الفهرس 
CHAPTER IOnly 14 pages are availabe for public view
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Abstract
”Letrozole (LET) (Femara®) is a third-generation non-steroidal oral aromatase inhibitor, which acts via binding non-covalently to aromatase heme moiety, causing a reversible aromatase enzyme inhibition, resulting in blocking the aromatization of testosterone and androstenedione into estradiol and estrone, respectively. It is widely used as a metastatic breast cancer-first line therapy, as well as an ovulation problems treatment in fertility clinics. LET has various side effects, including endocrinopathies, metabolic disturbances, acute inflammation, as well as impairments and cytotoxicity in ovaries, uteri, bone, livers and cerebella.Nowadays, coumarins as a secondary metabolite of citrus fruits are globally used as a natural medicine, for boosting immune system, prevention and treatment of acute and chronic diseases. They are rich in vitamins and macronutrients, and well known for exhibiting greater biological activity and lower toxicity than synthetic medicines. Auraptene (AUR) which is also known as 7-geranyloxycoumarin, is a natural, bioactive monoterpene coumarin ether isolated from Rutaceae and Apiacea family. AUR is well known for its ability to modulate intracellular signaling pathways, resulting in controlling inflammation, cell growth, and apoptosis.The main objective of this study was to estimate the role of AUR as an antioxidant, anti-inflammatory monoterpene coumarin against estrogen-deprived effects caused by LET in ovary, uterus, epiphyseal cartilage, liver and cerebellum of female rats.Materials and Methods:Twenty virgin female Wistar albino rats, weighing about 110±130g, divided into 4 groups (5 rats each group) as the following:1. group A (control group): female rats were received 0.9% NaCl (0.5 ml/rat/day) for 2 weeks.2. group B (Auraptene group): AUR was dissolved in saline and female rats were received daily oral doses of AUR (0.15 mg/kg) for 2 weeks.3. group C (Letrozole group): LET tablets containing 2.5 mg of active drug were dissolved in saline, female rats were received daily oral doses of LET (0.04 mg/kg) for 2 weeks.4. group D (Letrozole + Auraptene group): female rats were received daily oral doses of LET (0.04 mg/kg) + daily oral doses of AUR (0.15 mg/kg) for 2 weeks.Daily recordings of body weights for all rats were maintained. After 24 hours from the last dose, rats were anesthetized with an IP combination of 70 mg/kg ketamine hydrochloride and 5 mg/kg xylazine and euthanized by decapitation. At the time of sacrifice, blood samples were collected from rats’ neck vein, centrifuged, and the supernatant serum was stored at -20°C until assay. Then, rats’ abdominal cavities were opened, ovaries and uteri were dissected and weighted, for further analysis, epiphyseal cartilages, livers, cerebella were dissected, and histopathological changes for all dissected organs were examined.A. Biochemical analysis: Serum hormones (estrogen, progesterone, LH, FSH, AMH, testosterone and insulin), glucose, ALP, calcium, magnesium, lipid profile, antioxidative/oxidative parameters, inflammatory parameters were analyzed. B.Apoptotic markers: Annexine V-FITC//PI and cell cycle for ovary were determined by flow cytometric technique.C. Histopathological investigations:1.Histopathological examinations for ovaries, uteri, epiphyseal cartilages, livers and cerebellums.2. Morphological analysis for ovaries, uteri, epiphyseal cartilages and cerebellums.3. Immunohistochemical investigations for ovaries and uteri.
The present study showed that administration of LET daily for 14 days caused the following:1.Increased body weight, ovarian weight and uterine weight compared with the control group. 2.Serum estradiol, progesterone and FSH were decreased compared to the control group. Meanwhile, serum testosterone, LH, AMH, insulin and glucose were elevated compared to the control group. 3. There is an increment in serum ALP compared to the control group, while a decrement in Ca++, Mg++ is detected compared to the control group.4. Hyperlipidemia were observed compared to the control female rats; there is an elevation in serum total cholesterol, total glycerides, LDL-C, but HDL-C was decreased.5.High oxidative stress and low antioxidant levels were demonstrated by measurement of serum concentrations of MDA and NO, which were significantly increased, while serum concentration of GPx was decreased compared with the control group.6. There is an increment in serum pro-inflammatory cytokines, like TNF-α and IL-6 compared to the control group.7. Annexin V-FITC//PI analysis by flow cytometry showed decreasing in percentage of viable cells, and a significant increasing in early, late apoptosis and necrosis compared to the control rats.8. Cell cycle analysis by flow cytometry detected a decrement in G0/1 Phase, while there is an increment in Sub G1, S, G2/M phase compared with the control group.9. Histopathological examinations showed dysmorphic ovaries with cystic and atretic follicles, degeneration and reduction in numbers of devolving follicles and corpora lutea, apoptosis of granulosa cell and necrosis of luteal cells.10.Uterine histopathological observations revealed histological lesions; like reduction in uterine thickness and numbers of endometrial glands, luminal epithelium degeneration and apoptosis, endometrial glands hyperplasia, and myometrial edema mixed with moderate numbers of lymphocytes and macrophages were found.11. Histopathological examinations for epiphyseal cartilage showed a disturbance in histological criteria, there is an increment in growth plate thickness, chondrocyte hyperplasia and necrosis, linear fissures in superficial and middle zone, which is expanded by edema, decrement in the cartilage matrix and lack of mineralized calcium salts deposition.12. Hepatic histopathological investigations for liver revealed histo-hepatic lesions; including disorganized hepatic cords with abnormal hepatocytes microstructures and dilated sinusoids in-between, there was a degeneration in hepatic central vein surrounded by hepatocellular necrosis and inflammatory cellular infiltration.13. Histomorphological examinations for cerebellum revealed disturbance in cerebellar cortex, there was a reduction in Purkinje cells numbers, as well as an abnormal Purkinje cells distortion and shrinkage with observed empty areas in-between. As well, granular layer showed multiple neuronal vacuolation and granular cells clumping.14.Immunohistochemical analysis of ovary revealed a weak positive PCNA expression in granulosa and theca layers of cystic follicles, as well a weak Ki-67 expression in granulosa cell layer of antral, secondary, atretic and cystic follicles. Also, there was a weak uterine VEGF immuno-expression compared to the control group. On the other hand, AUR treatment for letrozole-treated rats ameliorated the above mentioned letrozole-alterations, through modifying imbalances of hormones and metabolic disturbances, reduction oxidative stress, inflammation, apoptosis and necrosis rates, as well restoring most normal histological criteria. As a result, it can be concluded that auraptene can be used as an effective treatment against cytotoxicity as well as metabolic disorders of estrogen insufficiency, as it has the potential to mitigate and ameliorate letrozole-induced toxicity in female rats.”