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Research Article
CYNARA SCOLYMUS FOR RELIEVING ON NONALCOHOLIC STEATOHEPATITIS INDUCED IN RATS
SAFAA H MOHAMED1, HANAA H AHMED1, ABDEL RAZIK H FARRAG 2, NAHLA S ABDEL-AZIM3,
ABDELAATY A SHAHAT3,4
1Department of Hormone, 2 Department of Pathology, 3Phytochemisrty Department, National Research Centre, El Bohous Street, 12311
Dokki, Cairo, Egypt. 4Medicinal, Aromatic and Poisonous Plants Research Center, College of Pharmacy, King Saud University, PO Box 2457,
Riyadh 11451, Saudi Arabia. Email: aashahat@hotmail.com, ashahat@ksu.edu.sa
Received: 12 Sep 2012, Revised and Accepted: 29 Oct 2012
ABSTRACT
Objective: The current study was undertaken to evaluate the efficacy of the total crude aqueous methanolic extract of Cynara scolymus and its
fraction against high fat diet-induced of nonalcoholic steatohepatitis (NASH) in adult female rats. Methods: Forty adult female Sprague Dawley rats
were classified into 4 groups. The first group was kept on standard rodent chow and served as healthy control. The other groups received high fat
diet (HFD) for 32 weeks for NASH induction. These animals were assigned as NASH-induced group, Cynara scolymus (CSM) extract-treated group
and purified fraction (CSF) -treated group. Results: The results revealed significant increase in serum ALT activity, cholesterol, LDL and triglycerides
levels as well as leptin and resistin levels. Additionally, serum NF-κB, TNF-α, Cox-2, CD 40 and HGF levels have been increased significantly, while,
serum HDL and adeponectin levels have been decreased significantly in NASH-induced group compared with healthy control group. Conversely,
treatment with CSM or CSF resulted in significant decrease in serum ALT activity, cholesterol, LDL and triglycerides levels as well as leptin and
resistin levels. Serum NF-κB, TNF-α, Cox-2, CD40 and HGF levels also showed significant decrease. While serum HDL and adiponectin levels were
significantly increased as a consequence of treatment with either CSM or CSF as compared to the untreated NASH-induced rats. The
photomicrogrophs of liver section of rats treated with CSM or CSF extract confirmed the present improvement in the studied biomarkers. The
results suggested that Cynara scolymus extract or its purified fraction possess hepatoprotective activity, hypolipidemic effect and anti-inflammatory
property. Conclusion: Thus, our findings reinforce current advice recommending the consumption of natural products to modulate nonalcoholic
steatohepatitis and its metabolic complications.
Keywords: Cynara scolymus, Nonalcoholic steatohepatitis, Insulin resistance, Inflammation, Hyperlipidemia, Rats.
INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD) is a clinic pathologic entity
increasingly recognized as a major health burden in developed as
well as in developing countries. It includes a spectrum of liver
damage ranging from simple steatosis to nonalcoholic
steatohepatitis (NASH), advanced fibrosis, and probable progression
to cirrhosis [1]. The presence of NASH with cirrhosis has been
documented in large series. Cirrhosis occurs in a minority of NASH
patients, but the overall incidence has been reported to be as high as
26%. Progression of fibrosis as detected by liver biopsy has been
reported to occur in 43% of NASH patients, while 54% of patients
remained unchanged and 3% showed histologic improvement
during a follow-up from 1 to 7 years [2]. In general, 30-50% of
individuals with NASH will develop fibrosis, 15% will develop
cirrhosis and 3% will progress to terminal liver failure [3]. Among
the many causative factors of NASH, oxidative stress, lipid
peroxidation and inflammation are considered the most probable
causative factors [4]. NASH is believed to be a feature of metabolic
syndrome because it is closely associated with visceral obesity,
dyslipidaemia, insulin resistance, and type 2 diabetes mellitus [5].
Artichoke (Cynara scolymus L.), Asteraceae family (Compositae) is a
plant that is widely grown in Mediterranean countries and is rich in
natural antioxidants. It is not only a good food, known for its
pleasant bitter taste, but also an interesting and widespread herbal
drug [6]. Artichoke leaf contains up to 2% phenolic acids, mainly 3-
caffeoylquinic acid (chlorogenic acid), plus 1.3-di-O-caffeoylquinic
acid (cynarin), and caffeic acid; 0.4% bitter sesquiterpene lactones
of which 47-83% is cynaropicrin; 0.11.0% flavonoids including the
glycosides luteolin-7-β-rutinoside (scolymoside), luteolin-7- β-D-
glucoside and luteolin-4-β-D-glucoside; phytosterols (taraxasterol);
sugars; inulin; enzymes; and a volatile oil consisting mainly of the
sesquiterpenes β-selinene and caryophyllene [7, 8].
The artichoke leaf extract has been used as hepatoprotective [9],
antimicrobial [10] and cholesterol reducing purposes [11].
Artichoke has been found to decrease the production of reactive
oxygen species, the oxidation of low-density lipoproteins [12], lipid
peroxidation [9], and protein oxidation and increase the activity of
glutathione peroxidase [13].
The aim of the present article is to investigate the efficacy of Cynara
scolymus total methanolic extract (CSM) and its fraction (CSF)
against high fat diet-induced NASH in adult female rats in attempt to
understand their mechanisms of action, which may pave the way for
possible therapeutic applications. This could be achieved through
conducting routine biochemical analysis for liver functions,
estimating the circulating levels of insulin resistance indices,
evaluating serum levels of inflammatory markers. Histopathological
investigation of liver sections was also carried out to confirm the
biochemical analyses.
MATERIALS AND METHODS
Plant materials
Preparation of Cynara scolomus total extracts (CSM)
The leaves of Cynara scolomus were collected from the experimental
farm at Nubaria. Alexandria, Egypt on October 2009, air dried (3 kg)
and extracted with 80 % methanol at room temperature for three
times , followed by the removal of solvent under reduced pressure to
obtain the crude aqueous methanolic extract (CSM) (26 % from the
dried leaves).
Preparation of Cynara scolomus fraction (CSF)
300 g of CSM was subjected to silica gel column chromatography and
eluted with solvent of increasing polarity
(hexane/ethylacetate/methanol). The fractions eluted with ethyl
acetate/methanol (1:1) were collected together to give a purified
fraction (CSF) (120 g).
Animals
The present study was conducted on forty adult female Sprague
Dawley rats weighing 120-150g obtained from the Animal House
Colony of the National Research Centre, Cairo, Egypt. The animals
were maintained on standard laboratory diet and water ad libitum
for two weeks before starting the experiment. All animals received
human care and use according to the guide lines for Animal
Experiments which were approved by the Ethical Committee of
Medical Research, National Research Centre, Egypt. Steatohepatitis
(NASH) was induced in rats by using high fat diet which provided
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 5, Suppl 1, 2013
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Int J Pharm Pharm Sci, Vol 5, Suppl 1, 57-66
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30% of its energy from fat, 35% from carbohydrate and 35% from
protein (casein) for 32 weeks. Supplements of vitamins and minerals
were also included [14].
Experimental set-up
The animals were classified into four groups with ten animals in
each: (1) Healthy control group which was fed ad-libitum with an
isocaloric regular rat chow [15], (2) Steatohepatitis (NASH) -
induced group which was fed ad-libitum with high fat diet [14], (3)
NASH -induced group orally treated with 150 mg/ kg b.wt. of CSM
daily for 8 weeks. This dose was calculated from the chronic toxicity
study for CSM (data not shown), and (4): NASH -induced group
orally treated with 150 mg/ kg b.wt. of CSF daily for 8 weeks. This
dose was calculated from the chronic toxicity study for CSF (data not
shown).
At the end of the experimental period, the rats were fasted overnight
and the blood samples were collected from the retro orbital plexus
under diethylether anaesthesia [16]. The blood samples were left to
clot and then centrifuged using cooling centrifuge at 1800 xg for ten
minutes to obtain sera. The clear serum samples were stored at -20
ºC until analysis. After blood collection, all animals were rapidly
killed and the liver tissues were dissected, washed in isotonic saline,
then cut into small pieces (0.5x0.5cm) and fixed in 10% saline
buffered formalin overnight for histological examination.
Biochemical assays
Serum alanine transaminase (ALT) activity was estimated
colorimetrically using kit purchased from Quimica Clinica Aplicada
S.A. Co., Spain, according to the method of Reitman and Frankel [17].
Serum cholesterol (Chol) concentration was determined
colorimetrically using kit purchased from Stanbio Laboratory,
Boerne, Texas, USA, according to the method of Allain et al. [18].
Serum LDL-cholesterol (LDL) concentration was assayed
colorimetrically using kit purchased from Quimica Clinica Aplicada
S.A. Co., Spain, according to the method of Assman et al. [19]. Serum
HDL-cholesterol (HDL) concentration was measured
colorimetrically using kit purchased from Stanbio Laboratory,
Boerne, Texas, USA, according to the method of Lopez-Virella et al.
[20]. Serum triglycerides (TG) level was determined colorimetrically
using kit purchased from Stanbio Laboratory, Boerne, Texas, USA,
according to the method of Fassati and Prencipe [21]. Serum
adiponectin concentration was measured by enzyme-linked
immunosorbent assay (ELISA) technique using kit purchased from
AssayPro, USA, according to the method of Pannacciulli et al. [22].
Serum leptin level was measured by ELISA procedure using kit
purchased from Ray Biotech Co., Georgia, USA, according to the
method described by Petridou et al. [23]. Serum resistin
concentration was determined by ELISA technique using kit
purchased from Glory Science Co., Ltd, Veterans Blvd, Suite, USA,
according to the method of Schaffler et al. [24]. Serum NF-κB p56
concentration was determined by ELISA technique using kit
purchased from Glory Science Co., Ltd, Veterans Blvd, Suite, USA,
according to the manufacturer’s instructions. Serum Cox-2
concentration was determined by ELISA technique using kit
purchased from Glory Science Co., Ltd, Veterans Blvd, Suite, USA,
according to the manufacturer’s instructions. Serum TNF-α
concentration was measured by ELISA procedure using kit
purchased from Ray Biotech Co., Georgia, USA, according to the
method of Brouckaert et al. [25]. Serum CD40 concentration was
measured by ELISA technique using kit purchased from Glory
Science Co., Ltd, Veterans Blvd, Suite, USA, according to the
manufacturer’s instructions. Serum hepatocyte growth factor (HGF)
level was quantified by ELISA procedure using kit purchased from
Glory Science Co., Ltd, Veterans Blvd, Suite, USA, according to the
method of Plum et al. [26].
Histopathological examination
Fragments of liver tissue previously fixed in 10% formalin saline were
processed and submitted to hematoxilin and eosin (H&E) stain.
SCHARLACH Rs stain was used for a more precise identification of
fatty change. Histological variables were semiquantitated from 0 to 4+,
including macro-and microvesicular fatty change, the foci of necrosis,
portal and perivenular fibrosis as well as the inflammatory infiltrate.
Statistical Analysis
In the present study, all results were expressed as Mean + S.E of the
mean. Data were analyzed by one way analysis of variance (ANOVA)
using the Statistical Package for the Social Sciences (SPSS) program,
version 11 followed by least significant difference (LSD) to compare
significance between groups [27]. Difference was considered
significant when P value was > 0.05. The percent difference was
calculated according to the following equation:
% difference = Treated group value – Control group value ∕ Control
group value X 100
RESULTS
(Table: 1) showed the effect of treatment with CSM and CSF on
serum ALT activity and lipid profile in NASH-induced rats. The
NASH-induced group showed significant increase in serum ALT
activity (60.8 %) in comparison with the healthy control group.
Conversely, treatment with CSM or with CSF produced significant
decrease in serum ALT activity (-40.9% and -39.6% respectively) in
comparison with the untreated NASH-induced group.
Table 1: Table shows the effect of treatment with CSM and CSF on serum ALT activity and lipid profile in NASH - induced rats.
Triglycerides (mg/dL)
LDL
(mg/dL)
HDL
(mg/dL)
Cholesterol
(mg/dL)
ALT
(U/L)
Parameters
Groups
64.8 ± 3.1
9.9 ± 0.2
41.3 ± 2.8
70.7 ± 1.7
35.4± 3.2
Healthy control group
95.8 ± 3.0a
(47.8 %)
18.8 ± 1.1a
(89.89 %)
20.8± 1.1a
(-49.6 %)
124.7 ± 3.7a
(76.3 %)
60.8±1.7a
(71.7%)
NASH – induced group
75.8 ± 3.7b
(-20.8 %)
12.8 ± 0.5
(-31.9 %)
30.9 ± 2.5b
(48.5%)
78.7 ± 2.5b
(-36.8 %)
40.9±1.7b
(-32.7%)
NASH +CSM treated group
72.5 ± 3.4b
(-24.36.9 %)
10.5± 0.5b
(44.14%)
33.7±0.9b
(62.01%)
75.47 ± 3.7b
(-35.4 %)
38.6±2.1b
(-36.5 %)
NASH +CSF-treated group
a: Significant change at P < 0.05 in comparison with the healthy control group.
b: Significant change at P < 0.05 in comparison with NASH-induced group
(%): percent difference with respect to the corresponding control value.
The induction of NASH produced significant elevation in serum
cholesterol, LDL and triglycerides levels (124.7%, 89.8% and 95.8%
respectively) associated with significant decline in serum HDL level
(-20.8%) in comparison with the healthy control group. On the other
hand, treatment of NASH-induced group with CSM resulted in
significant depletion in serum cholesterol, triglycerides levels and
insignificant decrease in serum LDL level (-78.7%, -75.8% and -
31.9% respectively) accompanied with significant rise in serum HDL
level (48.5%) in comparison with the untreated NASH-induced
group. Serum cholesterol, LDL and triglycerides levels were
significantly decreased by -75.4%, -44.14% and -72.5 %
respectively, while serum HDL level was significantly increased by
62.01% in NASH-induced group treated with CSF as compared to
untreated NASH-induced group.
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(Table: 2) showed the effect of treatment with CSM and its fraction
(CSF) on serum adiponectin, leptin and resistin levels in NASH-
induced rats. Significant increase in serum leptin and resistin levels
(121% and 79.2%) accompanied with significant decrease in serum
adiponectin level (-33.6%) were observed in NASH-induced group in
comparison with the healthy control group.
Table 2: Table shows the effect of treatment with CSM and CSF on serum adiponectin, leptin and resistin levels in NASH-induced rats
Resistin
(pg/mL)
Leptin
(pg/mL)
Adiponectin
(ng/mL)
Parameters
Groups
30.8 ± 0. 5
343.2 ± 2.6
10.4 ± 0.42
Healthy control group
55.2 ± 0.37a
(79.2 %)
761.2 ± 2.5a
(121 %)
6.9 ± 0.2a
(-33.6 %)
NASH – induced group
32.8 ± 0.37b
(-40.5 %)
580.8 ± 2.4b
(-23.6 %)
8.8 ± 0.3b
(27.5%)
NASH + CSM-treated group
30 ± 0.39b
(-45.6 %)
576.4 ± 2.8 b
(-24.2 %)
9.3 ± 0.2b
(34.7 %)
NASH +CSF-treated group
a: Significant change at P < 0.05 in comparison with the healthy control group.
b: Significant change at P < 0.05 in comparison with NASH-induced group
(%): percent difference with respect to the corresponding control value.
In contrast, treatment of NASH-induced group with CSM or CSF
resulted in significant decrease in serum leptin level (-23.6% and -
24.2 % respectively) and resistin level (-40.5% and -45.6 %
respectively) in concomitant with significant increase in serum
adiponectin level (27.5 and 34.7% respectively) as compared to
untreated NASH-induced group.
(Table: 3) showed the effect of treatment with CSM and CSF on
serum NF-κBp56, TNF-α levels and Cox-2 activity in NASH-induced
rats. Significant increase in serum NF-κBp56, TNF-α levels and Cox-2
activity (103.1%, 67.6% and 90.3% respectively) was recorded in
NASH-induced group in comparison with the healthy control group.
Conversely, the treatment of NASH-induced group with CSM or CSF
caused significant decrease in serum NF-κB p56 level (-44.6% and -
47.6 % respectively), TNF-α levels (-24.2% and -28.9 %
respectively) and Cox-2 activity (-25% and -65.1 % respectively) as
compared to the untreated NASH-induced group.
Table 3: Table shows the effect of treatment with CSM and CSF on serum NF-κB, TNF-α levels and Cox-2 activity in NASH-induced rats.
Cox-2
(U/L)
TNF-α
(Pg/mL)
NF-κB
(ng/mL)
Parameters
Groups
13.03 ± 0.4
58.1 ± 1.8
0.64 ± 0.04
Healthy control group
24.8 ± 1,1a
(90.3 %)
97.4± 1.2a
(67.6 %)
1.3 ± 0.1a
(103.1 %)
NASH–induced group
18.6 ± 0.3b
(-25 %)
73.8 ± 1.5b
(-24.2 %)
0.72± 0.02b
(-44.6 %)
NASH +CSM-treated group
16.2 ± 0.5b
(-34.3 %)
69.2 ± 1.2b
(-28.9 %)
0.68± 0.03b
(-47.6%)
NASH+CSF-treated group
a: Significant change at P < 0.05 in comparison with the healthy control group.
b: Significant change at P < 0.05 in comparison with NASH-induced group
(%): percent difference with respect to the corresponding control value.
The effect of treatment with CSM or CSF on serum CD40 and HGF levels
in NASH -induced rats was illustrated in (Table: 4). The data revealed
that the NASH–induced group showed significant increase in CD40 and
HGF levels (95.4% and 88.5% respectively) in comparison with the
healthy control group. Meanwhile, treatment of NASH-induced group
with CSM or CSF resulted in significant decrease in serum CD 40 (-31%
and -34.2% respectively) and HGF levels (-23.4% and-
28.1%respectively) as compared to the untreated NASH-induced group.
Table 4: Table shows the effect of treatment with CSM and CSF on serum CD40 and HGF levels in NASH-induced rats.
HGF
(ng/L)
CD40
(ng/L)
Parameters
Groups
102.40 ± 1.6
377.2 ± 1.8
Healthy control group
193.05 ± 1.4a (88.5 %)
737.2 ± 2.9a (95.4 %)
NASH – induced group
147.70 ± 1.6b (-23.4 %)
508.4 ± 2.6b (-31%)
NASH +CSM-treated group
138.70 ± 2.1b (-28.1 %)
485.0± 1.4b (-34.2%)
NASH +CSF-treated group
a: Significant change at P < 0.05 in comparison with the healthy control group.
b: Significant change at P < 0.05 in comparison with NASH-induced group
(%): percent difference with respect to the corresponding control value.
[[
Our histological study showed that there is no specific findings were
observed during the hepatohistological examination of the healthy
control rats (Fig.1-A). Histopathological investigation of liver tissue
slides stained with H&E in rats fed with high fat diet for induction of
NASH showed moderate to severe macrovesicular fatty changes, which
were diffusely distributed throughout the liver lobule. Parenchymal
inflammation with both acute and chronic inflammatory cells
accompanying focal necrosis was also observed (Fig. 1-B and 1-C).
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Fig. 1.A: It shows liver section of healthy control rat showing intact histological structure of the liver. Notice the central veins (CV),
hepatocytes, and blood sinusoids.
Fig. 1.B: It shows liver section of NASH induced rat showing a high degree of hepatocellular cytoplasmic vacuolation (macrovesicular and
microvesicular steatosis).
Fig. 1.c: It shows liver section of NASH–induced rat showing parenchymal inflammation with both acute and chronic inflammatory cells
accompanying focal necrosis.
Histological examination of liver tissues of NASH-induced group treated
with CSM, showed significant reduction in fatty infiltration as compared
with that in the untreated NASH-induced group (Fig. 1-D). Interestingly,
histological investigation of liver tissues of NASH-induced group treated
with the CSF revealed significant improvement in the degree of liver
fatty changes which appeared like the healthy control group (Fig. 1-E).
Fig. 1.D: It shows liver section of NASH-induced rat treated with CSM showing significant reduction in fat deposits in liver tissues.
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Fig. 1.E: It shows liver section of NASH –induced rat treated with CSF showing that the degree of liver steatosis was improved and the
histologic feature was restored to nearly normal (H & E x 300).
Histopathological examination of liver sections of healthy control
rats stained with CSHARLACH Rs stain showed negative stain (Fig. 2-
A). Moderate macro- and microvesicular fatty changes in the
periportal zone in the liver of NASH–induced rats were detected
(Fig. 2-B), whereas in NASH-induced rats treated with CSM, few of
macro and microvesicular fatty changes were observed (Fig. 2-C).
Meanwhile, no fatty infiltration was seen in liver of NASH-induced
rats treated with CSF (Fig. 2-D).
Fig. 2.A: It shows liver section of healthy control rat showing normal histological structure of the liver. The reaction is negative and the
hepatocytes are slightly swollen with centrally placed nuclei. No fatty change is seen.
Fig 2.B: It shows liver section of NASH–induced rat showing the positive reaction in the macro and microvesicular fatty infiltration.
Fig. 2.C: It shows liver section of NASH–induced rat treated with the CSM showing significant reduction in fatty deposits in liver tissues
and the reaction is negative in most areas of the lobules.
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Fig. 2.D: It shows liver section of NASH–induced rat treated with CSF showing that the reaction is negative indicating the improvement of
fatty infiltration (SCHARLACH Rs x 300).
Mean fatty infiltration in the NASH-induced group was 3 (Table: 5).
Fat deposit in this group was classified as macrovesicular. Mean
fatty infiltration in the NASH-induced group treated with CSM or CSF
was 1, and fat deposit was mixed. Fatty infiltration in the treated
groups was significantly lower than that in the untreated NASH-
induced group (P < 0.05).
Table 5: Grades of fatty infiltration in the different studied groups
Groups
Rats (n)
Steatosis grades
0
1
2
3
Healthy control group
10
10
-
-
-
NASH – induced group
10
-
-
3
8
NASH +CSM-treated group
10
8
-
2
-
NASH +CSF-treated group
10
9
-
1
-
DISCUSSION
The result of the present study revealed marked increase in serum
ALT activity in NASH group which is in agreement with Hooper et al.
[28]. Both aminotransferases (AST and ALT) are highly concentrated
in the liver and the increasing serum ALT activity is considered a
consequence of hepatocyte damage in NASH patients [29]. A growing
body of evidence supports the possibility that insulin resistance
associated with adipose tissue inflammation and hepatic
microvascular dysfunction as shown in our histological findings
might actually contribute to the development and/or progression of
ALT activity in serum [30].
Treatment of NASH group with CSM extract or CSF fraction induced
remarkable depletion in serum ALT activity. In addition, both of
these treatments led to an improvement in the histological feature of
the liver of the treated rats as shown in our results. These effects
could be attributed to the active ingredients in Cynara scolymus
crude extract and fraction which are known as caffeoylquinic acid
derivatives (cynarin and chlorogenic acid). These compounds have
been proved to be effective in decreasing serum ALT activity [31] via
their strong hepatoprotective effect and antioxidant capacity.
The current results showed marked increase in serum cholesterol,
triglycerides and LDL-cholesterol in concomitant with significant
decrease in serum HDL level in NASH group. These results coincide
with Adams et al. [32]. Cholesterol metabolism was associated with
liver fat content independent on body weight, implying that the
more fat the liver contains, the higher is cholesterol synthesis [33].
Cellular cholesterol synthesis is regulated by activation of
membrane bound transcription factors, designated sterol regulatory
element-binding proteins (SREBPs) which are the most abundant in
the liver [34] and the excess of cellular cholesterol is esterified by
the acyl CoA-cholesterol acyltransferase (ACAT) [35]. The high level
of cholesterol synthesis and the increased SREBP-2 activity has
paradoxically been shown in subjects with NASH [36].
In NASH disease, the ability of insulin to inhibit the production of
very low density lipoproteins (VLDL) is impaired [37]. This results
in hyperinsulinemia, and hypertriglyceridemia, which in turn lead to
lower HDL cholesterol concentration [38]. This explains the
diminished HDL serum level and the high triglycerides level in NASH
group in the current study. The histopathological results of the
present study showed macrovesicular and microvesicular steatosis.
Hepatic accumulation of triglycerides has been associated with the
development of macrovesicular steatosis of the liver. Since the
inhibition of mitochondrial fatty acid metabolism is considered to
result in microvesicular steatosis [39], secondary accumulation of
cytosolic triglycerides and phospholipids in the presence of initial
mitochondrial damage may explain the development of a mixed type
of liver steatosis over time.
The insufficient elimination of triglycerides, probably caused by
hepatic insulin resistance [40] may also contribute to the
development of NASH. Triglycerides are progressively reduced by
the action of lipoprotein lipase (LPL), eventually resulting in
intermediate-density lipoproteins (IDLs) and low-density
lipoproteins (LDL) with relatively high cholesterol content [41]. LDL
circulates and is absorbed by the liver through binding of LDL to
LDL receptor [42]. In addition, NAFLD ranging from simple steatosis
to nonalcoholic steatohepatitis (NASH) is strongly associates with
insulin resistance, which caused inflammatory cytkokine tumor
necrosis factor-alpha (TNF-α) to be over expressed in the liver. TNF-
α activates cholesterol synthesis and inhibits cholesterol elimination
through bile acids, which together contribute to increase LDL-
cholesterol and decrease HDL-cholesterol [37].
Treatment of NASH group with CSM or CSF produced marked
decrease in serum cholesterol, triglycerides and LDL levels
accompanied with significant increase in serum HDL. Additionally,
histopathological investigation of liver tissue of the treated groups
indicated a reduction in macrovesicular steatosis and microvesicular
steatosis. These results coincide with Lattanzio et al. [43] who
declared that, the active compunds in Cynara scolomus extract
represented by caffeic acid, chlorogenic acid, cynarin, cynaroside,
scolymoside and have been found to affect cholesterol metabolism.
Daniel, [44] reported that, Cynara scolomus extract has
anticholesterolemic action by decreasing rate of cholesterol
synthesis in the liver and other tissue and this may be due to that
Cynara scolomus contains some constituents as cynarin and luteolin
which play a crucial role in inhibiting cholesterol and triglycerides
synthesis. Luteolin by beta glucosidase in digestive tract could cause
inhibition up to 60٪ of cholesterol synthesis [45]. However, highly
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significant decrease of plasma LDL and an increase of HDL in the
treated groups are agreed with Cieslik et al. [46] who reported
decline tendency in total cholesterol, LDL and VLDL when diets were
supplemented with Cynara scolomus flour. Moreover, Taylor [47]
showed a decrease from 10% to 15% in total cholesterol LDL and
ratio of LDL to HDL cholesterol in serum due to treatment with
Cynara scolomus leaves extract. This could be explained as; this
extract contains active compounds as flavonoids and caffeoylquinic
acid which have hypolipidemic effect. These compounds could not
only increase the breakdown of cholesterol to bile salts and enhance
their elimination through increased bile production and flow but
they also inhibit the internal production of cholesterol in liver [48].
Furthermore, Cynara scolomus extract may work through the
indirect inhibition of enzyme hydroxyl methyle glutryle – CoA
(HMG-CoA) which avoid problems occur with strong direct
inhibitors of HMG-coA reductase during long treatment. The indirect
inhibition was supported by the fact that Cynara scolomus extract
effectively blocked insulin-dependent stimulation of HMG-coA-
reductase, a key enzyme in cholesterol synthesis and HMG-coA
reductase inhibitors generally reduce cholesterol, LDL and
triglycerides levels in serum [49].
The present data showed marked decrease in serum adiponectin
level in NASH group. It has been shown that adiponectin is found in
relatively high circulating levels but it is decreased in patients with
NASH and in clinical manifestations associated with insulin
resistance such as metabolic syndrome (MS) and type 2 diabetes
mellitus [50]. In addition, plasma adiponectin levels correlated
inversely with the markers of systemic oxidative stress, and
oxidative stress is known to be a feature of liver disease. Many
studies hypothesized that oxidative stress has been demonstrated in
conditions such as NAFLD and NASH due to the increased levels of
free fatty acids and consequent increased levels of free radicals [51].
In cultured adipocytes, under oxidative stress condition, the
suppressed mRNA expression and secretion of adiponectin were
detected. This could be attributed to the decreased gene expression
of adiponectin under this condition [52].
Treatment of NASH group with CSM or CSF showed marked increase
in serum adiponectin level. It has been demonstrated that Cynara
scolomus extract contains natural antioxidants such as caffeoylquinic
acid derivatives and flavonoids [53] that can regulate mRNA
expression and secretion of adiponectin [52].
Serum leptin level showed significant increase in NASH group in the
present study. Leptin is released into the circulation by mature
adipocytes in response to changes in body fat mass and nutritional
status. It has varied metabolic effects with the most significant of
these being related to body weight and energy expenditure [54]. In
NASH patients, leptin levels are elevated and are directly correlated
with the severity of steatosis [55]. The presence of hepatic steatosis
despite the presence of hyperleptinemia suggests the development
of leptin resistance [56]. In addition, leptin levels have been
reported to be associated with oxidative stress conditions which
enhance reactive oxygen species (ROS) formation in accumulated
fat. This leads to the elevated adipose nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase that leads to dysregulated
production of leptin [52].
Treatment of NASH group with CSM or CSF resulted in appreciable
decrease in serum leptin level as compared to the untreated NASH
group. Cynara scolomus active constituents (caffeic acid and
chlorogenic acid) could reduce plasma cholesterol and triglycerides
levels and this leads to a decrease in plasma leptin and an increase
in adiponectin levels [57].
Serum resistin level in NASH group showed significant increase in
comparison with the healthy control group. This result is in
agreement with Pagano et al. [58] who reported that patients with
NASH are characterised by high serum resistin level. A major target
organ of resistin is the liver, where resistin induces insulin
resistance and increases glucose production. Resistin is related to
hepatic fat content and insulin resistance [59]. It has been suggested
that resistin may contribute to hepatic steatosis by promoting
insulin resistance and the increased resistin levels in NASH patients
are related to histological severity of the disease [60]. Underlying
liver damage and the progression of pure fatty liver to NASH and
fibrosis, the hepatic stellate cells produce a variety of cytokines,
including resistin. Daniel reported that a genetic polymorphism in
the promoter region of the resistin gene may be an independent
predictor of circulating resistin level. Hence, it is possible that a gene
polymorphism(s) may be responsible for the high resistin levels in
NASH disease [58].
Treatment of NASH group with CSM or CSF produced remarkable
decrease in serum resistin level. Hepatoprotective effect of Cynara
scolomus leaves extract may be assumed to be related to inducing
glutathione peroxidase, besides its direct antioxidant properties
which may be useful for the prevention of oxidative stress that
exerts an impact on endogenous expression of resistin in the
adipocyte [31]. Polyphenolic compounds in Cynara scolomus extract
may be responsible for the suppression of hydrogen peroxide-
induced oxidative stress [61]. By this way, CSM and its fraction (CSF)
might reduce serum resistin level.
The present results showed significant increase in serum NF-κB p56
level in NASH group. High oxidative stress status in the liver of
NAFLD patients with steatohepatitis may lead to modulation of
Kupffer cell function, through activation of transcription factors such
as NF-κB [62]. NF-κB then translocates from the cytoplasm to the
nucleus to activate the inflammatory cytokines perturbing the
inflammatory cycle [63].
Treatment of NASH group with CSM or CSF recorded marked
decrease in serum NF-κB p56 level. The inhibition of NF-κB
activation correlated with suppression of inhibitor of NF-κB (IκBα)
phosphorylation and degradation, p65 nuclear translocation, and
NF-κB-dependent reporter gene transcription. Cynara scolomus
components mainly luteolin and apignen have been found to block
IκBα phosphorylation and degradation [64] and in turn it could
reduce NF-κB level. Serum TNF-α level showed significant elevation
in NASH group as compared to the healthy control group. This could
be attributed to the oxidative stress and stimulation of kupffer cell
as well as stellate cell to secrete inflammatory cytokines such as
TNF-α [65]. Moreover, it has been found that NAFLD patients have
elevated plasma levels of lipopolysaccharide-binding protein (LBP)
which are further increased in patients with NASH. This increase is
related to a rise in TNF-α gene expression in the hepatic tissue
which supports a role of endotoxemia in the development of
steatohepatitis [66].
Treatment of NASH group with CSM or CSF led to significant
decrease in serum TNF-α in comparison with the untreated NASH
group. This effect could be attributed to the presence of luteolin and
apignen in Cynara scolomus extract which could inhibit the
inflammatory cytokine production in lipopolysaccharide-induced
TNF- production [67].
Significant increase in serum Cox-2 activity was recorded in NASH
group in the current work. This could be explained as the oxidative
stress which triggers lipid peroxidation and cytokines production
such as TNF-α and interleukin (IL)-6 in the steatotic liver could
mediate inflammatory recruitment directly or indirectly via
activating NF-κB with upstream consequences that include
cyclooxygenase-2 activity [68].
Treatment of NASH group with CSM or CSF produced significant
decline in serum Cox-2 activity. Cynara scolomus extract with its
active consitutent (luteolin and apignen) has been found to block
NF-κB expression [64]. COX-2, which mediates prostaglandin
synthesis during inflammation, is induced by NF-κB [69]. Thus, the
inhibition of NF-κB by Cynara scolomus extracts contributes in the
inhibition of Cox-2 activity.
The present data showed significant increase in serum CD40 level in
NASH group. This result is in agreement with Ercin et al. [70].
Soluble CD40 was not only correlated with BMI, but was also more
strongly related to lipid peroxidation [71]. Circulating sCD40 was
believed to derive predominantly from platelets associated with
platelet activation and lipid peroxidation during oxidative stress
conditions. Thus oxidative stress plays a role in increasing platelet
Shahat et al.
Int J Pharm Pharm Sci, Vol 5, Suppl 1, 57-66
64
CD40 expression [72]. Besides that, the upregulation of CD40 is
mediated by TNF-α which stimules platelet activation via interaction
with its platelet receptors. TNF-α has been shown to enhance
oxidative stress via NADPH oxidase activation and TNF-α
upregulated platelet CD40 via arachidonic acid-mediated oxidative
stress [73]. Treatment of NASH group with CSM or CSF resulted in
significant deplation in serum CD40. Luteolin in Cynara scolomus
extract may be responsible for this effect. Luteolin could inhibit
CD40 ligand expression by activated basophils [74].
The present results showed significant increase in serum hepatocyte
growth factor (HGF) level in NASH group. This result is in consistent
with that of Koutsogiannis et al. [75]. It has been demonstrated that
HGF mRNA produced by nonparenchymal cells increases in NASH
patients [76]. In NASH, the activation of Kupffer cells and
macrophages within liver tissue increased the production of NF -κB
which induced the expression of HGF and consequently its level [4].
Treatment of NASH group with CSF resulted in marked decrease in
serum HGF as compared to the untreated NASH group. Luteolin and
apignen in Cynara scolomus have been found to block NF-κB
expression [64] and in turn they could indirectly reduce the
stimulant of HGF expression and consequently its level [4].
The current study shed lights on the potential role of CSM and CSF in
management of nonalcoholic steatohepatitis. The active contituents
of Cynara scolomus namely flavonoids and caffeoylquinic acid may
be responsible for this effect. These compounds have been proved to
have hepatoprotective activity, hypolipidemic effect, antioxidant
capacity and antiinflammatory property. Beside that, these
compounds could modulate insulin resistance status associated with
nonalcoholic steatohepatitis. Therefore, Cynara scolomus could have
possible therapeutic application in chronic diseases accompanied
with insulin resistance and severe inflammation.
ACKNOWLEDGEMENTS
Work is partially supported by Science and Technology
Development Fund (STDF), Egyptian Academy of Scientific Research
and Technology “ID# 245”
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