| Premature ovarian failure (POF) is a common endocrine disease in gynecologic.lt is confirmed that patients reduced ovarian secretion of estrogen due to premature ovarian failure can cause osteoporosis,lipid metabolism disorders,urogenital atrophy,cardiovascular disease and so on in recent studies. So women with pof have more difficulty with respect to their physical health and psychological aspects.The modern medical thinks pof for many factors may cause syndrome. Traditional chinese medicine holds that pof in nephrasthenia syndrome as the basic pathogenesis. Namely with innate endowment relevant,damage excessive can also lead to reduce shen yin tian gui. Secondly, the long-term disorder of heart, liver, spleen are influential.The method of replenishing kidney and strengthening spleen was drawing up on the grounding of professor ShiYanqiu. Wubie granules is replenishing kidney and strengthening spleen method reagent (turtle shell, medlar,dipsacus asperoides, dodder, fleece-flower root and so on) preparation of pure chinese medicine hospital, for the treatment of women in premature ovarian failure. This research use modern scientific methods to explore the premature ovarian function of animal models of preparation and to explore the mechanism of action of Wubie granules.The study was divided into 4 parts:Part 1 The acute oral toxicity test of Wubie granules.LDso is not detected.The maximum amount of administration by gavage shows that:20 mice were lavaged 148g/kg which was 269.1 times of adult clinical oral dose, no obvious toxic reaction, no deaths within 2 weeks.Part 2 To research the influence of Wubie granules to hormone levels of mice with premature ovarian failure. Methods:Take female SD rats, bilateral ovaries of rats were removed besides the normal group. Two weeks after surgery, grouping the successful model rats.After treated for 4 weeks, take blood from orbital vein. Measured serum E2, FSH and LH level.And using TTC reduction method measured the OD of reaction extract.The OD value to the activity of estrogen. Results:Wubie granula can rise the E2 level of ovariectomized rats. Compared with the model group has significant differences(p<0.05,p<0.01). Compared with model group,the level of FSH and LH existed a downward trend in the large dose of Wubie granula and the moderate dose of Wubie granula. Conclusions:Wubie granula can increase estrogen while promoted secreting vaginal angle of epithelial cells. Wubie granula with estrogen-like effects can regulate the level of hormone which changes the symptoms of POF.Part 3 To research the influence of Wubie granules to the different methods that induced POF model.In the first section, explore the feasibility of D(+)-galactose to premature ovarian failure mice and the mechanism of Wubie granules. In addition to the normal group, and the remaining mice were given 5% D(+)-galactose with 0.5ml each day cervicodorsal subcutaneous injection,42 days continuous injection.From the eleventh day,all mice were given the curative by ig.To determine the oestrous cycle by observing vaginal cells every timing.At the end of the experiment,taking the serum and testing the level of E2.Take and weight uterus and ovary. Then calculation ovarian and uterine index.Ovary and uterus for organization morphological observation. In the second section, to explore the effects of Wubie granules on mice with immune premature ovarian failure. Balb/c female mice model of pof was established by multiple sites subcutaneous injection of the mouse zona pellucida 3 and treated with Wubie granules after the establishment of the model, with diethylstilbestrol(des) as positive control. Each group were given the corresponding medicine for 4 weeks.The AzpAb were determined by enzyme-linked immunosorbent assay,and T lymphocyte cell subsets of the spleen were detected by immunohistochemistry. The fas and fasL protein expression of the ovarian were detected by immunohistochemistry. To analyse the pathological change of the ovary and spleen. In the third section, Observing the effects of Wubie granules to premature ovarian failure mice that caused by cisplatin. Cisplatin liquid(3.0mg/kg) was intraperitoneal injection to establish POF model. after 7 days,mice were given Wubie granules and cisplatin liquid continued intraperitoneal injection. To determine the oestrous cycle by observing vaginal cells every timing.At the end of the experiment,taking the serum and testing the level of E2.Take and weight uterus and ovary. Then calculation ovaries and uterine index. Ovarian for organization morphological observation. In the forth section, observing the effects of Wubie granules to premature ovarian failure mice that caused by GTW. In addition to the normal group, and the remaining mice were given GTW.From the fifth week, each group began to be given the corresponding medicine while continue giving GTW. At the end of the tenth week,the level of E2 was detected by radiation immune method. Take the ovaries, uterus weight, then calculation ovaries and uterine index. Ovarian for organization morphological observation,counted the number of various follicular and corpus luteum.Results:The first, third and fourth section of the study suggests that compared with model control group, ovarian index and uterine index of each dose group of Wubie granules were existing difference significantly. The morphology of ovarian tissue of every dose of Wubie granules improved obviously. The first, third and fourth section shows the level of serum E2 of high-dose of Wubie granules and moderate dose of Wubie granules increased obviously (p<0.05,p<0.01). Compared with the model group,the growth follicle and mature follicle of the large dose of Wubie granules increased significantly, atretic follicles was reduced significantly(p<0.05,p<0.01). The second section of the study suggests that the mice of pof model manifested such abnormalities as increased AzpAb in peripheral blood, increased CD4+/CD8+ in the spleen, decreased Fas protein expression and increased Fas-L protein expression in the ovary. No significant difference was found between the model group and adjuvant group.Compared with the model group,the concentration of AzpAb of Wubie granules (large group)were significantly decreased(P<0.01). The CD4+/CD8+ of Wubie granules was significantly lower than the model group(p<0.05,p<0.01).Wubie granules(large,medium dose) was significantly higher than that of Fas protein expression in model group(p<0.05, p <0.01).The expression of Fas-L protein of Wubie granules was significantly lower than the model group(p<0.05,p<0.01).After drug treatment,the pathological change of the ovary and spleen were improved.Part 4 To research the influence of Wubie granules on rats with thyroidism-type "Yin" deficiency.Female SD rats were are randomly assigned.Except the normal group,thyroidtablet were fed by ig every day,4mg/every rat,for four weeks.In the second week,we began to treat with Wubie granules,lasted for three weeks.Before treatment,we measure blood sugar,heart rate,weight,food intake,water intake,urine volume and temperature.We detection the same indicators,on the seventh,14th and twentieth day. At the end of the experiment,take serum for Cu/Zn. Measured serum T3, T4 and TSH level.Besides,organ(thyroid gland,adrenal gland,spleen,thymus gland) index were monitored.Results:The organ(thyroid gland,spleen,thymus gland)index of rats of Wubie granula high group were significantly lower than model group,compared with model group has significant differences(p<0.05,p<0.01).The concentration of Cu in serum of rats of Wubie granula high group was lower than model group,concentration of Zn was high than model group,and Cu/Zn has significant differences compared with model group(p<0.05).Wubie granula can reduce the level of T3T4 in serum,compared with model group (p<0.01).The concentrations of TSH were lower than model group,but it had no significant difference.Conclusion:(1) The rat model established by D(+)-galactose,ZP3,CDDP and GTW is effective and successful as a pof animal model and can apply for the durg research. (2) Wubie granules can promote the development of uterus and ovary, improve the morphology of ovarian tissue, adjust ovarian hormones.(3) Wubie granules can significantly decrease the concentration of AzpAb in peripheral blood,and regulate CD4+CD8+ and the Fas/FasL system of mice with immune pof.(4) Wubie granula has effects of sheng-jin,nourishing yin,and wubie granula can regulate the level of thyroid hormone. |
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