diabet.

Stud. 8 (2011) 454–67. doi:10.1900/RDS.2011.8.454.

[28] B.R. Bacon, M.J. Farahvash, C.G. Janney, B.A. Neuschwander-Tetri,

Nonalcoholic steatohepatitis: an expanded clinical entity., Gastroenterology.

107 (1994) 1103–9. http://www.ncbi.nlm.nih.gov/pubmed/7523217

[29] S.G. Sheth, F.D. Gordon, S. Chopra, Nonalcoholic steatohepatitis., Ann.

Intern. Med. 126 (1997) 137–45.


[30] T. Andersen, C. Gluud, Liver morphology in morbid obesity: a literature

study., Int. J. Obes. 8 (1984) 97–106.


[31] W.H. Kern, A.H. Heger, J.H. Payne, L.T. DeWind, Fatty metamorphosis of the

liver in morbid obesity., Arch. Pathol. 96 (1973) 342–6.


[32] S.M. Nasrallah, C.E. Wills, J.T. Galambos, Hepatic morphology in obesity.,

Dig. Dis. Sci. 26 (1981) 325–7. http://www.ncbi.nlm.nih.gov/pubmed/7238260.

[33] D. Bernuau, R. Guillot, A.M. Durand, N. Raoux, T. Gabreau, P. Passa, et al.,


166 | P a g e

Ultrastructural aspects of the liver perisinusoidal space in diabetic patients

with and without microangiopathy., Diabetes. 31 (1982) 1061–7.


[34] H.J. ZIMMERMAN, F.G. MacMURRAY, H. RAPPAPORT, L.K. ALPERT,

Studies on the liver in diabetes mellitus. II. The significance of fatty

metamorphosis and its correlation with insulin sensitivity., J. Lab. Clin. Med.


[35] T. Hano, Pathohistological study on liver cirrhosis in diabetes mellitus., Kobe

J. Med. Sci. 14 (1968) 87–106. http://www.ncbi.nlm.nih.gov/pubmed/5704884.

[36] L. Wideroff, G. Gridley, L. Mellemkjaer, W.H. Chow, M. Linet, S. Keehn, et

al., Cancer incidence in a population-based cohort of patients hospitalized

with diabetes mellitus in Denmark., J. Natl. Cancer Inst. 89 (1997) 1360–5.


[37] Z. Li, M. Berk, T.M. McIntyre, G.J. Gores, A.E. Feldstein, The lysosomal-

mitochondrial axis in free fatty acid-induced hepatic lipotoxicity, Hepatology.

47 (2008) 1495–1503. doi:10.1002/hep.22183.

[38] G. Serviddio, F. Bellanti, G. Vendemiale, E. Altomare, Mitochondrial

dysfunction in nonalcoholic steatohepatitis, Expert Rev. Gastroenterol.

Hepatol. 5 (2011) 233–244. doi:10.1586/egh.11.11.

[39] K. Gariani, K.J. Menzies, D. Ryu, C.J. Wegner, X. Wang, E.R. Ropelle, et al.,

Eliciting the mitochondrial unfolded protein response by nicotinamide adenine

dinucleotide repletion reverses fatty liver disease in mice., Hepatology. 63

(2016) 1190–204. doi:10.1002/hep.28245.

[40] M.A. Abdelmegeed, A. Banerjee, S.-H. Yoo, S. Jang, F.J. Gonzalez, B.-J. Song,

Critical role of cytochrome P450 2E1 (CYP2E1) in the development of high fat-

induced non-alcoholic steatohepatitis., J. Hepatol. 57 (2012) 860–6.

doi:10.1016/j.jhep.2012.05.019.

[41] A.E. Feldstein, A. Canbay, P. Angulo, M. Taniai, L.J. Burgart, K.D. Lindor, et


167 | P a g e

al., Hepatocyte apoptosis and fas expression are prominent features of human

nonalcoholic steatohepatitis., Gastroenterology. 125 (2003) 437–43.


[42] V.L. Gadd, R. Skoien, E.E. Powell, K.J. Fagan, C. Winterford, L. Horsfall, et

al., The portal inflammatory infiltrate and ductular reaction in human

nonalcoholic fatty liver disease, Hepatology. 59 (2014) 1393–1405.

doi:10.1002/hep.26937.

[43] N. Lanthier, Targeting Kupffer cells in non-alcoholic fatty liver disease/non-

alcoholic steatohepatitis: Why and how?, World J. Hepatol. 7 (2015) 2184–8.

doi:10.4254/wjh.v7.i19.2184.

[44] T. Strowig, J. Henao-Mejia, E. Elinav, R. Flavell, Inflammasomes in health

and disease, Nature. 481 (2012) 278–286. doi:10.1038/nature10759.

[45] A. Douhara, K. Moriya, H. Yoshiji, R. Noguchi, T. Namisaki, M. Kitade, et al.,

Reduction of endotoxin attenuates liver fibrosis through suppression of

hepatic stellate cell activation and remission of intestinal permeability in a rat

non-alcoholic steatohepatitis model., Mol. Med. Rep. 11 (2015) 1693–700.

doi:10.3892/mmr.2014.2995.

[46] J.A. Ehses, D.T. Meier, S. Wueest, J. Rytka, S. Boller, P.Y. Wielinga, et al.,

Toll-like receptor 2-deficient mice are protected from insulin resistance and

beta cell dysfunction induced by a high-fat diet, Diabetologia. 53 (2010) 1795–

1806. doi:10.1007/s00125-010-1747-3.

[47] T. Csak, A. Velayudham, I. Hritz, J. Petrasek, I. Levin, D. Lippai, et al.,

Deficiency in myeloid differentiation factor-2 and toll-like receptor 4

expression attenuates nonalcoholic steatohepatitis and fibrosis in mice., Am.

J. Physiol. Gastrointest. Liver Physiol. 300 (2011) G433-41.

doi:10.1152/ajpgi.00163.2009.

[48] M.R. Dasu, I. Jialal, Free fatty acids in the presence of high glucose amplify

monocyte inflammation via Toll-like receptors., Am. J. Physiol. Endocrinol.

Metab. 300 (2011) E145-54. doi:10.1152/ajpendo.00490.2010.


168 | P a g e

[49] P. Scaffidi, T. Misteli, M.E. Bianchi, Release of chromatin protein HMGB1 by

necrotic cells triggers inflammation, Nature. 418 (2002) 191–195.

doi:10.1038/nature00858.

[50] G. Szabo, T. Csak, Inflammasomes in liver diseases, J. Hepatol. 57 (2012)

642–654. doi:10.1016/j.jhep.2012.03.035.

[51] T. Csak, A. Pillai, M. Ganz, D. Lippai, J. Petrasek, J.-K. Park, et al., Both

bone marrow-derived and non-bone marrow-derived cells contribute to AIM2

and NLRP3 inflammasome activation in a MyD88-dependent manner in

dietary steatohepatitis., Liver Int. 34 (2014) 1402–13. doi:10.1111/liv.12537.

[52] P. Puri, F. Mirshahi, O. Cheung, R. Natarajan, J.W. Maher, J.M. Kellum, et

al., Activation and Dysregulation of the Unfolded Protein Response in

Nonalcoholic Fatty Liver Disease, Gastroenterology. 134 (2008) 568–576.

doi:10.1053/j.gastro.2007.10.039.

[53] Y. Wei, D. Wang, F. Topczewski, M.J. Pagliassotti, Saturated fatty acids

induce endoplasmic reticulum stress and apoptosis independently of ceramide

in liver cells, AJP Endocrinol. Metab. 291 (2006) E275–E281.

doi:10.1152/ajpendo.00644.2005.

[54] R.M. Evans, D.J. Mangelsdorf, Nuclear Receptors, RXR, and the Big Bang.,

Cell. 157 (2014) 255–66. doi:10.1016/j.cell.2014.03.012.

[55] S. Francque, A. Verrijken, S. Caron, J. Prawitt, R. Paumelle, B. Derudas, et

al., PPARα gene expression correlates with severity and histological treatment

response in patients with non-alcoholic steatohepatitis., J. Hepatol. 63 (2015)

164–73. doi:10.1016/j.jhep.2015.02.019.

[56] P. Angulo, GI epidemiology: nonalcoholic fatty liver disease., Aliment.

Pharmacol. Ther. 25 (2007) 883–9. doi:10.1111/j.1365-2036.2007.03246.x.

[57] R.J. Wong, R. Cheung, A. Ahmed, Nonalcoholic steatohepatitis is the most

rapidly growing indication for liver transplantation in patients with

hepatocellular carcinoma in the U.S., Hepatology. 59 (2014) 2188–95.


169 | P a g e

doi:10.1002/hep.26986.

[58] D. Barb, P. Portillo-Sanchez, K. Cusi, Pharmacological management of

nonalcoholic fatty liver disease, Metabolism. 65 (2016) 1183–1195.

doi:10.1016/j.metabol.2016.04.004.

[59] N. Chalasani, Z. Younossi, J.E. Lavine, A.M. Diehl, E.M. Brunt, K. Cusi, et

al., The diagnosis and management of non-alcoholic fatty liver disease:

practice Guideline by the American Association for the Study of Liver

Diseases, American College of Gastroenterology, and the American

Gastroenterological Association., Hepatology. 55 (2012) 2005–23.

doi:10.1002/hep.25762.

[60] S. Singh, B.B. Aggarwal, Activation of transcription factor NF-kappa B is

suppressed by curcumin (diferuloylmethane) [corrected]., J. Biol. Chem. 270

(1995) 24995–5000. http://www.ncbi.nlm.nih.gov/pubmed/7559628

[61] J.C. Kerihuel, Effect of activated charcoal dressings on healing outcomes of

chronic wounds., J. Wound Care. 19 (2010) 208, 210–2, 214–5.

doi:10.12968/jowc.2010.19.5.48047.

[62] H. Liang, P. Wang, X. Wang, N. Liu, X. Yue, D. Wang, et al., [Prospective

randomized trial of prophylaxis of postoperative peritoneal carcinomatosis of

advanced gastric cancer: intraperitoneal chemotherapy with mitomycin C

bound to activated carbon particles]., Zhonghua Wai Ke Za Zhi. 41 (2003) 274–

7. http://www.ncbi.nlm.nih.gov/pubmed/12882671.

[63] M.R. Afrin, S. Arumugam, M.A. Rahman, V. Karuppagounder, R. Sreedhar,

M. Harima, et al., 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., Int.

Immunopharmacol. 43 (2016) 70–78. doi:10.1016/j.intimp.2016.10.023.

[64] P. Kuusisto, H. Vapaatalo, V. Manninen, J.K. Huttunen, P.J. Neuvonen,

Effect of activated charcoal on hypercholesterolaemia., Lancet (London,

England). 2 (1986) 366–7. http://www.ncbi.nlm.nih.gov/pubmed/2874369.


170 | P a g e

[65] A. V Das, P.S. Padayatti, C.S. Paulose, Effect of leaf extract of Aegle

marmelose (L.) Correa ex Roxb. on histological and ultrastructural changes in

tissues of streptozotocin induced diabetic rats., Indian J. Exp. Biol. 34 (1996)

341–5. http://www.ncbi.nlm.nih.gov/pubmed/8698423.

[66] P. Latry, P. Bioulac-Sage, E. Echinard, H. Gin, L. Boussarie, J.A. Grimaud, et

al., Perisinusoidal fibrosis and basement membrane-like material in the livers

of diabetic patients., Hum. Pathol. 18 (1987) 775–80.


[67] S.A. Harrison, Liver disease in patients with diabetes mellitus., J. Clin.

Gastroenterol. 40 (2006) 68–76.


[68] N. Houstis, E.D. Rosen, E.S. Lander, Reactive oxygen species have a causal

role in multiple forms of insulin resistance., Nature. 440 (2006) 944–8.

doi:10.1038/nature04634.

[69] U. Ozcan, Q. Cao, E. Yilmaz, A.-H. Lee, N.N. Iwakoshi, E. Ozdelen, et al.,

Endoplasmic reticulum stress links obesity, insulin action, and type 2

diabetes., Science. 306 (2004) 457–61. doi:10.1126/science.1103160.

[70] V. Poitout, R.P. Robertson, Glucolipotoxicity: fuel excess and beta-cell

dysfunction., Endocr. Rev. 29 (2008) 351–66. doi:10.1210/er.2007-0023.

[71] I.C. West, Radicals and oxidative stress in diabetes., Diabet. Med. 17 (2000)

171–80. http://www.ncbi.nlm.nih.gov/pubmed/10784220.

[72] M.E.E. Shams, M.M.H. Al-Gayyar, E.A.M.E. Barakat, Type 2 Diabetes

Mellitus-Induced Hyperglycemia in Patients with NAFLD and Normal LFTs:

Relationship to Lipid Profile, Oxidative Stress and Pro-Inflammatory

Cytokines., Sci. Pharm. 79 (2011) 623–34. doi:10.3797/scipharm.1104-21.

[73] E.M. de Cavanagh, F. Inserra, J. Toblli, I. Stella, C.G. Fraga, L. Ferder,

Enalapril attenuates oxidative stress in diabetic rats., Hypertens. (Dallas,

Tex. 1979). 38 (2001) 1130–6. http://www.ncbi.nlm.nih.gov/pubmed/11711510.


171 | P a g e

[74] D. Scheuner, D. Vander Mierde, B. Song, D. Flamez, J.W.M. Creemers, K.

Tsukamoto, et al., Control of mRNA translation preserves endoplasmic

reticulum function in beta cells and maintains glucose homeostasis., Nat.

Med. 11 (2005) 757–64. doi:10.1038/nm1259.

[75] B. Yusta, L.L. Baggio, J.L. Estall, J.A. Koehler, D.P. Holland, H. Li, et al.,

GLP-1 receptor activation improves beta cell function and survival following

induction of endoplasmic reticulum stress., Cell Metab. 4 (2006) 391–406.

doi:10.1016/j.cmet.2006.10.001.

[76] C. Huang, C. Lin, L. Haataja, T. Gurlo, A.E. Butler, R.A. Rizza, et al., High

expression rates of human islet amyloid polypeptide induce endoplasmic

reticulum stress mediated beta-cell apoptosis, a characteristic of humans with

type 2 but not type 1 diabetes., Diabetes. 56 (2007) 2016–27. doi:10.2337/db07-

0197.

[77] D.R. Laybutt, A.M. Preston, M.C. Akerfeldt, J.G. Kench, A.K. Busch, A. V

Biankin, et al., Endoplasmic reticulum stress contributes to beta cell apoptosis

in type 2 diabetes., Diabetologia. 50 (2007) 752–63. doi:10.1007/s00125-006-

0590-z.

[78] B. Song, D. Scheuner, D. Ron, S. Pennathur, R.J. Kaufman, Chop deletion

reduces oxidative stress, improves beta cell function, and promotes cell

survival in multiple mouse models of diabetes., J. Clin. Invest. 118 (2008)

3378–89. doi:10.1172/JCI34587.

[79] D. Ron, P. Walter, Signal integration in the endoplasmic reticulum unfolded

protein response., Nat. Rev. Mol. Cell Biol. 8 (2007) 519–29.

doi:10.1038/nrm2199.

[80] A.P. Lakshmanan, M. Harima, K. Suzuki, V. Soetikno, M. Nagata, T.

Nakamura, et al., The hyperglycemia stimulated myocardial endoplasmic

reticulum (ER) stress contributes to diabetic cardiomyopathy in the transgenic

non-obese type 2 diabetic rats: a differential role of unfolded protein response

(UPR) signaling proteins., Int. J. Biochem. Cell Biol. 45 (2013) 438–47.


172 | P a g e

doi:10.1016/j.biocel.2012.09.017.

[81] D.T. Rutkowski, J. Wu, S.-H. Back, M.U. Callaghan, S.P. Ferris, J. Iqbal, et

al., UPR pathways combine to prevent hepatic steatosis caused by ER stress-

mediated suppression of transcriptional master regulators., Dev. Cell. 15

(2008) 829–40. doi:10.1016/j.devcel.2008.10.015.

[82] X.Z. Wang, B. Lawson, J.W. Brewer, H. Zinszner, A. Sanjay, L.J. Mi, et al.,

Signals from the stressed endoplasmic reticulum induce C/EBP-homologous

protein (CHOP/GADD153)., Mol. Cell. Biol. 16 (1996) 4273–80.


[83] G.S. Hotamisligil, Role of endoplasmic reticulum stress and c-Jun NH2-

terminal kinase pathways in inflammation and origin of obesity and diabetes.,

Diabetes. 54 Suppl 2 (2005) S73-8.


[84] T. Nakagawa, H. Zhu, N. Morishima, E. Li, J. Xu, B.A. Yankner, et al.,

Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and

cytotoxicity by amyloid-beta., Nature. 403 (2000) 98–103. doi:10.1038/47513.

[85] H. Hatcher, R. Planalp, J. Cho, F.M. Torti, S. V Torti, Curcumin: from ancient

medicine to current clinical trials., Cell. Mol. Life Sci. 65 (2008) 1631–52.

doi:10.1007/s00018-008-7452-4.

[86] J.Y. Liu, S.J. Lin, J.K. Lin, Inhibitory effects of curcumin on protein kinase C

activity induced by 12-O-tetradecanoyl-phorbol-13-acetate in NIH 3T3 cells.,

Carcinogenesis. 14 (1993) 857–61.


[87] Y.A. Mahmmoud, Curcumin modulation of Na,K-ATPase: phosphoenzyme

accumulation, decreased K+ occlusion, and inhibition of hydrolytic activity.,

Br. J. Pharmacol. 145 (2005) 236–45. doi:10.1038/sj.bjp.0706185.

[88] H. Choi, Y.-S. Chun, S.-W. Kim, M.-S. Kim, J.-W. Park, Curcumin inhibits

hypoxia-inducible factor-1 by degrading aryl hydrocarbon receptor nuclear


173 | P a g e

translocator: a mechanism of tumor growth inhibition., Mol. Pharmacol. 70

(2006) 1664–71. doi:10.1124/mol.106.025817.

[89] S. Mito, R.A. Thandavarayan, M. Ma, A. Lakshmanan, K. Suzuki, M. Kodama,

et al., Inhibition of cardiac oxidative and endoplasmic reticulum stress-

mediated apoptosis by curcumin treatment contributes to protection against

acute myocarditis., Free Radic. Res. 45 (2011) 1223–31.

doi:10.3109/10715762.2011.607252.

[90] K. Reyes-Gordillo, J. Segovia, M. Shibayama, P. Vergara, M.G. Moreno, P.

Muriel, Curcumin protects against acute liver damage in the rat by inhibiting

NF-kappaB, proinflammatory cytokines production and oxidative stress.,

Biochim. Biophys. Acta. 1770 (2007) 989–96.

doi:10.1016/j.bbagen.2007.02.004.

[91] K. Watanabe, Y. Ohta, M. Nakazawa, H. Higuchi, G. Hasegawa, M. Naito, et

al., Low dose carvedilol inhibits progression of heart failure in rats with

dilated cardiomyopathy., Br. J. Pharmacol. 130 (2000) 1489–95.

doi:10.1038/sj.bjp.0703450.

[92] S.K. Jain, J. Rains, J. Croad, B. Larson, K. Jones, Curcumin supplementation

lowers TNF-alpha, IL-6, IL-8, and MCP-1 secretion in high glucose-treated

cultured monocytes and blood levels of TNF-alpha, IL-6, MCP-1, glucose, and

glycosylated hemoglobin in diabetic rats., Antioxid. Redox Signal. 11 (2009)

241–9. doi:10.1089/ars.2008.2140.

[93] V. Thallas-Bonke, S.R. Thorpe, M.T. Coughlan, K. Fukami, F.Y.T. Yap, K.C.

Sourris, et al., Inhibition of NADPH Oxidase Prevents Advanced Glycation

End Product-Mediated Damage in Diabetic Nephropathy Through a Protein

Kinase C- -Dependent Pathway, Diabetes. 57 (2008) 460–469.

doi:10.2337/db07-1119.

[94] A. Guven, O. Yavuz, M. Cam, F. Ercan, N. Bukan, C. Comunoglu, et al.,

Effects of melatonin on streptozotocin-induced diabetic liver injury in rats.,

Acta Histochem. 108 (2006) 85–93. doi:10.1016/j.acthis.2006.03.005.


174 | P a g e

[95] Vivian Soetikno, Meilei Harima, Kenji Suzuki, Kenichi Watanabe,

Amelioration of Liver Injury by Curcumin in Streptozotocin-Induced Diabetic

Rats, International Conference on Medical and Pharmaceutical Sciences;

Bangkok, June 16-17, 2012.

[96] V. Soetikno, F.R. Sari, P.T. Veeraveedu, R.A. Thandavarayan, M. Harima, V.

Sukumaran, et al., Curcumin ameliorates macrophage infiltration by

inhibiting NF-κB activation and proinflammatory cytokines in streptozotocin

induced-diabetic nephropathy., Nutr. Metab. (Lond). 8 (2011) 35.

doi:10.1186/1743-7075-8-35.

[97] M. Chanpoo, H. Petchpiboonthai, B. Panyarachun, V. Anupunpisit, Effect of

curcumin in the amelioration of pancreatic islets in streptozotocin-induced

diabetic mice., J. Med. Assoc. Thai. 93 Suppl 6 (2010) S152-9.


[98] K. Meghana, G. Sanjeev, B. Ramesh, Curcumin prevents streptozotocin-

induced islet damage by scavenging free radicals: a prophylactic and

protective role., Eur. J. Pharmacol. 577 (2007) 183–91.

doi:10.1016/j.ejphar.2007.09.002.

[99] H.Y. Chung, B. Sung, K.J. Jung, Y. Zou, B.P. Yu, The molecular inflammatory

process in aging., Antioxid. Redox Signal. 8 (2006) 572–81.

doi:10.1089/ars.2006.8.572.

[100] J.F. Navarro-González, C. Mora-Fernández, M. Muros de Fuentes, J. García-

Pérez, Inflammatory molecules and pathways in the pathogenesis of diabetic

nephropathy., Nat. Rev. Nephrol. 7 (2011) 327–40.

doi:10.1038/nrneph.2011.51.

[101] D.L. Eizirik, M.L. Colli, F. Ortis, The role of inflammation in insulitis and

beta-cell loss in type 1 diabetes., Nat. Rev. Endocrinol. 5 (2009) 219–26.

doi:10.1038/nrendo.2009.21.

[102] S. Gordon, P.R. Taylor, Monocyte and macrophage heterogeneity., Nat. Rev.

Immunol. 5 (2005) 953–64. doi:10.1038/nri1733.


175 | P a g e

[103] P.R. Taylor, L. Martinez-Pomares, M. Stacey, H.-H. Lin, G.D. Brown, S.

Gordon, Macrophage receptors and immune recognition., Annu. Rev.

Immunol. 23 (2005) 901–44. doi:10.1146/annurev.immunol.23.021704.115816.

[104] G. Boden, X. Duan, C. Homko, E.J. Molina, W. Song, O. Perez, et al., Increase

in endoplasmic reticulum stress-related proteins and genes in adipose tissue

of obese, insulin-resistant individuals., Diabetes. 57 (2008) 2438–44.

doi:10.2337/db08-0604.

[105] M.F. Gregor, L. Yang, E. Fabbrini, B.S. Mohammed, J.C. Eagon, G.S.

Hotamisligil, et al., Endoplasmic reticulum stress is reduced in tissues of

obese subjects after weight loss., Diabetes. 58 (2009) 693–700.

doi:10.2337/db08-1220.

[106] P. Puri, F. Mirshahi, O. Cheung, R. Natarajan, J.W. Maher, J.M. Kellum, et

al., Activation and dysregulation of the unfolded protein response in

nonalcoholic fatty liver disease., Gastroenterology. 134 (2008) 568–76.

doi:10.1053/j.gastro.2007.10.039.

[107] N.K. Sharma, S.K. Das, A.K. Mondal, O.G. Hackney, W.S. Chu, P.A. Kern, et

al., Endoplasmic reticulum stress markers are associated with obesity in

nondiabetic subjects., J. Clin. Endocrinol. Metab. 93 (2008) 4532–41.

doi:10.1210/jc.2008-1001.

[108] F. Gu, D.T. Nguyên, M. Stuible, N. Dubé, M.L. Tremblay, E. Chevet, Protein-

tyrosine phosphatase 1B potentiates IRE1 signaling during endoplasmic

reticulum stress., J. Biol. Chem. 279 (2004) 49689–93.

doi:10.1074/jbc.C400261200.

[109] C. Ji, N. Kaplowitz, ER stress: can the liver cope?, J. Hepatol. 45 (2006) 321–

33. doi:10.1016/j.jhep.2006.06.004.

[110] M. Schröder, R.J. Kaufman, The mammalian unfolded protein response.,

Annu. Rev. Biochem. 74 (2005) 739–89.

doi:10.1146/annurev.biochem.73.011303.074134.


176 | P a g e

[111] A.-H. Lee, E.F. Scapa, D.E. Cohen, L.H. Glimcher, Regulation of hepatic

lipogenesis by the transcription factor XBP1., Science. 320 (2008) 1492–6.

doi:10.1126/science.1158042.

[112] K. Yamamoto, K. Takahara, S. Oyadomari, T. Okada, T. Sato, A. Harada, et

al., Induction of liver steatosis and lipid droplet formation in ATF6alpha-

knockout mice burdened with pharmacological endoplasmic reticulum stress.,

Mol. Biol. Cell. 21 (2010) 2975–86. doi:10.1091/mbc.E09-02-0133.

[113] D.T. Rutkowski, R.J. Kaufman, P.C. Stirling, et al., H.P. Harding, et al., et

al., A trip to the ER: coping with stress., Trends Cell Biol. 14 (2004) 20–8.

doi:10.1016/J.TCB.2003.11.001.

[114] K. Zhang, R.J. Kaufman, From endoplasmic-reticulum stress to the

inflammatory response, Nature. 454 (2008) 455–462.

doi:10.1038/nature07203.

[115] T. Gotoh, M. Mori, Nitric Oxide and Endoplasmic Reticulum Stress,

Arterioscler. Thromb. Vasc. Biol. 26 (2006).

[116] S. Oyadomari, M. Mori, Roles of CHOP/GADD153 in endoplasmic reticulum

stress., Cell Death Differ. 11 (2004) 381–9. doi:10.1038/sj.cdd.4401373.

[117] T. Nakagawa, J. Yuan, Cross-talk between two cysteine protease families.

Activation of caspase-12 by calpain in apoptosis., J. Cell Biol. 150 (2000) 887–

94. http://www.ncbi.nlm.nih.gov/pubmed/10953012.

[118] N. Morishima, K. Nakanishi, H. Takenouchi, T. Shibata, Y. Yasuhiko, An

endoplasmic reticulum stress-specific caspase cascade in apoptosis.

Cytochrome c-independent activation of caspase-9 by caspase-12., J. Biol.

Chem. 277 (2002) 34287–94. doi:10.1074/jbc.M204973200.

[119] K. Homma, K. Katagiri, H. Nishitoh, H. Ichijo, Targeting ASK1 in ER stress-

related neurodegenerative diseases., Expert Opin. Ther. Targets. 13 (2009)

653–64. doi:10.1517/14728220902980249.

[120] K. Hattori, I. Naguro, C. Runchel, H. Ichijo, The roles of ASK family proteins


177 | P a g e

in stress responses and diseases., Cell Commun. Signal. 7 (2009) 9.

doi:10.1186/1478-811X-7-9.

[121] M. Schröder, R.J. Kaufman, ER stress and the unfolded protein response.,

Mutat. Res. 569 (2005) 29–63. doi:10.1016/j.mrfmmm.2004.06.056.

[122] H.P. Harding, D. Ron, Endoplasmic reticulum stress and the development of

diabetes: a review., Diabetes. 51 Suppl 3 (2002) S455-61.


[123] H.P. Harding, I. Novoa, Y. Zhang, H. Zeng, R. Wek, M. Schapira, et al.,

Regulated translation initiation controls stress-induced gene expression in

mammalian cells., Mol. Cell. 6 (2000) 1099–108.


[124] J. Hollien, J.S. Weissman, Decay of endoplasmic reticulum-localized mRNAs

during the unfolded protein response., Science. 313 (2006) 104–7.

doi:10.1126/science.1129631.

[125] J. Hollien, J.H. Lin, H. Li, N. Stevens, P. Walter, J.S. Weissman, Regulated

Ire1-dependent decay of messenger RNAs in mammalian cells., J. Cell Biol.

186 (2009) 323–31. doi:10.1083/jcb.200903014.

[126] C. Hetz, L.H. Glimcher, Fine-tuning of the unfolded protein response:

Assembling the IRE1alpha interactome., Mol. Cell. 35 (2009) 551–61.

doi:10.1016/j.molcel.2009.08.021.

[127] P. Hu, Z. Han, A.D. Couvillon, R.J. Kaufman, J.H. Exton, Autocrine tumor

necrosis factor alpha links endoplasmic reticulum stress to the membrane

death receptor pathway through IRE1alpha-mediated NF-kappaB activation

and down-regulation of TRAF2 expression., Mol. Cell. Biol. 26 (2006) 3071–84.

doi:10.1128/MCB.26.8.3071-3084.2006.

[128] D.T. Nguyên, S. Kebache, A. Fazel, H.N. Wong, S. Jenna, A. Emadali, et al.,

Nck-dependent activation of extracellular signal-regulated kinase-1 and

regulation of cell survival during endoplasmic reticulum stress., Mol. Biol.


178 | P a g e

Cell. 15 (2004) 4248–60. doi:10.1091/mbc.E03-11-0851.

[129] Y. Li, R.F. Schwabe, T. DeVries-Seimon, P.M. Yao, M.-C. Gerbod-Giannone,

A.R. Tall, et al., Free Cholesterol-loaded Macrophages Are an Abundant

Source of Tumor Necrosis Factor- and Interleukin-6: MODEL OF NF- B-

AND MAP KINASE-DEPENDENT INFLAMMATION IN ADVANCED

ATHEROSCLEROSIS, J. Biol. Chem. 280 (2005) 21763–21772.

doi:10.1074/jbc.M501759200.

[130] P.S. Gargalovic, N.M. Gharavi, M.J. Clark, J. Pagnon, W.-P. Yang, A. He, et

al., The Unfolded Protein Response Is an Important Regulator of

Inflammatory Genes in Endothelial Cells, Arterioscler. Thromb. Vasc. Biol. 26

(2006).

[131] A.A. Nanji, K. Jokelainen, G.L. Tipoe, A. Rahemtulla, P. Thomas, A.J.

Dannenberg, Curcumin prevents alcohol-induced liver disease in rats by

inhibiting the expression of NF-kappa B-dependent genes., Am. J. Physiol.

Gastrointest. Liver Physiol. 284 (2003) G321-7. doi:10.1152/ajpgi.00230.2002.

[132] R. Stienstra, F. Saudale, C. Duval, S. Keshtkar, J.E.M. Groener, N. van

Rooijen, et al., Kupffer cells promote hepatic steatosis via interleukin-1beta-

dependent suppression of peroxisome proliferator-activated receptor alpha

activity., Hepatology. 51 (2010) 511–22. doi:10.1002/hep.23337.

[133] M. Rouse, A. Younès, J.M. Egan, Resveratrol and curcumin enhance

pancreatic β-cell function by inhibiting phosphodiesterase activity., J.

Endocrinol. 223 (2014) 107–17. doi:10.1530/JOE-14-0335.

[134] M.I. Shariff, I.J. Cox, A.I. Gomaa, S.A. Khan, W. Gedroyc, S.D. Taylor-

Robinson, Hepatocellular carcinoma: current trends in worldwide

epidemiology, risk factors, diagnosis and therapeutics, Expert Rev.

Gastroenterol. Hepatol. 3 (2009) 353–367. doi:10.1586/egh.09.35.

[135] A. Mouralidarane, J. Soeda, C. Visconti-Pugmire, A.-M. Samuelsson, J.

Pombo, X. Maragkoudaki, et al., Maternal obesity programs offspring

nonalcoholic fatty liver disease by innate immune dysfunction in mice.,


179 | P a g e

Hepatology. 58 (2013) 128–38. doi:10.1002/hep.26248.

[136] J. Ertle, A. Dechêne, J.-P. Sowa, V. Penndorf, K. Herzer, G. Kaiser, et al.,

Non-alcoholic fatty liver disease progresses to hepatocellular carcinoma in the

absence of apparent cirrhosis., Int. J. Cancer. 128 (2011) 2436–43.

doi:10.1002/ijc.25797.

[137] G.C. Farrell, D. van Rooyen, L. Gan, S. Chitturi, NASH is an Inflammatory

Disorder: Pathogenic, Prognostic and Therapeutic Implications., Gut Liver. 6

(2012) 149–71. doi:10.5009/gnl.2012.6.2.149.

[138] S.I. Grivennikov, F.R. Greten, M. Karin, Immunity, Inflammation, and

Cancer, (n.d.). doi:10.1016/j.cell.2010.01.025.

[139] G.P. Sims, D.C. Rowe, S.T. Rietdijk, R. Herbst, A.J. Coyle, HMGB1 and RAGE

in inflammation and cancer., Annu. Rev. Immunol. 28 (2010) 367–388.

doi:10.1146/annurev.immunol.021908.132603.

[140] R.A. DeMarco, M.P. Fink, M.T. Lotze, Monocytes promote natural killer cell

interferon gamma production in response to the endogenous danger signal

HMGB1., Mol. Immunol. 42 (2005) 433–44. doi:10.1016/j.molimm.2004.07.023.

[141] P. Scaffidi, T. Misteli, M.E. Bianchi, Release of chromatin protein HMGB1 by

necrotic cells triggers inflammation., Nature. 418 (2002) 191–5.

doi:10.1038/nature00858.

[142] A. Tsung, J.R. Klune, X. Zhang, G. Jeyabalan, Z. Cao, X. Peng, et al., HMGB1

release induced by liver ischemia involves Toll-like receptor 4 dependent

reactive oxygen species production and calcium-mediated signaling., J. Exp.

Med. 204 (2007) 2913–23. doi:10.1084/jem.20070247.

[143] A. Tsung, R. Sahai, H. Tanaka, A. Nakao, M.P. Fink, M.T. Lotze, et al., The

nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-

reperfusion, J. Exp. Med. JEM. 0 (2005) 1135–1143.

doi:10.1084/jem.20042614.

[144] K. Watanabe, V. Karuppagounder, S. Arumugam, R.A. Thandavarayan, V.


180 | P a g e

Pitchaimani, R. Sreedhar, et al., Pruni cortex ameliorates skin inflammation

possibly through HMGB1-NFκB pathway in house dust mite induced atopic

dermatitis NC/Nga transgenic mice., J. Clin. Biochem. Nutr. 56 (2015) 186–

94. doi:10.3164/jcbn.14-75.

[145] V. Karuppagounder, S. Arumugam, R.A. Thandavarayan, V. Pitchaimani, R.

Sreedhar, R. Afrin, et al., Modulation of HMGB1 translocation and

RAGE/NFκB cascade by quercetin treatment mitigates atopic dermatitis in

NC/Nga transgenic mice., Exp. Dermatol. 24 (2015) 418–23.

doi:10.1111/exd.12685.

[146] A. Dela Peña, I. Leclercq, J. Field, J. George, B. Jones, G. Farrell, NF-kappaB

activation, rather than TNF, mediates hepatic inflammation in a murine

dietary model of steatohepatitis., Gastroenterology. 129 (2005) 1663–74.

doi:10.1053/j.gastro.2005.09.004.

[147] M. Chen, Y. Liu, P. Varley, Y. Chang, X.-X. He, H. Huang, et al., High-

Mobility Group Box 1 Promotes Hepatocellular Carcinoma Progression

through miR-21-Mediated Matrix Metalloproteinase Activity., Cancer Res. 75

(2015) 1645–56. doi:10.1158/0008-5472.CAN-14-2147.

[148] M.M. Soliman, M. Abdo Nassan, T.A. Ismail, Immunohistochemical and

molecular study on the protective effect of curcumin against hepatic toxicity

induced by paracetamol in Wistar rats., BMC Complement. Altern. Med. 14

(2014) 457. doi:10.1186/1472-6882-14-457.

[149] Y. Fu, S. Zheng, J. Lin, J. Ryerse, A. Chen, Curcumin protects the rat liver

from CCl4-caused injury and fibrogenesis by attenuating oxidative stress and

suppressing inflammation., Mol. Pharmacol. 73 (2008) 399–409.

doi:10.1124/mol.107.039818.

[150] R. Afrin, S. Arumugam, V. Soetikno, R.A. Thandavarayan, V. Pitchaimani, V.

Karuppagounder, et al., Curcumin ameliorates streptozotocin-induced liver

damage through modulation of endoplasmic reticulum stress-mediated

apoptosis in diabetic rats, Free Radic. Res. 49 (2015) 279–289.


181 | P a g e

doi:10.3109/10715762.2014.999674.

[151] J.U. Marquardt, L. Gomez-Quiroz, L.O. Arreguin Camacho, F. Pinna, Y.-H.

Lee, M. Kitade, et al., Curcumin effectively inhibits oncogenic NF-κB

signaling and restrains stemness features in liver cancer., J. Hepatol. 63

(2015) 661–9. doi:10.1016/j.jhep.2015.04.018.

[152] Q. Gu, H. Guan, Q. Shi, Y. Zhang, H. Yang, Curcumin attenuated acute

Propionibacterium acnes-induced liver injury through inhibition of HMGB1

expression in mice., Int. Immunopharmacol. 24 (2015) 159–65.

doi:10.1016/j.intimp.2014.12.005.

[153] C. Tu, Q. Yao, B. Xu, J. Wang, C. Zhou, S. Zhang, Protective effects of

curcumin against hepatic fibrosis induced by carbon tetrachloride: modulation

of high-mobility group box 1, Toll-like receptor 4 and 2 expression., Food

Chem. Toxicol. 50 (2012) 3343–51. doi:10.1016/j.fct.2012.05.050.

[154] C. Wang, H. Nie, K. Li, Y. Zhang, F. Yang, C. Li, et al., Curcumin inhibits

HMGB1 releasing and attenuates concanavalin A-induced hepatitis in mice.,

Eur. J. Pharmacol. 697 (2012) 152–7. doi:10.1016/j.ejphar.2012.09.050.

[155] R.D. Lillie, Further Experiments with the Masson Trichrome Modification of

Mallory’s Connective Tissue Stain, Stain Technol. (2009).

http://www.tandfonline.com/doi/abs/10.3109/10520294009110327.

[156] V. Soetikno, F.R. Sari, A.P. Lakshmanan, S. Arumugam, M. Harima, K.

Suzuki, et al., Curcumin alleviates oxidative stress, inflammation, and renal

fibrosis in remnant kidney through the Nrf2-keap1 pathway, Mol. Nutr. Food

Res. 57 (2013) 1649–1659. doi:10.1002/mnfr.201200540.

[157] M. Kinoshita, T. Uchida, A. Sato, M. Nakashima, H. Nakashima, S. Shono, et

al., Characterization of two F4/80-positive Kupffer cell subsets by their

function and phenotype in mice., J. Hepatol. 53 (2010) 903–10.

doi:10.1016/j.jhep.2010.04.037.

[158] L. Cominacini, Oxidized Low Density Lipoprotein (ox-LDL) Binding to ox-LDL


182 | P a g e

Receptor-1 in Endothelial Cells Induces the Activation of NF-kappa B through

an Increased Production of Intracellular Reactive Oxygen Species, J. Biol.

Chem. 275 (2000) 12633–12638. doi:10.1074/jbc.275.17.12633.

[159] C.Z. Larter, M.M. Yeh, Animal models of NASH: Getting both pathology and

metabolic context right, J. Gastroenterol. Hepatol. 23 (2008) 1635–1648.

doi:10.1111/j.1440-1746.2008.05543.x.

[160] J.A. Davila, Diabetes and hepatocellular carcinoma: what role does diabetes

have in the presence of other known risk factors?, Am. J. Gastroenterol. 105

(2010) 632–4. doi:10.1038/ajg.2009.715.

[161] C.D. Lao, M.T. Ruffin, D. Normolle, D.D. Heath, S.I. Murray, J.M. Bailey, et

al., Dose escalation of a curcuminoid formulation., BMC Complement. Altern.

Med. 6 (2006) 10. doi:10.1186/1472-6882-6-10.

[162] T. Csak, A. Velayudham, I. Hritz, J. Petrasek, I. Levin, D. Lippai, et al.,

Deficiency in myeloid differentiation factor-2 and toll-like receptor 4

expression attenuates nonalcoholic steatohepatitis and fibrosis in mice., Am.

J. Physiol. Gastrointest. Liver Physiol. 300 (2011) G433-41.

doi:10.1152/ajpgi.00163.2009.

[163] L. Mollica, F. De Marchis, A. Spitaleri, C. Dallacosta, D. Pennacchini, M.

Zamai, et al., Glycyrrhizin binds to high-mobility group box 1 protein and

inhibits its cytokine activities., Chem. Biol. 14 (2007) 431–41.

doi:10.1016/j.chembiol.2007.03.007.

[164] K. Ikeda, Y. Arase, M. Kobayashi, S. Saitoh, T. Someya, T. Hosaka, et al., A

long-term glycyrrhizin injection therapy reduces hepatocellular carcinogenesis

rate in patients with interferon-resistant active chronic hepatitis C: a cohort

study of 1249 patients., Dig. Dis. Sci. 51 (2006) 603–9. doi:10.1007/s10620-

006-3177-0.

[165] G. Shiota, K. Harada, M. Ishida, Y. Tomie, M. Okubo, S. Katayama, et al.,

Inhibition of hepatocellular carcinoma by glycyrrhizin in diethylnitrosamine-

treated mice., Carcinogenesis. 20 (1999) 59–63.


183 | P a g e


[166] B. Rendon-Mitchell, M. Ochani, J. Li, J. Han, H. Wang, H. Yang, et al., IFN-

gamma induces high mobility group box 1 protein release partly through a

TNF-dependent mechanism., J. Immunol. 170 (2003) 3890–7.


[167] H. Wang, O. Bloom, M. Zhang, J.M. Vishnubhakat, M. Ombrellino, J. Che, et

al., HMG-1 as a late mediator of endotoxin lethality in mice., Science. 285

(1999) 248–51. http://www.ncbi.nlm.nih.gov/pubmed/10398600.

[168] G.A. Michelotti, M. V. Machado, A.M. Diehl, NAFLD, NASH and liver cancer,

Nat. Rev. Gastroenterol. Hepatol. 10 (2013) 656–665.

doi:10.1038/nrgastro.2013.183.

[169] V. Racanelli, B. Rehermann, The liver as an immunological organ.,

Hepatology. 43 (2006) S54-62. doi:10.1002/hep.21060.

[170] M. Inoue, T. Ohtake, W. Motomura, N. Takahashi, Y. Hosoki, S. Miyoshi, et

al., Increased expression of PPARgamma in high fat diet-induced liver

steatosis in mice., Biochem. Biophys. Res. Commun. 336 (2005) 215–22.

doi:10.1016/j.bbrc.2005.08.070.

[171] V. Gazit, J. Huang, A. Weymann, D.A. Rudnick, Analysis of the role of hepatic

PPARγ expression during mouse liver regeneration., Hepatology. 56 (2012)

1489–98. doi:10.1002/hep.25880.

[172] D. Tang, R. Kang, H.J. Zeh Iii, M.T. Lotze, High-Mobility Group Box 1,

Oxidative Stress, and Disease, Antioxid. Redox Signal. 14 (2011) 1315–1335.

[173] Y. Wang, J. Shan, W. Yang, H. Zheng, S. Xue, High Mobility Group Box 1

(HMGB1) Mediates High-Glucose-Induced Calcification in Vascular Smooth

Muscle Cells of Saphenous Veins, Inflammation. 36 (2013) 1592–1604.

doi:10.1007/s10753-013-9704-1.

[174] B. Li, L. Wang, Q. Lu, W. Da, Liver injury attenuation by curcumin in a rat

NASH model: an Nrf2 activation-mediated effect?, Ir. J. Med. Sci. (2014).


184 | P a g e

doi:10.1007/s11845-014-1226-9.

[175] S. Reuter, S.C. Gupta, M.M. Chaturvedi, B.B. Aggarwal, Oxidative stress,

inflammation, and cancer: how are they linked?, Free Radic. Biol. Med. 49

(2010) 1603–16. doi:10.1016/j.freeradbiomed.2010.09.006.

[176] R.S. Warren, H. Yuan, M.R. Matli, N.A. Gillett, N. Ferrara, Regulation by

vascular endothelial growth factor of human colon cancer tumorigenesis in a

mouse model of experimental liver metastasis., J. Clin. Invest. 95 (1995)

1789–97. doi:10.1172/JCI117857.

[177] M. Capurro, I.R. Wanless, M. Sherman, G. Deboer, W. Shi, E. Miyoshi, et al.,

Glypican-3: a novel serum and histochemical marker for hepatocellular

carcinoma., Gastroenterology. 125 (2003) 89–97.


[178] T. Fujikawa, H. Shiraha, K. Yamamoto, Significance of des-gamma-carboxy

prothrombin production in hepatocellular carcinoma., Acta Med. Okayama. 63


[179] G.C. Farrell, V.W.-S. Wong, S. Chitturi, NAFLD in Asia—as common and

important as in the West, Nat. Rev. Gastroenterol. Hepatol. 10 (2013) 307–

318. doi:10.1038/nrgastro.2013.34.

[180] G.C. Farrell, C.Z. Larter, Nonalcoholic fatty liver disease: From steatosis to

cirrhosis, Hepatology. 43 (2006) S99–S112. doi:10.1002/hep.20973.

[181] G.C. Farrell, D. van Rooyen, L. Gan, S. Chitturi, NASH is an Infl ammatory

Disorder: Pathogenic, Prognostic and Therapeutic Implications, Gut Liver. 6

(2012) 149–171. doi:10.5009/gnl.2012.6.2.149.

[182] A.P. Rolo, J.S. Teodoro, C.M. Palmeira, Role of oxidative stress in the

pathogenesis of nonalcoholic steatohepatitis., Free Radic. Biol. Med. 52 (2012)

59–69. doi:10.1016/j.freeradbiomed.2011.10.003.

[183] X. Hou, S. Xu, K.A. Maitland-Toolan, K. Sato, B. Jiang, Y. Ido, et al., SIRT1

Regulates Hepatocyte Lipid Metabolism through Activating AMP-activated


185 | P a g e

Protein Kinase, J. Biol. Chem. 283 (2008) 20015–20026.

doi:10.1074/jbc.M802187200.

[184] M.A. Suter, A. Chen, M.S. Burdine, M. Choudhury, R.A. Harris, R.H. Lane, et

al., A maternal high-fat diet modulates fetal SIRT1 histone and protein

deacetylase activity in nonhuman primates., FASEB J. 26 (2012) 5106–14.

doi:10.1096/fj.12-212878.

[185] Y. Colak, O. Ozturk, E. Senates, I. Tuncer, E. Yorulmaz, G. Adali, et al.,

SIRT1 as a potential therapeutic target for treatment of nonalcoholic fatty

liver disease., Med. Sci. Monit. 17 (2011) HY5-9.


[186] D.G. Hardie, AMPK: a key regulator of energy balance in the single cell and

the whole organism, Int. J. Obes. 32 (2008) S7–S12. doi:10.1038/ijo.2008.116.

[187] C. Cantó, J. Auwerx, PGC-1α, SIRT1 and AMPK, an energy sensing network

that controls energy expenditure, Curr. Opin. Lipidol. 20 (2009) 98–105.

doi:10.1097/MOL.0b013e328328d0a4.

[188] A. Purushotham, T.T. Schug, Q. Xu, S. Surapureddi, X. Guo, X. Li,

Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results

in hepatic steatosis and inflammation., Cell Metab. 9 (2009) 327–38.

doi:10.1016/j.cmet.2009.02.006.

[189] T. Yamauchi, J. Kamon, H. Waki, K. Murakami, K. Motojima, K. Komeda, et

al., The mechanisms by which both heterozygous peroxisome proliferator-

activated receptor gamma (PPARgamma) deficiency and PPARgamma agonist

improve insulin resistance., J. Biol. Chem. 276 (2001) 41245–54.

doi:10.1074/jbc.M103241200.

[190] S. Francque, A. Verrijken, S. Caron, J. Prawitt, R. Paumelle, B. Derudas, et

al., PPARα gene expression correlates with severity and histological treatment

response in patients with non-alcoholic steatohepatitis, J. Hepatol. 63 (2015)

164–173. doi:10.1016/j.jhep.2015.02.019.


186 | P a g e

[191] W.N. Hannah, S.A. Harrison, Lifestyle and Dietary Interventions in the

Management of Nonalcoholic Fatty Liver Disease, Dig. Dis. Sci. 61 (2016)

1365–1374. doi:10.1007/s10620-016-4153-y.

[192] H. Yamazaki, M. Fujieda, M. Togashi, T. Saito, G. Preti, J.R. Cashman, et al.,

Effects of the dietary supplements, activated charcoal and copper

chlorophyllin, on urinary excretion of trimethylamine in Japanese

trimethylaminuria patients., Life Sci. 74 (2004) 2739–47.

doi:10.1016/j.lfs.2003.10.022.

[193] R. Afrin, S. Arumugam, M.I.I. Wahed, V. Pitchaimani, V. Karuppagounder, R.

Sreedhar, et al., Attenuation of Endoplasmic Reticulum Stress-Mediated Liver

Damage by Mulberry Leaf Diet in Streptozotocin-Induced Diabetic Rats, Am.

J. Chin. Med. 44 (2016) 87–101. doi:10.1142/S0192415X16500063.

[194] M.S. Seo, J.H. Kim, H.J. Kim, K.C. Chang, S.W. Park, Honokiol activates the

LKB1-AMPK signaling pathway and attenuates the lipid accumulation in

hepatocytes., Toxicol. Appl. Pharmacol. 284 (2015) 113–24.

doi:10.1016/j.taap.2015.02.020.

[195] X.-N. Li, J. Song, L. Zhang, S.A. LeMaire, X. Hou, C. Zhang, et al., Activation

of the AMPK-FOXO3 pathway reduces fatty acid-induced increase in

intracellular reactive oxygen species by upregulating thioredoxin., Diabetes.

58 (2009) 2246–57. doi:10.2337/db08-1512.

[196] J. Ertle, A. Dechêne, J.-P. Sowa, V. Penndorf, K. Herzer, G. Kaiser, et al.,

Non-alcoholic fatty liver disease progresses to hepatocellular carcinoma in the

absence of apparent cirrhosis., Int. J. Cancer. 128 (2011) 2436–43.

doi:10.1002/ijc.25797.

[197] P.J. Neuvonen, P. Kuusisto, H. Vapaatalo, V. Manninen, Activated charcoal in

the treatment of hypercholesterolaemia: dose-response relationships and

comparison with cholestyramine., Eur. J. Clin. Pharmacol. 37 (1989) 225–30.


[198] B.B. Zhang, G. Zhou, C. Li, AMPK: an emerging drug target for diabetes and


187 | P a g e

the metabolic syndrome., Cell Metab. 9 (2009) 407–16.

doi:10.1016/j.cmet.2009.03.012.

[199] D.B. Shackelford, R.J. Shaw, The LKB1-AMPK pathway: metabolism and

growth control in tumour suppression., Nat. Rev. Cancer. 9 (2009) 563–75.

doi:10.1038/nrc2676.

[200] N.B. Ruderman, X.J. Xu, L. Nelson, J.M. Cacicedo, A.K. Saha, F. Lan, et al.,

AMPK and SIRT1: a long-standing partnership?, Am. J. Physiol. Endocrinol.

Metab. 298 (2010) E751-60. doi:10.1152/ajpendo.00745.2009.

[201] L. Zheng, W. Yang, F. Wu, C. Wang, L. Yu, L. Tang, et al., Prognostic

Significance of AMPK Activation and Therapeutic Effects of Metformin in

Hepatocellular Carcinoma, Clin. Cancer Res. 19 (2013) 5372–5380.

doi:10.1158/1078-0432.CCR-13-0203.

[202] Z. Xie, J. Zhang, J. Wu, B. Viollet, M.-H. Zou, Upregulation of mitochondrial

uncoupling protein-2 by the AMP-activated protein kinase in endothelial cells

attenuates oxidative stress in diabetes., Diabetes. 57 (2008) 3222–30.

doi:10.2337/db08-0610.

[203] K. Abdelmohsen, R. Pullmann, A. Lal, H.H. Kim, S. Galban, X. Yang, et al.,

Phosphorylation of HuR by Chk2 Regulates SIRT1 Expression, Mol. Cell. 25

(2007) 543–557. doi:10.1016/j.molcel.2007.01.011.

[204] A. Salminen, J.M.T. Hyttinen, K. Kaarniranta, AMP-activated protein kinase

inhibits NF-κB signaling and inflammation: impact on healthspan and

lifespan., J. Mol. Med. (Berl). 89 (2011) 667–76. doi:10.1007/s00109-011-0748-

0.

[205] J.T. Rodgers, C. Lerin, W. Haas, S.P. Gygi, B.M. Spiegelman, P. Puigserver,

Nutrient control of glucose homeostasis through a complex of PGC-1α and

SIRT1, Nature. 434 (2005) 113–118. doi:10.1038/nature03354.

[206] Y. Zhang, L.W. Castellani, C.J. Sinal, F.J. Gonzalez, P.A. Edwards,

Peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-


188 | P a g e

1alpha) regulates triglyceride metabolism by activation of the nuclear receptor

FXR., Genes Dev. 18 (2004) 157–69. doi:10.1101/gad.1138104.

[207] T.C. Leone, J.J. Lehman, B.N. Finck, P.J. Schaeffer, A.R. Wende, S. Boudina,

et al., PGC-1alpha deficiency causes multi-system energy metabolic

derangements: muscle dysfunction, abnormal weight control and hepatic

steatosis., PLoS Biol. 3 (2005) e101. doi:10.1371/journal.pbio.0030101.

[208] X. Qiang, L. Xu, M. Zhang, P. Zhang, Y. Wang, Y. Wang, et al.,

Demethyleneberberine attenuates non-alcoholic fatty liver disease with

activation of AMPK and inhibition of oxidative stress., Biochem. Biophys. Res.

Commun. 472 (2016) 603–9. doi:10.1016/j.bbrc.2016.03.019.

[209] J.M. Ajmo, X. Liang, C.Q. Rogers, B. Pennock, M. You, Resveratrol alleviates

alcoholic fatty liver in mice., Am. J. Physiol. Gastrointest. Liver Physiol. 295

(2008) G833-42. doi:10.1152/ajpgi.90358.2008.

[210] R. Rahimian, E. Masih-Khan, M. Lo, C. van Breemen, B.M. McManus, G.P.

Dubé, Hepatic over-expression of peroxisome proliferator activated receptor

gamma2 in the ob/ob mouse model of non-insulin dependent diabetes

mellitus., Mol. Cell. Biochem. 224 (2001) 29–37.


[211] N. Kubota, Y. Terauchi, H. Miki, H. Tamemoto, T. Yamauchi, K. Komeda, et

al., PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and

insulin resistance., Mol. Cell. 4 (1999) 597–609.


[212] P.D. Miles, Y. Barak, W. He, R.M. Evans, J.M. Olefsky, Improved insulin-

sensitivity in mice heterozygous for PPAR-gamma deficiency., J. Clin. Invest.

105 (2000) 287–92. doi:10.1172/JCI8538.

[213] L. Malaguarnera, R. Madeddu, E. Palio, N. Arena, M. Malaguarnera, Heme

oxygenase-1 levels and oxidative stress-related parameters in non-alcoholic

fatty liver disease patients, J. Hepatol. 42 (2005) 585–591.

doi:10.1016/j.jhep.2004.11.040.


189 | P a g e

[214] C. Zou, C. Zou, W. Cheng, Q. Li, Z. Han, X. Wang, et al., Heme oxygenase-1

retards hepatocellular carcinoma progression through the microRNA

pathway., Oncol. Rep. 36 (2016) 2715–2722. doi:10.3892/or.2016.5056.

[215] S.-M. Kim, J.P. Grenert, C. Patterson, M.A. Correia, CHIP(-/-)-Mouse Liver:

Adiponectin-AMPK-FOXO-Activation Overrides CYP2E1-Elicited JNK1-

Activation, Delaying Onset of NASH: Therapeutic Implications., Sci. Rep. 6

(2016) 29423. doi:10.1038/srep29423.

[216] H. Nakagawa, A. Umemura, K. Taniguchi, J. Font-Burgada, D. Dhar, H.

Ogata, et al., ER stress cooperates with hypernutrition to trigger TNF-

dependent spontaneous HCC development., Cancer Cell. 26 (2014) 331–43.

doi:10.1016/j.ccr.2014.07.001.

[217] H. Fujii, Y. Ikura, J. Arimoto, K. Sugioka, J.C. Iezzoni, S.H. Park, et al.,

Expression of perilipin and adipophilin in nonalcoholic fatty liver disease;

relevance to oxidative injury and hepatocyte ballooning., J. Atheroscler.

Thromb. 16 (2009) 893–901. http://www.ncbi.nlm.nih.gov/pubmed/20032580.

[218] T. Tsujimoto, H. Kawaratani, T. Kitazawa, T. Hirai, H. Ohishi, M. Kitade, et

al., Decreased phagocytic activity of Kupffer cells in a rat nonalcoholic

steatohepatitis model., World J. Gastroenterol. 14 (2008) 6036–43.


[219] B.C. Tieu, C. Lee, H. Sun, W. Lejeune, A. Recinos, X. Ju, et al., An adventitial

IL-6/MCP1 amplification loop accelerates macrophage-mediated vascular

inflammation leading to aortic dissection in mice., J. Clin. Invest. 119 (2009)

3637–51. doi:10.1172/JCI38308.

[220] G. He, D. Dhar, H. Nakagawa, J. Font-Burgada, H. Ogata, Y. Jiang, et al.,

Identification of liver cancer progenitors whose malignant progression

depends on autocrine IL-6 signaling., Cell. 155 (2013) 384–96.

doi:10.1016/j.cell.2013.09.031.

[221] S. Zhong, L. Zhao, Y. Wang, C. Zhang, J. Liu, P. Wang, et al., CD36 deficiency

aggravates macrophage infiltration and hepatic inflammation by up-


190 | P a g e

regulating MCP-1 expression of hepatocytes through HDAC2-dependant

pathway., Antioxid. Redox Signal. (2016) ars.2016.6808.

doi:10.1089/ars.2016.6808.

[222] A.M. Bode, Z. Dong, Post-translational modification of p53 in tumorigenesis.,

Nat. Rev. Cancer. 4 (2004) 793–805. doi:10.1038/nrc1455.

[223] R.G. Jones, D.R. Plas, S. Kubek, M. Buzzai, J. Mu, Y. Xu, et al., AMP-

activated protein kinase induces a p53-dependent metabolic checkpoint., Mol.

Cell. 18 (2005) 283–93. doi:10.1016/j.molcel.2005.03.027.

[224] Y. Fazel, A.B. Koenig, M. Sayiner, Z.D. Goodman, Z.M. Younossi,

Epidemiology and natural history of non-alcoholic fatty liver disease,

Metabolism. 65 (2016) 1017–1025. doi:10.1016/j.metabol.2016.01.012.

[225] B.W. Smith, L.A. Adams, Nonalcoholic fatty liver disease and diabetes

mellitus: pathogenesis and treatment., Nat. Rev. Endocrinol. 7 (2011) 456–65.

doi:10.1038/nrendo.2011.72.

[226] C. Nugent, Z.M. Younossi, Evaluation and management of obesity-related

nonalcoholic fatty liver disease., Nat. Clin. Pract. Gastroenterol. Hepatol. 4

(2007) 432–41. doi:10.1038/ncpgasthep0879.

[227] R. Afrin, S. Arumugam, A. Rahman, M.I.I. Wahed, V. Karuppagounder, M.

Harima, et al., Curcumin ameliorates liver damage and progression of NASH

in NASH-HCC mouse model possibly by modulating HMGB1-NF-κB

translocation, Int. Immunopharmacol. 44 (2017) 174–182.

doi:10.1016/j.intimp.2017.01.016.

[228] E. Bugianesi, N. Leone, E. Vanni, G. Marchesini, F. Brunello, P. Carucci, et

al., Expanding the natural history of nonalcoholic steatohepatitis: from

cryptogenic cirrhosis to hepatocellular carcinoma., Gastroenterology. 123


[229] S.E. Shoelson, J. Lee, A.B. Goldfine, Inflammation and insulin resistance., J.

Clin. Invest. 116 (2006) 1793–801. doi:10.1172/JCI29069.


191 | P a g e

[230] C.J. Rhodes, Type 2 Diabetes-a Matter of -Cell Life and Death?, Science 307

(5708) (2005) 380–384. doi:10.1126/science.1104345.

[231] M.E. Guicciardi, G.J. Gores, Apoptosis as a mechanism for liver disease

progression., Semin. Liver Dis. 30 (2010) 402–10. doi:10.1055/s-0030-1267540.

[232] C.P. Day, O.F. James, Steatohepatitis: a tale of two "hits"?,

Gastroenterology. 114 (1998) 842–5.



192 | P a g e

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.


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

http://www.worldscientific.com/doi/abs/10.1142/S0192415X16500063

http://www.worldscientific.com/doi/abs/10.1142/S0192415X16500063


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


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


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.



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


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


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.




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.





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.


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.

non-alcoholic steatohepatitis onset of mechanisms under … · 2018-02-02 · non-alcoholic fatty...

Documents