| Research backgroundPremature Ovarian Failure (POF) is a syndrome that persistent amenorrhoea and atrophy of the sexual organs with increase in follicle-stimulating hormone and luteinizing hormone and decrease in estrogen before forty years old occur to women whose menstruation menarche is normal or puberty is postponed and secondary sex characteristics are well developed. Epidemiological studies have established that morbidity of POF is1/100 before 40,1/1000 before 30,1/10000 before 20 and it is increasing annually. POF can cause infertility, low estradiol level and other diseases which can affect women’s life and physiological and mental health.So POF has become a hot but difficult medical research.Er-xian decoction can improve clinical symptoms of POF and it is a famous and useful prescription by clinical practice.lt is composed of Xianmao, Yinyanghuo, Bajitian, Danggui, Zhimu and Huangbai. Xianmao and Yinyanghuo can warm the kidney-yang. Bajitian can invigorate kidney and strengthen yang, enrich and benefit essence and blood. Danggui can strengthen liver and kidney, activate and nourish blood. Zhimu and Huangbai can nourish kidney-yin and clear away ministerial fire. Moreover, Er-xian decoction is useful for climactic syndrome, amenorrhoea and other chronic diseases. Therefore Er-xian decoction becomes a wide-used prescription in gynaecopathia treatment.Experiment One Study of establishing the rat animal model of premature ovarian failure caused by cisplatinObjective:To investigate the common method of animal model of chemotherapy-induced premature ovarian failure caused by cisplatin, we intraperitoneally injected different volume of cisplatin to cause the damage of ovarian function of rats, so that we could ensure the most suitable concentration for establishment of rat animal model of premature ovarian failure caused by cisplatin.The result can be used in next experiment and helpful for later study.Methods:60 healthy female rats with normal estrous cycle were randomly divided into normal group, low-dose group and high-dose group.Each group has 20 rats. The low-dose group and high-dose group were intraperitoneally injected with cisplatin of 4 mg/kg-1 and 6 mg/kg-1, while normal group were intraperitoneally injected with 1ML normal saline on the 1st and 8th day. During the days, we observed vaginal exfoliated cells of rats to study the change of estrous cycle and vital signs, recorded the weights.7 days after the 2nd injection, we took rat serum for determination of FSH, E2, Pro and LH and ovary for pathological section to observe ovarian tissue morphology by HE coloration.Results:(1) Before injection, weight of rats is not obviously different. After injection, weight of rats in low-dose group and high-dose group were descreased in different degrees. They drumbled, liked arching back and huddling up, lost appetite and their fur was dull. Compared with low-dose group, weight of rats in high-dose group is lighter after 2nd injection, while weight of rats in normal group grows stably.(2) After injection, estrous cycle of rats in normal group was normal, while estrous cycle of rats in low-dose group was disordered like longer diestrus and shorter oestrus. But estrous cycle of some rats recovered a week after 2nd injection. However, estrous cycle of rats in high-dose group was longer and even stayed at diestrus without oestrus. Estrous cycle of rats in high-dose group didn’t recover.(3) Compared with normal group, level of FSH in rats in high-dose group elevated (P<0.05), level of FSH in rats in low-dose group elevated not obviously(P>0.05). Level of Pro is not different in each group before and after injection (P>0.05). Compared with normal group, level of E2 in rats in high-dose group and low-dose group decreased (P<0.05).(4) Ovaries of rats in normal group were bigger, follicles of different developing stages were developing well, atretic follicles were few and granulosa cells were full. Primordial follicles and primary follicles in low-dose group decreased, atretic follicles increased, and corpus luteums decreased. Secondary follicles and mature follicles were few and there were many atretic follicles. Granulosa cells were fewer and corpus luteums decreased obviously.Conclusion:After injection of different doses of cisplatin, ovarian function of rats was damaged in different degrees and the effection in high-dose group was more. Our experiment showed an easier and more reliable way to establish animal model of premature ovarian failure.Experiment Two Effect of Er-xian decoction in follicular granulosa cell apoptosis in chemotherapy-induced ovarian failure of ratsObjective:This experiment was on the base on follicle apoptosis related cytokines and genes for the study of the main line, according to Experiment One to establish the animal model of premature ovarian failure, to investigate the Er-xian decoction on the effects of follicle growth, development and apoptosis. Through the whole animal experiment, mechanism of action of treatment of Er-xian decoction on ovarian premature failure was elucidated on the the molecular level of cell apoptosis, theory and experimental basis for the study of new drugs for ovarian premature failure was provided.Methods:Healthy female SD rats were randomly divided into normal group, Erxian Decoction group, model group and positive group. Besides normal group, other groups were intraperitoneally injected with cisplatin of 6 mg/kg. After that, normal group and model group were intragastric infusion with 1ML distilled water, positive group was with 1ML progynova and Erxian Decoction group was with 1ML Erxian Decoction for four weeks at the same time every morning and afternoom. We observed general condition and estrous cycle of rats; took rats serum for determination of FSH, E2, Pro, LH, AMH and INH-B; observation of ovarian tissue morphology by HE coloration and count of all levels of follicle; analysis of granulosa cell apoptosis by TUNEL; the expression of immunohistochemical detection of BAX, Bcl-2 and VEGF protein.; the expression of IL-2 by in situ hybridization.Results:(1) Weight of rats in normal group grows fast and stably. Before injection, weight of rats is not obviously different. After injection, weight of rats in Erxian Decoction group, model group and positive group descreased obviously. After intragastric infusion with distilled wate, weight of rats didn’t change obviously. After intragastric infusion with Erxian Decoction, weight of rats gained slightly. After intragastric infusion with progynova, weight of rats gained obviously.(2) Compared with normal group, LH elevated and E2 decreased in other groups(P<0.05). Compared with model group, E2 elevated and FSH decreased in Erxian Decoction group. FSH and LH in model group and positive group were not different obviously, while E2 of model group decreased(P<0.05).(3) The total number of follicles decreased in model group compared to normal group, there are differences between groups on the whole. Compared with normal group, there were rarely secondary follicles or mature follicles, mostly atresia follicles, follicular granulosa cells layer was significantly thin, corpus luteum decreased significantly in model group. Compared with model group, Erxian Decoction group had more follicles, mostly were secondary follicles.(4) Judging from apoptotic cells form, the model group compared with the normal group, the number of apoptotic granulosa cells increased obviously, and the cytoplasm visible tan, follicular dysplasia. After Erxian Decoction treatment, positive expression decreased significantl. There was no obvious difference between positive group and model group.(5) The expression of BAX significantly increased. The expression of Bcl-2 significantly decreased in model group compared to normal group. After treatment of Er-Xian decoction, the expression of BAX decreased, expression of Bcl-2 increased. There was no significant difference between positive group and Er-Xian decoction group. The expression of VEGF protein in model group was obviously lower than the normal group, which in positive group and Er-Xian decoction group was higher. The expression of IL-2 was little, it is more in model group and less in positive group and Er-Xian decoction group compared to model group.Conclusion:The experiment proves that Er-Xian decoction can reduce autoimmune and ovarian antigen target cell damage in ovarian premature failure rats, to protect the ovarian function. By adjusting the hormone content in serum and changing the expression of related proteins in apoptotic cells, Er-Xian decoction can reduce the damage to the ovaries by cisplatin, promote the follicular development and improve and enhance the ovarian function. |
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