| Objective:Endometriosis is a kind of aggressive, hormone-dependent benign disease in the female reproductive system, refers to the presence of normal endometrial mucosa (glands and stroma) abnormally implanted in locations other than the uterine cavity. As common gynecopathy, the treatment of endometriosis is directed toward surgical excision and drug therapy. Medications currently recommended is Hormone therapy; include gonadotropin-releasing hormone (GnRH) agonists, progestins, oral contraceptive pills, androgens and so on. There are some data supporting the use of aromatase inhibitors for refractory or recurrent endometriosis. Endometriosis requires estrogen to grow; aromatase is the enzyme which synthesizes estrogen. Aromatase inhibitors work by inhibiting the action of the enzyme aromatase; converts androgens into estrogens by a process called aromatization; reduces local levels of estrogen; provides a new approach for the treatment of estrogen-dependent diseases. Letrozole is a new generation aromatase inhibitor, has been shown for the treatment of endometriosis. It not only affects the local estrogen levels, but inhibits angiogenesis, cell proliferation, apoptosis and other mechanisms to promote treatment endometriosis. In this study, we use the autologous transplantation of abdominal wall to establish rats endometriosis model, utilize different doses of letrozole and mifepristone to treat endometriosis model. Through observation and comparison of change in some dates, the study seeks to clarify the treatment effectiveness and feasibility of letrozole in animal endometriosis models, provides an objective basis for its clinical application; explores its mechanism of action; provides the basis experimental evidence for scientific research; further enriches the theoretical system on the treatment of endometriosis.Methods:There was not to detect rat estrous cycle. To make rats in estrogen period in unity, all the rats were given0.02mg/kg diethylstilbestrol with intragastric administration for five days before the operation. In the tenth day after the operations, all the rats were given the same dose of diethylstilbestrol for five days to promote the growth of ectopic endometrial. According to Vernon's methods, all SD rats were established endometriosis model of by surgically transplanting autologous uterine tissues to abdominal sites. After4weeks, there were the second laparotomy to measure the volume of transplant and confirmed the success of modeling by pathology. The models were divided randomly into5groups: model control group:saline5ml/kg.d; mifepristone group:2.5mg/kg.d; low-dose letrozole group:0.1mg/kg.d; medium-dose letrozole group:0.25mg/kg.d; high-dose letrozole group:0.5mg/kg.d. Each group was fed by this dose, once a day. After4weeks, rats were killed by bleeding at femoral artery. The blood was collected, levels of E2, P, T, FSH, LH in serum were measured by radioimmunoassay; the volume size of ectopic endometrial tissue was measured; and ectopic tissues were obtained, separately were done: HE stained biopsy; MMP-9, ICAM-1, VEGF, IL-6, TNFα, NF-κB expression by immunohistochemical staining; IL-6, TNF-α and NF-KBmRNA content by RT-PCR; NE-κB protein by Western Blot.The results were expressed as the (X±S).One-way ANOVA was used to compare the outcomes by statistical package for the social science13.00. It is a statistically significant difference (P<0.05).Results:1. The model of endometriosis in rats was established successfully. The survival rate of ectopic endometrium was76.67%. The ectopic lesions showed good growth for the ridgy cystic structure within the fluid, the surface of cystic structure has blood vessels. The histological appearance showed endometrial epithelial cells, glands and stroma by pathological examination.2. In each group, there was no significant difference in the volume of ectopic endometrium before treatment (P>0.05). But the volume of each group was reduced differently after the treatment. Compared with before treatment, the difference was statistically significant (P<0.01). The volume of each group was less than model group, the difference was significant (P<0.05). No significant difference was found in the volume between mifepristone group and low-dose of letrozole group (P>0.05). It was similar to happen between medium-dose group and high-dose group (P>0.05). In mifepristone group and low-dose group, the volume was larger than medium-dose group and high-dose group (P<0.05).3. In each group, there were no significant difference in levels of E2, P, FSH, LH (P>0.05). However, levels of T were significantly different among groups (P<0.05), the outcome was showed by paired comparison:the mifepristone group didn't affect T levels in serum, there was no statistical significance with the control group (P>0.05); in letrozole groups, levels of T appeared dose-dependent changes, there were differences among the groups (P <0.05).4. Each group of ectopic endometrium in rats was done morphological observation by the light microscope:Endometriotic vesicles of the control group grew well; the layers of ectopic tissue were endometrium, myometrium and serosa from the inside to the outside; the most of the epithelial cells appeared secretory phenomena (formation of vacuoles under cell nucleus). In other groups, the ectopic endometrial tissue showed different degrees of epithelial atrophy and reduction of glands, part of ectopic endometrium performed atrophy or degeneration completely and only the surrounding muscle fibers.5. In each group, protein's expressions of ICAM-1, MMP-9, VEGF, IL-6, TNF-a and NF-kB were detected by Immunohistochemical method:The ectopic endometrium mainly manifested different degrees of staining in cytoplasm of epithelial cells. The expression of kinds of6cytokine in model control group was stronger than each drug group, the difference was statistically significant (P<0.05). The effect of high-dose letrozole group was the strongest, the expression of cytokines was the weakest, and compared with other groups, there were significant difference (P<0.05). 6. After treatment, every group, the content of IL-6mRNA, TNF-amRNA, NF-KBmRNA were assayed by RT-PCR in ectopic endometrial tissue:All kinds of3dates, each drug group was lower than the model control group, the difference was statistically significant (P<0.05); and the expression were reduced gradually in the mifepristone, low-dose, medium-dose and high-dose letrozole group, the outcome was showed significant differences by paired comparison (P<0.05).7. In ectopic endometrial tissue, the protein of NF-kB was dedectd by Western Blot:In the model control group, NF-kB was significantly higher than other drug group, the difference was statistically significance (P<0.05). Letrozole for NF-kB expression appeared dose-dependent changes, to mifepristone group, differences were statistically significant (P<0.05).Conclusion:Letrozole can significantly inhibit the rats'ectopic endometrium and not change the rat circulating E2, P, FSH, LH levels. The mechanism may be related to the demotion of expression of some cytokines, such as ICAM-1, MMP-9, VEGF, IL-6, TNFa and NF-kB. In the ectopic site, those cytokines influence the adhesion, invasion and angiogenesis; reduce expression of aromatase; inhibit the immune response of EMs, so as to achieve the treatment of endometriosis. |
