Study On Letrozole Treating Endometriosis Model In Rats

Endometriosis (EMs) is a common, benign and chronic gynecological disorder in women of reproductive age that can result in progressive dysmenorrheal, pelvic pain and infertility. The morbidity of this disease is increasing year by year. The prevalence of pelvic endometriosis approachesl0%-15%in the reproductive female population.Although it is a benign disease, it has malignant biological behavior such as invasion, metastasis and recurrence. Its etiology and pathogenesis is still unclear, which leads to little effect in treatment and high recurrence. EMs significantly affects Patients’health and life qualities.It has been a difficult and hot matter in the gynecological field.Endometriosis is an estrogen-dependent disease, estrogens play a crucial role in the establishment and development of this condition. Cytochrome P-450 aromatase (Aromatase P450, P450arom) is the speed-limit enzyme in the anabolism of estrogen, which can catalyze androstenedione and testosterone into estrone and estradiol respectively. There are transcriptions of P450arom mRNA and its protein expression in the ectopic and eutopic endometrium in endometriosis, however, normal endometrial tissue does not express aromatase gene, there are indications that in addition to ovarian estrogens, ectopic endometrial tissues can also produce estrogens.further more, there is a positive feedback loop among prostaglandin, P450arom and estrogen in local endometriotic tissues, that favors continuous formation of E(2) and estrogen level increasing in the local ectopic tissues, thus promoting the growth and implantation of endometriosis. On behalf of the current medical therapies for endometriosis, gonadotropin-releasing hormone agonists (GnRHa) can only suppress the synthesis of estrogen in ovary, but they can’t inhibit the synthesis of estrogen in endometriotic focus itself, however, aromatase inhibitor can suppress aromatase activity not only of ovary but also besides ovary include skin, Lipid, adrenal gland and local ectopic tissues, consequently,the synthesis of estrogen were effectively inhibited. This is exactly the theoretical basis for aromatase inhibitor treating endometriosis.Using aromatase inhibitor is a new therapeutic Method.It is showed that aromatase inhibitors have obvious effect on progressive and recurrent postmenopausal endometriosis, but most of patients with EMs are premenopausal cases whose aromatase locate in ovary. It is thought that serum estrogen diminishing will increase the secretion of LH and FSH through negative feedback, accordingly, LH will excite ovary to produce more androstenedione which is aromatase substrate, FSH as well as will enlarge the aromatase contents. Aromatase inhibitors’therapeutic effects decrease consequently, and this feedback will also lead to ovarian excessive stimulate. Therefore, theoretically, aromatase inhibitors should not be used alone in premenopausal women, the combination of aromatase inhibitors and ovarian-suppressant treatment should be administered. However, there are also other points of view which indicate that although the level of LH and FSH increase, aromatase inhibitors can still decrease the serum estrogen level, thereby treating EMs effectively. The therapeutic effects, mechanism of action as well as reasonable therapeutic regimen for aromatase inhibitors treating premenopausal endometriosis have yet to be studied.In this study, endometriosis model in rats were established successfully, the following items were tested includes:the ectopic lesions’volume before and after the treatment, the aromatase mRNA expression and the protein expression of P450arom, COX2、VEGF and PCNA in the endometriotic tissues, endometriotic apoptotic cells,the serum levels of FSH、LH and E2, weights and morphological changes of the other uterine horn and ovary. In order to compare aromatase inhibitor alone with aromatase inhibitor combined with ovarian-suppressant treatment of the treating effect, mechanism of action and effect on reproductive system.This study provided reasonable therapeutic regimen and experimental basis for aromatase inhibitors treating premenopausal endometriosis. This study included two parts.Part I Establishment of a rat model of endometriosisObjective To establish an endometriosis model in Sprague-davley (SD) rat using Surgically transplanting autologous endometrium, and to provide an ideal animal model for the next experiment. Methods 65 sexual maturity unpregnant female SD rats were used, the rats’estrous cycles were monitored by observing the morphological changes of vaginal cast-off cells, the rats were chosen which had at least two continuous estrous cycles, and in the third estrus, according to Jones’method, autologous endometrium of 5mmx5mm in size were satured on the rats’abdominal wall. Four weeks after the model induced, the animals were again laporatomized and the condition as well as the size of the explants were tested.Then successfully induced model rats were randomly divided into five groups as the experimental objects for part II. Seven weeks after the model induced, that was three weeks after grouping treatments, the rats were laporatomized for the third time to observe the shape and the size, then the transplants were cut off to do the tests. Results 1.All of the experimental rats had regular and continuous estrous cycle which was 4-5days.The estrous cycle included four phases which were preestrusN estrus、postestrus and diestrous.2.50 rats of total 60rats formed endometriotic lesions, the successful ratio was 76.92%(50/65) when the animals were laporatomized at the second time four weeks after model establishment. The appearance of the successful transplant looked like a clear cyst in which some clear or faint yellow liquid accumulated,and blood vessels were clearly legible on the surface. The structures of the lesions were clear and having no adhesion with surrounding tissues. The ectopic endometrial implants had the same fundamental structure as normal endometrial tissue:glands、epithe Lium and mesenchyma.3. The ectopic endometrium growed well in the control group when the rats were laporatomized for the third time, both the size and morphology had no remarkable changes(.P>0.05), these showed that the models were stabile and trusted. Conclusion The establishment of endometriosis model in rats’abdominal wall by Surgically transplanting autologous endometrium in estrus were economicak simple and convenient、effective and liable, it provided favorable experimental models for the medical therapy for endometriosis.PartⅡStudy on letrozole treating endometriosis model in ratsObjective To compare aromatase inhibitor alone with aromatase inhibitor combined with ovarian-suppressant treatment of the treating effect, mechanism of action and effect on reproductive system by testing the ectopic lesions volume before and after the treatment, the aromatase mRNA expression and the protein expression of P450arom、COX2、VEGF and PCNA in the endometriotic tissues, endometriotic apoptotic cells,the serum levels of FSH、LH and E2, weights and morphological changes of the other uterine horn and ovary. Methods Surgically transplanted autologous uterine tissues to ectopic site beside the uterine in rats were used as animal models to study endometriosis.50 EMs model rats were randomly divided into 5 groups(n=10):A:letrozole-treated group:letrozole was administered at a dose of 1 mg/kg by oral lavage every day for three weeks; B:MPA-treated group: Medroxyprogesterone acetate (MPA)8 mg/kg was administered by oral lavage every day for three weeks;C:letrozole and MPA combination-treated group:letrozole 1 mg/kg combined with MPA 8 mg/kg was administered by oral lavage every day for three weeks; D:ovariectomized group:bilateral ovaries were excised at the time of the second laporatomy and were observed for three weeks; E:saline solution control group:saline solution of the same volume was given every day for three weeks. The model rats were laporatomized the third time three weeks after grouping treatments. The changes of ectopic lesions’volumes in each group were compared before and after the treatment by using vernier caliper to measure the length, width and height; expressions of aromatase mRNA were examined by hybridisation in situ; Immunohistochemistry was used to detect the expression of P450arom、COX2、VEGF and PCNA proteins in the endometriotic tissues of the rat models; Apoptotic cells were assessed by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling (TUNEL) assay;The serum levels of FSH、LH and E2 were tested by radioimmunoassay.The other uterine horn and ovary were weighted and were observed by optical microscopy to study the change of morphology after HE stained.Results 1.There were no difference in the lesions’volumes among each groups (P>0.05). The volumes of the endometriotic tissues of the four group reduced markedly compared with the control group (P<0.01), among them, the greatest changes in the volume were of the combination group and ovariectomized group (P<0.01).2.Expressions of P450arommRNA and proteins of P450arom、COX2、VEGF and PCNA decreased in tissues of endometriosis on rat models among A,C and D groups(P<0.05). expressions of VEGF and PCNA proteins decreased(P<0.05) but P450arommRNA and proteins of P450arom and COX2 had no change (P>0.05) in B group.3.The apoptotic rates increased clearly in A、B、C and D groups (P<0.0l) and apoptotic level in C group was the highest (P<0.05).4.The level of FSH、LH and E2 reduced in B and C groups (P<0.05),moreover, the E2 level of C group was lower than D group(P<0.05),it equaled to the E2 level of D group. However, the serum levels of FSH、LH and E2 had no difference in A group compared with E group (P>0.05).5. The ovarian weights in A group increased greatly, and the ovaries showed Polycystic ovaries. The weights in B and C groups had no difference compared with the control group (P>0.05), and the ovaries were in the inhibition of activity.6. The uterine weights decreased in the four groups, and the endometriums presented atrophy or proliferation inhibitition.Conclusion 1. Letrozole alone had effect on EMs rat models,but it could arise ovarian hyperstimulation which lead to the increase of ovarian weight and ovarian polycyst.2. Letrozole associated with MPA was more effective on EMs rat models than letrozole or MPA alone. Moreover, it wouldn’t bring about ovary overstimulated which was due to using letrozole alone. 3. Letrozole cooperated with MPA playing the role of treating EMs by means of decreasing the serum E2 level and reducing estrogen and prostaglandin along with angiogenesis in the local endometriotic tissues.They as well as increased the apoptosis while decreasing the proliferation of the ectopic tissues.4. The influence of letrozole combined with MPA on reproductive system should be come into notice.