The Protective Effects Of GnRH Agonist Associate With Estrogen Against Cisplatin-induced Ovarian Damage In Mice.

With the development of medical technology, the survival rate of cancer patients improved significantly. Chemotherapy as the primary means of treatment has been widely used in clinical, but premature ovarian failure (POF) of adolescent girls caused by chemotherapy seriously affect the quality of life of childbearing age women, therefore, applicating chemotherapy while protecting the function of ovarian is extremely important.In order to reduce the damage of chemotherapy-induced ovarian, domestic and international has made a number of methods. Most are still in the experimental stage, and the majority of invasive technology. In recent years, gonadotropin-releasing hormone agonist (GnRHa) research has become a hot research. The role of GnRHa protecte ovarian damage caused by chemotherapy have been positive from animal experiments to clinical studies, but GnRHa induced hot flashes, vaginal dryness, menstrual irregularities, osteoporosis and other serious symptoms of low estrogen troubled patients. GnRHa associated with estrogen can improve symptoms caused by low estrogen. A large number of studies found that estrogen also has anti-apoptosis. If a large number of scientific experiments through the study confirmed that estrogen can improve the GnRHa combined result of low estrogen symptoms, ovarian function can have a synergistic protective effect. POF caused by the chemotherapy can be resolved. For this experimental research direction, to provide more breadboard evidence for further clinical research.ObjectiveTo investigate the protective effects of GnRHa associated with estradiol against Cisplatin (CDDP)-induced ovarian failure in female mice.Materials and Methods1 Select 5-6 week old SPF female inbred Balb/c mice 40, which were purchased from Experimental Animal Center of Henan Province, Permit No.:SCXK (Henan) 2005-0001, weight 18±2g. Feed granular food and free feed in room with temperature 20℃to 25℃, light 12 hours a day. Raised one week to be familiar with the environment, and then collected the cells of the female mice at 9:00 am cells smeared on the slide vaginal daily. Observe the estrous cycle of female mice by microscopic. Select female mice with normal estrous cycle into the experiment.2 Cisplatin (CDDP, 10mg×10) was purchased from Jinzhou Kowloon Tai Pharmaceutical Company Limited.3 GnRHa (Alarelin, 0.1mg×10) was purchased pharmaceutical factory belonged to AnHui Feng Yuan pharmaceutical company Limited.4 progynova (estradiol valerate,1mg×21) was purchased from Bayer Healthcare Company Limited.5 40 female Balb/c mice selected into the experiment were divided into 4 groups randomly:(1) The control group:to give lml-d saline intraperitoneally and 1ml·d-1 saline intragastric for 20 days.(2) CDDP groups:normal saline intraperitoneal injection for 10 days, followed by cisplatin 2.0 mg·(kg·d)-1 intraperitoneal injection of 10 days, to give 1ml·d-1 saline intragastric for 20 days.(3) GnRHa+E2 group:progynova 0.1 mg·(kg·d)-1 intragastric for 20 days straight, Alarelin 0.1 mg·(kg·d)-1 subcutaneously for 20 days.(4) CDDP+GnRHa+E2 group:by GnRHa+E2 group and CDDP combination therapy group program.The vaginal cells of the animals above were smeared on the slides daily for observating the estrous cycle. All mice were killed at diestrus when groups administration was stopped. Collecoted blood by cardiac puncture. The serum follicle-stimulating hormone (FSH) and estradiol (E2) were measured by radioimmunoassay (RIA). Bilateral ovaries and uterus were obtained to weigh the wet weight and were fixed in 4% paraformaldehyde in phosphatebuffered saline. The ovaries were embedded by paraffin wax and sliced continuously in 3um. Care was taken to ensure that bilateral ovaries were removed from each mouse in their entirety for histological processing. The sections were stained with haematoxylin and eosin(HE). One slicing in ten slicings was selected to be observed the difference in pathology. The number of bilateral ovarian follicles with normal shape were counted(The plus of bilateral ovarian follicles). Mice in each group were measured primordial follicles, growing follicles (including primary follicles, secondary follicles and sinus of the follicle) and mature follicles.Results1 The changes of estrous cycle of mice(1) 4 groups mice were all in normal estrous cycle before administration,4-5 days for a cycle, including proestrus, estrus, metestrus and diestrus.(2) The mice in the control group had normal estrous cycle.(3) The mice in CDDP group showed persistent diestrus or metestrus, occasional estrus.(4) The mice in GnRHa+E2 group had disorder of estrous cycle, estrous cycle during the treatment extended to 6-7 days, estrus, or the performance of the continuous extension of estrus.(5) The mice in CDDP+GnRHa+E2 group, estrous cycle extended to 8-9 days, estrus and diestrus were extended. 2 The changes of serous concentrations of FSH and E2Concentration of FSH of the mice in CDDP group were significantly increased after administration, E2 levels were significantly decreased, compared with the other 3 groups, the difference was statistically significant (P<0.05). CDDP+GnRHa+E2 group compared with the control group, the difference was not statistically significant (P>0.05). CDDP alone and single with GnRHa+E2 on serum FSH and E2 had significant effects (P<0.01), and CDDP and GnRHa+E2 has a good interaction.3 The changes of ovarian and uterine weightIn CDDP group the weight of ovarian and uterine were significantly reduced, compared with the other 3 groups, the difference was statistically significant (P<0.05). CDDP+GnRHa+E2 group and the control group and GnRHa+E2 group, the difference was not statistically significant (P>0.05). CDDP alone and single with GnRHa+E2 on the mice ovaries and uterus weight were significantly (P<0.01), mainly the role of CDDP, and CDDP and GnRHa+E2 interaction was statistically significant (P<0.05).4 The changes in the number of folliclesIn CDDP group the total number of follicles is at least compared with the remaining 3 groups, the differences were statistically significant (P<0.05). CDDP+GnRHa+E2 group the total number of follicles in the control group and CDDP groups, but significantly more than the original follicles CDDP group, the difference was statistically significant (P<0.05). CDDP and GnRHa+E2 on mise primordial follicles and total follicles showed significant effects (P<0.01), while the interaction between the two is also statistically significant (P<0.05).5 The morphologic changes of ovarian tissue(1) control group:see primordial follicles, growing follicles, mature follicle, a larger number of corpus luteum, Less interstitial, vascular rich.(2) CDDP group:Displays the number of follicles at all levels are significantly reduced, granular cell layer decreased. Confusing organizational structure, blood vessels less Less interstitial fibrosis.(3) GnRHa+E2 group:shows a large number of primordial follicles, growing follicles and mature follicle decrease, no fibrosis.(4) CDDP+GnRHa+E2 group:the proportion of primordial follicles seen increased fibrosis compared with CDDP group reduced, no significant bleeding, necrosis.Conclusions1 2.0 mg-(kg-d)-'CDDP can affect ovarian and uterine weight, it also can alter hormone levels, damage ovarian structural and reduce the number of follicles.2 GnRHa associate with estrogen administration maybe provide a protection against CDDP-induced ovarian damage in mice.