1. INTRODUCTION:
Hepatitis has been caused by viral infections, toxic agents or
drugs and may be by an autoimmune response also. The disease presents as acute,
chronic and fulminant hepatitis as well as healthful carrier state [1]. The
viral hepatitis particularly, Hepatitis B is the most common form of acute
hepatitis. Hepatitis B virus (HBV) is a major cause of acute hepatitis,
cirrhosis and hepatocellular carcinoma worldwide. HBV continues to be the
single most important cause of viral hepatitis in the developing and
underdeveloped world. To date there are nearly 370 million HBV carriers in the
world with highest incidence of 10-20% in the tropical countries. Liver
diseases due to HBV infection is considered to be the fourth or fifth important
cause of mortality in the most productive period of life (15 to 45 years).
Hepatitis remains as clinical challenge and a problem of great importance.
Acute hepatitis can have serious health effects including mortality.
Despite considerable progress in the treatment of liver diseases
by oral hepatoprotective agents, search for newer drugs continues because the
existing synthetic drugs have several limitations [2]. Hence crude drugs or
natural food diet which possesses antioxidant or free radical scavenging
activity has become a central focus for research designed to prevent or
ameliorate tissue injury and may have a significant role in maintaining health.
Carotenoids are a class of more than 600 natural pigments that are
present in fruits and vegetables [3]. These carotenoids are ubiquitous in the
plant kingdom, fruits and vegetables that are a rich source of carotenoids are
thought to provide health benefits by decreasing the risk of various diseases
[4]. Lycopene is a potent antioxidant and member of the carotenoid family. It
is the naturally occurring compound that gives the characteristic red color to
the tomato, watermelon, pink grapefruit, orange, and apricot. A number of
studies have indicated the health benefits of consuming lycopene [5-10]. As a
major carotenoid in human blood, lycopene protects against oxidative damage to
lipids, proteins and DNA [11-12]. Lycopene is a potent quencher of singlet
oxygen which suggests that it may have comparatively stronger antioxidant properties,
than other major plasma carotenoids [13]. We have reported that the antioxidant
potential of lycopene proves to be valid reason for its hepatoprotective role
in experimental animals [14].
Among the numerous models of experimental hepatitis, D-GalN induced
liver damage is very similar to human viral hepatis in its morphological and
functional features [15]. Because of its specificity for hepatocytes D-GalN is
very well suited and is widely used for investigations and pharmacokinetic
studies of hepatoprotective, shock-preventing and therapeutic effectors in
inflammatory processes of the liver [16-17]. D-GalN given at the time of
endotoxin challenge sensitizes mice and other species to the lethal effects of
endotoxin [18]. This liver injury model has been used to evaluate the efficacy
of several hepatoprotective agents [19]. Furthermore, D-GalN - induced liver
injury served as a model for testing and elucidating the protective and even
therapeutic value of flavones, quinines and carotenoids that are known as
antioxidants and of other plant products that are claimed to be
hepatoprotective [20-21]. The scientific research to date has demonstrated an
array of health benefits clearly associated with lycopene. It offers important
health benefits particularly in regard to prostate, lung, heart and skin
health. To add the numerous health benefits of lycopne, our previous report
also demonstrated its hepatoprotective role in experimental animals [14].
Thus the present investigation further explores the effect lycopene
on hematological parameters during D-galactosamine/lipopolysaccharide induced
hepatitis in rats.
2. MATERIALS AND METHODS:
2.1 Chemicals
D-GalN and LPS (Sero type 011.B4 extracted by phenol water method
from E. Coli) were obtained from Sigma Chemical Co. (St. Louis, MO,
USA). All other chemicals (acids, bases, solvents and salts) used were of
analytical grade obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai,
India and Glaxo Laboratoreis, CDH division, Mumbai, India. Jagsonpal
Pharmaceuticals, New Delhi, India, kindly provided Lycopene.
Lycopene stock solution
Lycopene (100 mg) was mixed in 2 ml Tween-80 at room temperature
until a homogeneous paste was obtained. Physiologic saline at room temperature
was added, drop wise and with vigorous stirring, to a final concentration of 10
mg lycopene/ml of suspension [22].
2.2 Animals
Adult male albino rats of Wistar strain weighing around 120 to 150
g obtained from Tamil Nadu Veterinary and Animal Sciences University (TANUVAS),
Madhavaram, Chennai, India were used in this study. They were housed in
polypropylene cages over husk bedding and a 12 hour light and dark cycle was
maintained throughout the experimental period. Rats were fed a commercial
pelleted diet (Hindustan Lever Limited, Bangalore, India) and water ad
libitum.
The experiments were conducted according to the ethical norms
approved by Ministry of Social Justices and Empowerment, Government of India
and Institutional Animal Ethics Committee guidelines (IAEC No.01/026/08).
2.3 Experimental design
The animals were divided into four groups of
six animals each.
Group 1: Served as vehicle control and was administered with tween
80 in saline
Group 2: Rats were given lycopene alone (10 mg/kg body weight for
6 days intraperitoneally).
Group 3: Rats were induced with D-GalN and LPS (300 mg/kg body
weight and 30 µg/kg body weight, i.p 18 hours before the experiment) [23].
Group 4: Rats were pretreated with lycopene for 6 days prior to
the induction of D-GalN/LPS
2.4 Collection of samples
for biochemical analysis
After the experimental period, the animals were anaesthetized by
intraperitoneal injection of pentobarbital sodium (30 mg/kg body weight) and
sacrificed.
Blood was collected and the liver tissue was excised quickly. The
tissues were washed in physiological saline to remove blood clot and other
tissue materials.
2.5 Separation of serum
The blood samples collected in plain centrifuge tubes were kept in
inclined position to allow complete clotting of blood and then centrifuged at
2,500 rpm for 30 minutes. The resultant clear supernatant was pipetted out and
preserved in small vials in the freezer for the purpose of biochemical
investigations.
2.6 Preparation of liver homogenate
Within 3 hours after sacrifice, liver samples were blotted to
dryness. From this, a piece weighing about 100 mg was taken and homogenized at
4oC in Tris-HCl buffer (0.1M, pH 7.4). The tissue homogenates were
centrifuged at 2,500 rpm for 30 minutes. The resultant supernatant was kept
under refrigeration until further biochemical analysis. All the assay
procedures were carried out within 48 hours after sample collection.
2.7 Haematological parameters
Haemoglobin was measured by the method of Drabkin and Austin [24].
Blood haemoglobin levels were expressed as mg/dl. The total erythrocyte count
was determined accurately by diluting a measured quantity of blood with a fluid
isotonic solution by the method of Huxtable [25]. To count white blood cells
(WBC), the procedure of Raghuramulu et al. was followed [26]. WBC diluting
fluid or Turk’s fluid was used as the dilutant which can destroy RBCs. Packed
cell volume (PCV) was determined by centrifugation using Wintrobe tubes [27].
PCV volume was expressed in percentage. Plasma prothrombin time (PTT) was done
by the Quick’s one stage method [28]. Prothrombin time was expressed in
seconds. Erythrocyte sedimentation rate (ESR) was determined by the
method of Bottiger and Svedberg [29]. The erythrocyte sedimentation rate was
expressed as mm/h.
2.8 General biochemical parameters
Protein content was estimated by the method of Lowry et al.
[30]. The levels of protein values were expressed as mg/dl for serum and mg/g
for tissue. The albumin and globulin content of the serum was estimated as
described by Natelson [31]. The values were expressed as mg/dl.
2.9 Statistical analysis
All the grouped data were statistically evaluated with Statistical
Package for Social Sciences (SPSS), Version 10.0. Hypothesis testing methods
included one way analysis of variance (ANOVA) followed by least significant
difference (LSD) test. A ‘P’ value of less than 0.05 was considered to
indicate statistical significance. All the results were expressed as mean +
S.D. for six animals in each group.
3. RESULTS
3.1 Haematological
parameters
The haemotological changes observed in control and experimental
groups of rats were presented in Table 1. A significant decrease (P<0.05)
in Hb, RBC, WBC and PCV was noted with a marked increase (P<0.05) in
PTT and ESR in rats injected with D-GalN/LPS (Group 3) when compared to control
(Group 1). Prior oral administration of lycopene (Group 4) reversed the above
changes and near normal levels was attained. Rats treated with lycopene alone
(Group 2) did not show any significant changes when compared to control, which
implicates non-toxic nature of the lycopene.
3.2 General biochemical parameters
3.2.1 Protein
Total protein content in serum and liver of control and
experimental groups of rats has been depicted in Figure 1 and 2 respectively.
Protein content was significantly decreased (P<0.05) in rats induced
with D-GalN/LPS (Group 3) when compared to control rats (Group 1). The
recoupment of protein level to near normal was observed in rats pretreated with
lycopene (Group 4) as that of control. Lycopene alone treated rats (Group 2)
showed no significant alteration in protein content in comparison with control.
Figure 1. Levels of total protein in the serum of control and
experimental group of animals
Values are expressed as mean + SD for six rats in each group aAs compared with Group
1 (control), b As compared with Group 3 (D-GalN/LPS); a,b
represent P < 0.05
Figure 2. Levels of total protein in the
liver of control and experimental group of animals
Values are expressed as mean + SD for six rats in each group a As compared with Group
1 (control), b As compared with Group 3 (D-GalN/LPS); a,b
represent P < 0.05
3.2.2 Albumin and globulin
The levels of albumin and globulin in control and experimental
groups of rats were depicted in table 2.There was a significant reduction (P<0.05)
in albumin level and A/G ratio with substantial increase (P<0.05) in globulin
in rats challenged with D-GalN/LPS (Group 3) when compared to control (Group
1). The above alterations were found to be at near normal when pretreated with
lycopene (Group 4).
Table 2. Levels of albumin, globulin and albumin/globulin ratio
in liver of control and experimental groups of rats.
|
Parameters
|
Group- 1
Control
|
Group- 2
Lycopene
alone
|
Group -3
DGalN/LPS
|
Group- 4
Lycopene+DGalN/
LPS
|
Albumin
|
4.88+
0.42
|
4.87 +
0.42
|
2.25 +
0.21a
|
4.61 +
0.41b
|
|
Globulin
|
3.17 +
0.29
|
3.15 +
0.29
|
4.94 +
0.43a
|
3.38 +
0.27b
|
|
Albumin/Globulin
ratio
|
1.56 +
0.15
|
1.55 +
0.15
|
0.56 +
0.05a
|
1.32 +
0.13b
|
Units - mg/g
Values are expressed as mean + SD for six rats in each
group aAs compared with Group 1, bAs compared with Group
3 a,brepresent P < 0.05
4. DISCUSSION
D-GalN/LPS induced hepatoceullar damage, a well-established model
of hepatitis takes advantage of the ability of D-GalN to potentiate the toxic
effects of LPS producing hepatitis within a few hours of administration [32].
The liver plays a key role in haemostasis and its regulation. The results of
our present study suggests that the normal regulation of haemostasis is altered
upon by the toxic insult of D-GalN/LPS and that results in the derangements of
the haematological parameters in group 3 rats in comparison with that of group
1 control animals. Diseases affecting any normal physiological state can have
secondary influence on haematogical profile. Screening of various
haematological parameters depicts its significance in diagnosis to estimate the
degree of hepatocellular function [33]. In our study the decreased Hb levels
and RBC count indicate the severity of hepatic damage induced by D-GalN/LPS.
The reduction in Hb levels might be due to increased catabolism and degradation
of Hb to bilirubin. This reflects the defects in the intrinsic and extrinsic
pathways of coagulation system due to D-GalN/LPS induction. The reduced WBC
count indicates decreased resistance of the body to infection caused by the
administration of D-GalN/LPS. This finding matches with the general observation
that the hepatitis often associated with other secondary infections due to the
poor immunity of the affected individual.
The observed changes in PCV show the primary haematological
derangements. PTT measures the rate at which prothrombin is converted to
thrombin in the presence of thromboplastin, Ca2+, fibrinogen and
other coagulation factors (V, VII and X). The prolongation of PTT in hepatitis
induced rats might be due to the fact that the liver may be so damaged that it
cannot adequately synthesize the clotting factors [34]. Our finding coincides
with the previous reports that have showed prolonged partial thromboplastin
time in liver diseases according to the degree of hepatic failure [35]. A
decrease in plasma protein (ceruloplasmin, fibrinogen) may have an impact on
the sedimentation rate. A vast majority of acute or chronic infections and most
neoplastic and degenerative diseases are associated with changes in plasma
proteins, which lead to an acceleration of sedimentation rate [36]. Decreased
albumin synthesis due to liver disease also increases the ESR [37]. The results
of our investigation reveals that D-GalN/LPS - induced changes in
haemotological parameters were normalised upon prior treatment with lycopene in
group 4 animals indicating its prophylactic action in maintaining haemostasis
during liver injury.
Liver is an important site of protein synthesis and it has the
highest rate of synthesis/g tissue. Many toxic compounds that provoke liver
injury have been found to interfere with hepatic protein synthesis. The
decrease in protein levels due to the toxic insult of D-GalN/LPS is consistent
with previous studies. Accumulation of
UDP-sugar nucleotides [38-39] may contribute to the changes in RER and to the
disturbance of protein metabolism. The disaggregation of polyribosomes in
D-GalN/LPS - induced hepatitis is associated with the inhibition of protein
synthesis [40]. This may be due to the interference of D-GalN/LPS with the
incorporation of amino acids into liver proteins. Dean et al.
(1991) has suggested that oxidative damage in addition to causing lipid
peroxidation can lead to modification of proteins through the introduction of
carbonyl groups [41]. Apart from the well documented inhibition of protein
synthesis, it has been suggested that reactive oxygen species produced by
activated macrophages might be the primary cause in D-GalN-induced liver damage
[42-43]. The present findings of our study matches with the previous reports by
exhibiting a marked decrease in protein level both of serum and liver fraction
in group 3 animals administered with
D-GalN/LPS when compared with that of normal control of group 1 animals.
The depleted protein levels in group 3 animals are normalized in lycopene
pretreated group 4 rats and this may be due to the well known antioxidant defense of lycopne against free radical induced liver damage by D-GalN/LPS.
Albumin is the most abundant circulatory protein and its synthesis
is a typical function of normal liver cells. The serum albumin level has been
used as a test of liver function because it is affected by hepatic protein
synthesis [44]. Increased protein catabolism in drug - induced hepatitis might
have a direct adverse effect on the synthesis and secretion of albumin. The
significant decrease in albumin levels in hepatitis – induced group 3 rats in
comparison with that of group 1 control could be attributed to suppress protein
synthesis in liver and subsequent impaired hepatic function following
D-GalN/LPS administration [45]. The results of our finding coincides with the
previous studies that reports hypoalbuminemia during hepatic dysfunction
[46-47]. The elevated globulin content in the hepatitis - induced rats appears
to be compensatory as the A/G ratio showed a significant drop in group 3
animals when compared to that of control group 1 rats. Thus decreased A/G ratio
observed may be due to the impaired synthesis of albumin as seen in hepatic
disease. The above changes were reverted to their normal values upon
pretreatment with lycopene. This observation suggests the protective ability of
the lycopene on impaired hepatic function which in turn improves the synthetic
capability of the liver in D-GalN/LPS - induced rats. Thus the present finding
clearly suggests that lycopene helps to restore the hematological derangements
during hepatitis induced by D-GalN/LPS through its antioxidant property and
requires furthers exploration to establish the complete mechanism.
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