الفهرس 
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Abstract
Methotrexate, a folic acid antagonist is a chemotherapeutic drug utilized for
treating different types of cancers such as breast cancer, lymphoma, acute
lumphoblastic leukemia and cancer of the neck and head, methotrexate also used
for treatment of psoriasis and rheumatoid arthritis. It is undoubtedly effective but
its toxic effects severely limit its use. It has many toxic effects on different body
organs such as liver, kidney and testis.
Pimpinella anisum (anise) has been shown to possess several medicinal
properties; it is widely reported as, a stimulating drink for digestion, nutritive, antiparasitic,
anti- bacterial, anti- fungal and anti-pyretic agent. In clinical studies, it
has shown anti- inflammatory and antioxidant activity.
In recent years, public attention in benefits of natural plants to prevent or
help in treating disease has driven the scientific committee to explode the health
efficiency of many plants. Therefore, the present study examined the side effects of
methotrexate in liver, kidney and testes and the modulation of toxicity caused by
concomitant exposure to MTX by anise oil.
Animal groups:
Animals were divided into 4 groups as follows:
group 1 (Control group)
Animals of this group (7 rats) were orally given 1 ml saline daily for four weeks.
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group 2 (Anise oil group)
Animals of this group (7 rats) were orally given with 0.5 ml /kg body weight of
anise oil 4 days/week for four weeks.
group 3 (Methotrexate group)
Animals of this group (7 rats) were orally given 0.04 mg /kg body weight of
methotrexate dissolved in saline daily for 30 days.
group 4 (Anise +Methotrexate group)
Animals of this group (7 rats) were orally given 0.04 mg /kg body weight of
methotrexate dissolved in saline daily and after 1 hour were orally given0.5 ml/kg
anise oil 4 days/ week for 4 weeks.
Immediately after dissection, small pieces of liver, kidney and testes were
removed and fixed either in alcoholic Bouin՚s for histopathological study and
general carbohydratesor 10% formaline for immunohistochemical study and total
protein. Parraffin sections were prepared and stained by haematoxylin and eosin
for histopathological examination. For histochemical purposes, carbohydrates was
demonstrated by periodic acid schiff՚s (PAS) technique and total protein was
demonstrated by mercury bromophenol blue method. For immunohistochemical
study, Avidin- biotin peroxidase method was used for immunohistochemical
demonstration of α-SMA and PCNA. For biochemical analysis, the liver function
enzymes (AST, ALT), kidney function enzymes (urea, creatinine) and testosterone
and LH for testes function. In addition, antioxidant enzymes super oxide dismutase
(SOD), catalase (CAT) and MDA were determined in sera of animals of all groups
after treatment.
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The results can be summarized as follows:
I- Liver
Histopathologically, specimens examined from animals treated with
anise oil didn’t show any histopathological change and revealed the same
picture of the control animals. Animals treated with methotrexate showed
many histopathological changes in liver, e.g. destruction of liver
architecture, enlarged and congested blood vessels, leucocytic
infiltration, cytoplasmic vacuolization of hepatocytes, pyknosis and fatty
infiltration. When animals treated with both methotrexate and anise oil at
the same time, marked improvement was observed in the hepatic tissue,
and the histopathological changes were less prominent when compared
with those in the methotrexate group.
Histochemically, general carbohydrates in liver of animals treated
with anise oil showed the same content of the control group. Animals
treated with methotrexate showed depletion in liver carbohydrate content.
When animals treated with both MTX and anise oil at the same time,
marked increase in carbohydrate content was observed when compared
with MTX group.
Total protein content in liver of animals treated with anise oil showed
normal content. Liver sections obtained from animals treated with
methotrexate have shown decrease in total protein content. On the other
hand, animals treated with methotrexate and anise oil showed a marked
restoration in the protein content in the most liver cells.
Concerning immunohistochemical results, animals treated with
anise oil showed normal PCNA expression in few cell and normal α-
SMA expression similarly to control group. Animals treated with MTX
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showed strong PCNA expression in the most of hepatocytes and strong α-
SMA expression. On the other hand, liver sections obtained from animals
treated with both MTX and anise oil showed restoration of both normal
PCNA and α- SMA expression.
II- Kidney:
Histopathologically, kidney sections obtained from rats treated with
anise oil didn’t show any histopathological changes and revealed the
same picture of the control rats. Animals treated with methotrexate
showed many histopathological changes in renal tissue such as, dilatation
in renal tubules, atrophy of glomeruli, degeneration of large number of
tubular cells, congestion of blood vessels, edema in some sections and
nuclear condensation were also observed. When animals treated with
both MTX and anise oil, marked improvement was observed in renal
tissue.
In respect histochmical results, general carbohydrates and total
protein contents in kidney of animals treated with anise oil showed the
same contents of control group. On the other hand, kidney sections
obtained from animals treated with methotrexate group showed marked
depletion in general carbohydrates and total protein content. When
animals treated with methotrexate and anise oil, a marked restoration of
general carbohydrates and total protein was observed in renal tissue.
Immunohistochemically, kidney sections obtained from rats treated
with anise oil showed normal expression of PCNA and α- SMA which
similar to control group. On the other hand animals administrated with
methotrexate showed strong expression of PCNA and α- SMA, and
animals administrated with both methotrexate and anise oil showed
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marked decrease in PCNA and α- SMA expression when compared with
methotrexate group.
III- Testis:
Histopathologically, testes sections obtained from animals treated with
anise oil didn’t show any histological alternations and give the same
picture of control animals. Animals treated with methotrexate showed
many histopathological changes e.g. decrease in the diameter of
seminiferous tubules and in the height of epithelium, congestion of blood
vessel, degeneration of the seminiferous tubules, exfoliate of germ cells,
decrease in the number of spermatozoa in the lumen of seminiferous
tubules. On the other hand, animals treated with both methotrexate and
anise oil showed marked improvement in testicular tissue and
seminiferous tubules.
Histochemically, tests sections of rats treated with anise oil showed the
same general carbohydrate and total protein content of control group. On
the other hand, testis sections obtained from rats treated with
methotrexate showed marked depletion in both general carbohydrate and
total protein content. When animals treated with methotrexate and anise
oil, a marked restoration of normal general carbohydrate and total protein
content were observed.
Concerning immunohistochemical results, PCNA expression of testes
of animals treated with anise oil was the same expression of control
group, where spermatogonia showed strong expression. When animals
treated with methotrexate, the testes sections showed decrease in PCNA
expression. On the other hand, animals treated with methotrexate and
anise oil showed marked restoration of normal expression of PCNA.
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Weak expression of α- SMA in the basal lamina of seminiferous tubules
of rats treated with anise oil was observed as those in control group.
While strong expression of α- SMA was observed in testis of rats treated
with methotrexate. When animals treated with methotrexate and anise oil,
the α-SMA expression decreased and retained to normal expression.
Biochemical analysis:
Anise oil restores the elevated liver function enzymes (AST, ALT) and
kidney function enzymes (Urea, CREAT). It also restores the deficiency
of testicular hormones (LH, TESTO). In addition, anise oil improved the
depletion of antioxidant enzymes (SOD, CAT and MDA) induced by
methotrexate.
Finally in conclusion, anise oil has a curative effect on the
histopathological, histochemical , immunohistochemical , oxidative and
biochemical changes caused by methotrexate- induced in the liver,
kidney and testis of albino rats.