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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. 104 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. 105 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 106 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 107 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. 108 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. |