15018752330
发表时间:2015-11-12 浏览次数:515次
Introduction
Hepatotoxicants, including viruses, fungal products, bacterial metabolites,
minerals, environmental pollutants, and chemotherapeutic agents, can induce
various disorders of the organ. Azathioprine (AZA) is a common immunosuppressant
drug used in medicine to treat different diseases. It is now widely used in
oncology, dermatology, gastroenterology, and rheumatology for its anti-leukemic
and immunosuppressive properties. AZA (6-(1-methyl-4-nitroimidazol-5-yl)
thiopurine) is also used for the prevention of rejection in organ transplants
and the treatment of auto-immune diseases. It is indicated as an adjunct for the
prevention of rejection in the renal transplantations. AZA is also used in the
prevention of rejection in cardiac, hepatic, and pancreatic
transplantations.
The therapeutic use of AZA is associated with many
complications. It induces a range of toxic effects that may ultimately result in
the discontinuation of treatment. These toxic effects include gastrointestinal
disturbances, pancreatitis, reversible alopecia, rashes, fever, tachycardia,
pneumonitis, hypotension, and renal dysfunction.Many studies have frequently
reported the hepatotoxicity of AZA in vitro and in vivo. Many of
these studies related the mechanism of hepatotoxicity and liver injury to the
oxidative stress. AZA toxicity to rat hepatocytes in vitro and the
mechanism of AZA toxicity to hepatocytes and decreasing its viability involves
the depletion of glutathione (GSH) leading to mitochondrial injury with profound
depletion of adenosine triphosphate (ATP) and cell death by necrosis were
reported.
Plants rich in natural polyphenolic compounds were intensely
studied in recent years due to their potent anticarcinogenic, antioxidant, and
immunomodulatory properties. Chamomile (Matricaria recutita L.) is one of
the most widely used medicinal plants in the world. Aqueous chamomile extract is
used as herbal medicine, in the form of tea, demonstrated to possess
anti-inflammatory and antioxidant properties.Chamomile preparations are commonly
used for many human ailments such as hay fever, muscle spasms, menstrual
disorders, insomnia, ulcers, wounds, gastrointestinal disorders, rheumatic pain,
and hemorrhoids,and also used to treat anxiety, hysteria, nightmares, insomnia,
and other sleep problems.The useful effects of chamomile are related to the
presence of several flavonoid constituents (the most abundant phenolic compounds
in herbs), and the core structure consists of either flavone (apigenin and
luteolin) or flavonol derivatives (quercetin and patuletin). These occur in
various forms such as aglyco- mono- and di-glycosides and/or acyl-derivatives.
Other principal components are essential oils such as terpenoids, α-bisabolol
and its oxides, and azulenes including chalmuzene and acetylene
derivatives.
Fennel (Foeniculum vulgare Mill.), a plant belonging
to the family Apiaceae, has a long history of herbal uses. Fennel seeds
are used as analgesic, carminative, anti-inflammatory, diuretic, and
antispasmodic agents. The antioxidant potential and antimicrobial activity of
fennel seed extracts and essential oil have been reported. Fennel seeds
methanolic extract contains (FSMEs) high amount of polyphenols, including
flavonoids as a major component of polyphenols, gallic acid, caffeic acid,
ellagic acid, quercetin, and kaempferol.Trans-anethole, fenchone, estragole,
4-terpineol, sabinene, alpha-terpinene and monoterpene hydrocarbons (limonene)
as the major compounds, were identified in the essential oil.
The present
study was undertaken to elucidate the ability of oral chamomile flowers
methanolic extract (CFME) and FSME to alleviate the adverse effects of AZA on
the liver through biochemical and histological examinations in albino rats.
Methods
Experimental animals
Male albino rats (Sprague-Dawley
strains) weighing 150-200 g were used in this study. They were obtained
from the animal house of National Research Centre, Giza, Egypt and
acclimatized for 1 week prior to the experiment. Animals were housed in
stainless steel cages at room temperature (20-25 °C) and a photoperiod
of 12 h light-dark cycle. Animals were allowed free standard laboratory
diet and drinking tap water ad libitum.
Chemicals
AZA
B.P uncoated tablets 50 mg manufactured by RPG Life Sciences Ltd., Ceat
Mahal, 463, Dr. A B Road, Worli, Mumbai: 400 025, India. Methyl alcohol
(98%) was obtained from El-Nasr Pharmaceutical Chemicals Co., "ADWIC"
(Egypt). Perchloric acid and trichloroacetic acid (extra pure 99%) were
manufactured by SISCO Research Laboratories PVT LTD (Mumbai, India).
Thiobarbituric acid was purchased from MERCK (Darmstadt, Germany). Other
solvents and chemicals used were either analar or of analytical grade
unless otherwise specified. Plant materials: Dried chamomile (Matricaria chamomilla) flowers and dried fennel (F. vulgare) seeds were purchased from Abd El-Rahman Harraz (Bab El-Khalk Zone, Cairo, Egypt).
Preparation and extraction of the plant materials
Three
samples of dried chamomile flowers and fennel seeds were ground. Eighty
grams of each ground sample were transported into 1 L Erlenmeyer
flasks, and then 800 mL of 80% methanol (80:20, methanol:water, v/v)
were added to the samples. Extraction was carried out using an orbital
shaker at a room temperature for 8 h, they were filtrated through filter
paper (Whatman No. 1), the residue was re-extracted twice for complete
extraction, and then, the combined extracts of every sample were
evaporated at 45 °C, using a rotary vacuum evaporator (Rotavapor R-114
BÜCHI, Switzerland) and stored at -4 °C until use.[15]
Animal grouping
Sixty
rats were divided into six groups (10 rats each) based on their body
weight and treated daily for 28 consecutive days as follows. Group 1:
rats were administered with normal saline by gastric intubation and
served as control group; group 2: rats received CFME at a dose of 200
mg/kg (dissolved in normal saline) by gastric intubation;[18] group 3: rats received FSME at a dose of 200 mg/kg (dissolved in normal saline) by gastric intubation;[19] group 4: rats were orally administrated with AZA at a dose of 25 mg/kg (dissolved in normal saline) by gastric intubation;[20]
group 5: rats were orally administrated with CFME at a dose of 200
mg/kg followed by AZA, after 15 min, at a dose of 25 mg/kg by gastric
intubation; and group 6: rats were orally administrated with FSME at a
dose of 200 mg/kg followed by AZA, after 15 min, at a dose of 25 mg/kg.
During
the experiment, the animals were weighed twice every week. Body weight
gain (BWG) of each control and respective treated rats was calculated
with reference to the initial body weight recorded at the beginning of
the experiment and the final body weight at the end of the experiment.
Collection of blood samples
At
the end of the experimental period, animals were fasted overnight. They
were slightly anesthetized with diethyl ether. Blood samples were
withdrawn from the retro-orbital venous plexus into serum tubes and left
to clot and then centrifuged at 3000 g for 15 min at 4 °C where
the clear sera were separated for the determination of alanine
aminotransferase (ALT), aspartate aminotransferase (AST), alkaline
phosphatase (ALP) activities, and total cholesterol, triglycerides, and
total- and direct-bilirubin levels.
Tissue sampling
At
the end of blood collection, each animal was rapidly sacrificed, and
the liver was dissected out and weighed then apart from its left lobe
was immediately kept in 10% buffered formalin-saline solution for a
later histopathological examination. Another part from the same lobe of
the liver was washed with saline, dried, weighed, and homogenized in 50
mmol/L phosphate buffer (ice-cold) solution (pH 7.4) to give 20%
homogenate (w/v).The homogenate was centrifuged at 3000 g
for 20 min. The supernatant was separated and stored at -70 °C until
the determination of the levels of malondialdehyde (MDA), total
antioxidant capacity (TAC), and reduced GSH content. Furthermore, the
relative liver weight of each animal was then calculated as follows:
Relative organ weight = (absolute organ weight [g] × 100)/(body weight
of rat on sacrifice day [g]).
Analytical determinations
Colorimetric
determinations of serum AST and ALT activities were carried out using
UV-160 1PC UV-visible spectrophotometer (Shimadzu, Japan) for reading
the absorbance. The assay was performed according to the instruction
manual of RANDOX reagent kits manufactured by RANDOX Laboratories Ltd.
(Admore, Diamond road, Crumlin, Co., Antrim, UK BT29 4QY). Colorimetric
determination of serum ALP activity was performed according to the
instruction manual of Reactivos GPL kits manufactured by Reactivos GPL
(Barcelona, Espana, Spain). Serum total- and direct- bilirubin levels,
triglycerides, and total cholesterol levels were determined
colorimetrically using UV-160 1PC UV-visible spectrophotometer for
reading the absorbance. The assays were performed according to the
instruction manual of Reactivos GPL kits manufactured by Reactivos GPL
(Barcelona, Espana, Spain). MDA as an indirect index for lipid
peroxidation was determined in liver, based on its reaction with
thiobarbituric acid which forms a pink complex that can be measured
photometrically.
Colorimetric determination of hepatic GSH content and TAC were carried
out using UV-160 1PC UV-visible spectrophotometer for reading the
absorbance using Kits produced by Biodiagnostic Co., Egypt.
Histopathological examination
Pieces
of the livers from rats of control and treated groups were fixed in 10%
formalin saline for 24 h. More washing in tap water overnight was
followed by dehydration in graded alcohol, clearing in xylene for 20
min, and embedding in paraffin wax. Transverse serial sections were then
cut at 5 µm thickness and mounted on albuminized slide.Sections were stained with hematoxylin and eosin and investigated by light microscopy.
Statistical analysis
The
obtained data were subjected to one-way analysis of variance. The
analysis was performed using Statistical Analysis System (SAS) program
software; copyright (c) 1998 by SAS Institute Inc., Cary, NC, USA. Tukey
test was used to evaluate the significance between the individual
groups at P < 0.05.The values in this study were expressed as a mean ± standard error.
Results
AZA treatment resulted in a significant increase in hepatic MDA level concomitant with a significant decline in hepatic GSH content and TAC as compared to control rats However, the administration of either CFME or FSME alone revealed insignificant changes in the mentioned parameters when compared to control rats except in rats that received FSME where a significant increase in hepatic GSH was observed. Pre-administration with CFME or FSME significantly reversed the elevation in hepatic MDA level and also reversed the decrease in hepatic GSH content and TAC-induced by AZA treatment toward the normal values of the controls.
The selected and specialized serum markers of liver functions among the different groups are shown in and . It is clearly indicated that CFME or FSME had no effect on AST, ALT, and ALP activities as well as total- and direct-bilirubin levels when compared with the control group. The treatment of rats with AZA alone resulted in significant increases in AST, ALT, and ALP activities, direct-bilirubin levels and a non-significant increase in total-bilirubin level. However, the administration of CFME or FSME to rats succeeded significantly in preventing the AZA-induced changes in the above mentioned parameters. In addition, the more prominent preventive effect was observed in the group pre-treated with FSME.
In the present study, serum triglyceride level showed insignificant change among the different studied groups, but serum cholesterol level increased significantly in AZA-treated rats as compared with the control group. The animals those were administrated with CFME or FSME alone showed an insignificant change in serum cholesterol level as compared with the control group. The administration of CFME in combination with AZA offered little protection against AZA-induced changes in cholesterol level, whereas the administration of FSME to rats succeeded in ameliorating significantly the AZA-induced changes in the mentioned parameter
AZA treatment resulted in a significant decrease in BWG (%), when compared to control rats [Figure 5]a. The administrated with CFME produced a non-significant decrease in BWG, but FSME administration caused a significant decrease in BWG when compared to control rats. Pre-treatment with CFME before AZA treatment did not ameliorate the decrease in BWG induced by AZA treatment, while as pre-administration with FSME before AZA treatment induced a significant increase in BWG when compared to the AZA-treated group.
Data in [Figure 5]b
show that AZA treatment induced a significant increase in mean relative
liver weight (RLW) when compared to normal control rats. However, the
oral administration of CFME or FSME did not significantly affect the
mean RLW. The pre-treatment with CFME did not improve the increase in
mean RLW ratio induced by AZA treatment, but the administration with
FSME ameliorated the increase in mean RLW induced by AZA treatment.
The light microscopical examination of the liver sections from the control rats revealed normal hepatocytes architecture [Figure 6].
The liver sections obtained from FSME and CFME treated rats showed more
or less normal hepatocytes architecture, but some congested blood
vessels were seen in FSME-treated rats [Figure 7] and mild inflammation around the portal tract in the CFME-treated rats [Figure 8].
In contrast, the liver sections obtained from the AZA-treated rats
revealed hepatocytes disorganization, and fatty degeneration as
indicated by large and microvesicular fat droplets. The hepatocytes
nuclei were shrinked and pyknotic or apoptotic. There were areas of
hemorrhages in blood vessels and in between hepatocytes. Hepatocytes
were seen congested and fibrosed with pyknotic nuclei, microscpical
examination also revealed bile duct necrosis around the portal tract [Figure 9].
The liver sections of rats treated with FSME prior to AZA treatment
showed marked improvement and regeneration in the periportal and central
zone. Some hepatocytes revealed acidophilic and granular cytoplasm with
central rounded vesicular nuclei [Figure 10].
The liver sections of rats administrated with CFME prior to AZA
treatment showed the normal hepatocytes architecture with normal central
vein and portal tract, the fatty degeneration, and fibrosis or nuclear
damage disappeared. Acidophilic cytoplasm with central rounded vesicular
nuclei was observed [Figure 11].
Discussion
AZA is a common immunosuppressant used in medicine to treat different diseases.[25]
However, AZA use has been complicated by a high incidence of hepatic
injury which was found to be associated with oxidative damage.[26]
Hepatic injury is a common pathological feature which exists in many
liver diseases. Liver fibrosis, cirrhosis, and even liver cancer could
result from the long existence of hepatic injury.[27]
Chamomile flower and fennel seeds were reported to have antioxidant
effects. Therefore, in this study, we investigated the protective
effects of CFME and FSME, as natural products, against AZA-induced liver
injury.
In recent years, there has been an increased interest in
the possible role of reactive oxygen species (ROS) in the pathogenesis
of tissue injury.[26] Status of the oxidative/
anti-oxidative profile was the mechanistic approach to assess the
toxicity of AZA and/ or protection to its toxic implications by using
free radical scavengers.[28] After administration, AZA is rapidly cleaved non-enzymatically within erythrocytes depending on GSH,[29] to yield 6-mercaptopurine (6-MP) and an imidazole side chain.[30] AZA is also metabolized in the liver by the conversion of AZA to 6-MP catalyzed largely and enzymatically by GSH S-transferase [31] using GSH as a substrate.[28] AZA metabolism in rat hepatocytes leads to GSH depletion, mitochondrial injury, decreased ATP levels, and cell death.[8]
6-MP is further converted into 6-thiouric acid by xanthine oxidases
(XO). It has been reported that XO has the potential to generate ROS in
human hepatocytes [32] and that the oxidation of 6-MP by XO is involved in the AZA-induced liver injury in patients with inflammatory bowel disease.[33]
Another metabolic pathway converts 6-MP into 6-thioinosine
monophosphate via hypoxanthine-guanine phosphoribosyl transferase, and
this intermediate is then metabolized into active 6-thioguanine
nucleotides (6-TGNs).[34] 6-TGNs are also responsible for the cytotoxic side effects.[35]
The
metabolic conversion of 6-MP into 6-thiouric acid via XO, which is a
critical source of ROS, potentially leads to hepatotoxicity.[32] It has been suggested that ROS production by mitochondria caused by thiopurines could damage the membranes and macromolecules.[8]
In the present study, the oxidative injury in AZA-treated animals was
evident from the significant decline in GSH and TAC levels. The
oxidative stress was further confirmed by increased lipid peroxidation
and histopathological changes in the liver tissue. These findings are in
agreement with previous studies, which recorded the involvement of
oxidative stress and lipid peroxidation in AZA-induced liver injury.[32],[36]
In
hepatocytes, GSH is consumed during the metabolism of AZA to 6-MP. The
mechanism of AZA toxicity to hepatocytes involves the depletion of GSH
leading to mitochondrial injury with profound depletion of ATP and cell
death by necrosis.[9] Furthermore, GSH is
responsible for ROS scavenging. Therefore, the decrease in GSH induced
by AZA administration may be caused by the exhaustion of GSH during ROS
scavenging.[32] Lipid peroxidation, as well as altered levels of some endogenous scavengers, are taken as indirect in vivo reliable indices for the contribution of free radical generation and in turn oxidative stress.[37] It has already been demonstrated that the depletion of GSH precedes the induction of lipid peroxidation.[38]
In
this study, AZA-induced hepatotoxicity is evidenced by significant
increments in the values of serum ALT, AST, ALP, direct-bilirubin, and
cholesterol that may be attributed to the liver injury and also
confirmed by pathological changes in the liver of AZA-treated rats. The
increase of serum ALT, AST, and ALP activities may be mainly due to the
leakage of these enzymes from the liver cytosol into the blood.[39]
In necrosis or membrane damage, ALT and AST are released into
circulation, and it can, therefore, be measured in serum as markers of
hepatic damage.[40]
Furthermore, our results are in agreement with a study which reported
the cholestatic type of liver injury in a man treated with AZA, as
developed after 16 days of starting AZA therapy.[41]
Serum
ALP and total-bilirubin levels are also related to the status and
function of hepatic cells. The increase in serum ALP is due to increased
synthesis in the presence of biliary pressure.[42]
Bilirubin has been used to evaluate chemically-induced hepatic injury.
It is one of the most useful clinical clues to the severity of necrosis,
and its accumulation is a measure of binding, conjugation, and
excretory capacity of hepatocytes.[43]
Lipids
concentration is determined by metabolic functions, which are
influenced by the integrity of vital organs such as the liver and
kidney. Therefore, lipid profile such as cholesterol and triglycerides
are increased in hepatopathy. The lipid content of hepatocytes is
regulated by the integrated activities of cellular enzymes that catalyze
lipid uptake, synthesis, oxidation, and export.[44]
In
the current study, the observed decrease in BWG in rats treated with
AZA are in agreement with the observations which reported the weight
loss in rats [45] and mouse [46]
exposed to cytotoxic agents such as AZA and methotrexate which could
possibly be due to the inhibition of DNA synthesis and increased
oxidative stress with consequent cellular damage of the body organs in
affected rats.[45]
Since
oxidative stress has been recognized to be involved in etiology of
several liver diseases and because liver is very susceptible to toxic
effects, natural antioxidants, and plant extracts have been proposed as
therapeutic agents to protect against liver damage.[47]
Administration of chamomile flowers and fennel seeds extracts to
AZA-treated animals was potentially effective in reducing the lipid
peroxidation and enhancing antioxidant capacity in the liver of
AZA-treated animals. This appeared from the amelioration of MDA to near
normal level and the significant improvement of the GSH and TAC
contents. These results agree with that reported for chamomile flowers
extract [48],[49] and for fennel seeds extract.[16],[50]
In
addition, chamomile flower and fennel seed extract could also
significantly decrease serum ALT, AST, and ALP, suggesting their
hepatoprotective activity. The mechanisms by which chamomile flower and
fennel seed extracts offered their protective effects against AZA
hepatotoxicity are based on their antioxidant abilities, which may be
responsible for protecting the hepatic cells against the oxidative
stress, possibly by increasing the endogenous defensive capacity of the
liver to combat oxidative stress induced by AZA. This in turn improves
the liver integrity and function and consequently improves the hepatic
excretory function of bilirubin and also improves lipid metabolism. This
improvement was more pronounced in the animals that were received
fennel seed extract. Chamomile flower and fennel seed extracts also
significantly ameliorated the decrease in the BWG. This may be
attributed to the antioxidant effect of these extracts.
Several
reports demonstrated that chamomile flowers and fennel seeds extracts
contain important nutrients and exhibit antioxidant functions. The
results of our recently published study revealed that the methanolic
extract of both plants possesses considerable amounts of phenolic
compounds and radical scavenging activity,[51] which were in agreement with those reported recently.[52]
Some phenolic compounds have the capacity to quench lipid peroxidation
products, prevent DNA oxidative damage, and scavenge ROS.[53]
Flavonoids
isolated from chamomile, such as apigenin and luteolin, have been shown
to possess antioxidant, anticarcinogenic, carminative, antispasmodic,
and mild sedative properties.[54]
Fennel seed extract contains, by chromatographic analysis,
trans-anethole, fenchone, methylchavicol, limonene, α-pinene, camphene,
ß-pinene, ßmyrcene, α-phellandrene, 3-carene, camphor, and cisanethole.[55]
Among these, dlimonene and ß-myrcene have been shown to affect the
liver function. D-limonene increases the concentration of reduced GSH in
the liver.[56]
The
biochemical investigations were confirmed by the histopathological
results of the liver tissue. In our study, light microscopic examination
of AZA-treated rats revealed hepatocytes disorganization, fatty
degeneration indicated by large and microvesicular fat droplets and
shrinkage, pyknotic, or apoptotic nuclei. Furthermore, large nodules
with eosinophilic cytoplasm were present. Liver mean relative weight of
AZA-treated rats was significantly increased as compared to control
rats. These findings are in agreement with the findings of other
investigators that reported an increase in mean relative liver weight in
mice treated with AZA.[4]
In addition, disorganization of the liver architecture with multiple
focal areas of necrosis in AZA-treated mice and other small hepatocytes
with deeply stained acidophilic cytoplasm and dark nuclei were reported.[57]
Furthermore degenerated mitochondria, dilated cisterns of rough
endoplasmic reticulum and multiple lipid droplets were noticed. A study
reported that AZA-induced cell death (apoptosis), hydropic degeneration,
portal fibrosis, and inflammation.[58]
It has been suggested that AZA induces hepatotoxicity and mitochondrial
dysfunction owing to the stimulation of stress-activated protein kinase
pathways and intracellular GSH reduction.[59]
On the other hand, the experimental evidence pointed to increased lipid
peroxidation leading to the induction of a necrotic or apoptotic effect
in hepatocytes.[8] Furthermore, increase in ROS (as hydroxyl radical) could be involved in AZA toxicity.[32]
In
contrast, mean relative liver weights were decreased in the rats
treated with CFME or FSME before AZA treatment. Furthermore, marked
improvements in the histopathological changes were noticed, due to their
antioxidant abilities. Polyphenols and flavonoids in chamomile flowers
extracts and apigenin-7-O-glucoside as the major constituent of chamomile which inhibited cancer cell growth were recorded.[60] Al-Musa and Al-Hashem [49]
reported that the administration of ethanolic extract of chamomile
flowers to streptozotocin-induced diabetic rats significantly
ameliorated the morphological changes in the livers of treated rats.
They attributed these effects to its potent antioxidant potential
resulting in membrane stability. The increase in the antioxidant enzyme
activity and the reduction in the lipid peroxidation by fennel
methanolic extract may result in reducing a number of deleterious
effects due to the accumulation of oxygen radicals, and could exert a
beneficial action against pathological alterations, especially in
inflammatory diseases.[19]
From
the results of the current study, it can be concluded that oral
administration of either CFME or FSME has a beneficial effect in
modulating liver injury induced by AZA treatment probably through their
potent antioxidative and radical scavenging activity and due to their
higher content of total phenolic compounds. Both plants should be
considered as accessible sources of natural hepatoprotective compounds.
Financial support and sponsorship
Nil.
Conflict of interest
There is no conflict of interest.
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