| Objective On the basis of Gengnian Anyi Decoction,professor Li Lirong promoted the Cuyun Anyi Decoction for the fertility decline of the elderly women.In the early stage,clinical observations and researches have been carried out,and good results have been achieved.The effects of the traditional Chinese medicine Cuyun Anyi Decoction on the ovarian reserve function and endometrial receptivity of the aging model rats were observed with the natural aging young female rats(10-12 months old).And it’s mechanism were also studied.These could supplied a reliable experimental data and solid theoretical basis for the clinical application of Cuyun Anyi Decoction in the elderly women with fertility decline.Method The 10-12 month old rats with natural aging were selected as the research objects for modeling.The rats were randomly divided into two groups: the early-aged model group and the early-aged drug group,and the normal rats were as the normal control group(4-6 months old).The administration methods of each group were as follows:Group A(normal control group): 12.73ml/kg of purified water of equal volume was given by gavage once a day for 15 consecutive days;Group B(early-aged model group): 10.82ml/kg purified water of equal volume was given by gavage once a day for 15 consecutive days;Group C(early-aged drug group): 10.82ml/kg of Cuyun Anyi Decoction was given by gavage once a day for 15 days;This experiment was divided into four parts.The first part: To establish and evaluate the early-aged rat model.Through the observation of vaginal exfoliated cells smears in 15 days of natural aging female rats(10-12 months old),we selected the ones with prolonged oestrus interval or disorder of oestrus cycle as the model of early-aged female rats.The second part: To study the effect of Cuyun Anyi Decoction on the regulation of reproductive endocrine in the early-aged model rats.Rats in each group were anesthetized.Then we opened their stomachs and took blood from aorta 15 days after administration.The levels of AMH,INH-B,E2,FSH and LH in serum of rats were detected by enzyme linked immunosorbent assay(ELISA).The third part:To study the effect of "Cuyun Anyi Decoction" on the ovarian histomorphology and the expression of ovarian stem cell factor(SCF)and growth differentiation factor-9(GDF-9)in the early-aged model rats.The ovarian tissue of the rats after taking blood from part two was dissected and weighed.The organ index was calculated.The ovarian tissue was taken pathological section of hematoxylin eosin(HE)and the microstructure of it were observed under microscope.Western blot was used to detect the quantitative expression of ovarian stem cell factor(SCF)and growth differentiation factor-9(GDF-9).The fourth part: To study the effect of Cuyun Anyi Decoction on endometrial histomorphology and expression of LIF and HOXa-10 m RNA in the early-aged female rats.The uterus tissue of the rats after taking blood from part two was dissected and weighed.The organ index was calculated.The uterus tissue was taken pathological section of hematoxylin eosin(HE)and the microstructure of it were observed under microscope.The transmission electron microscope sections were prepared to observe the cytoplasmic processes.The expression of LIF in endometrium was measured by immunohistochemistry HRP,and the content of Hoxa-10 m RNA was measured by real-time fluorescence quantitative PCR.Enzyme linked immunosorbent assay(ELISA),which is based on immunological reaction,makes antigen or antibody bind to the surface of a solid-phase carrier,and maintains its immune activity.Then,the excess free reactants are removed by washing,the optical density(OD value)of each pore is measured at the wavelength of 450 nm by enzyme reader,and the concentration of the reactants to be measured is calculated by drawing the standard curve.ELISA can be divided into several methods.In this paper,AMH,INH-B,FSH and LH in serum of rats were detected by double antibody sandwich method,and E2 was detected by competitive method.The paraffin sections were treated with hematoxylin eosin staining(HE)and immunohistochemical staining,and the images were observed and recorded under the microscope.In this paper,this method was mainly used to observe the cell morphology of ovary and endometrium and the expression of LIF protein in ovary.Western blotting is a method of transferring proteins to the membrane and detecting them with antibodies.In this study,we used this method to detect the quantitative expression of ovarian stem cell factor(SCF)and growth differentiation factor-9(GDF-9).Real-time PCR is a method that adds fluorescent groups to the PCR system in the DNA amplification reaction,monitors the whole PCR process in real time by using fluorescence signal accumulation,and finally analyzed unknown template by standard curve.This method was used to detect Hoxa-1 mRNA in endometrium.All data were processed with SPSS19.0 software package.The data were expressed as(?).The one way ANOVA was used to analyze the differences among groups,P<0.05 the difference was statistically significant.Result1.Establishment and evaluation of the early-aged rat model The regular estrous cycle was observed in the smear of vaginal exfoliated cells of 4-6 months old female rats.In 10-12 months old female rats,we can observe the prolongation of oestrus interval or disorder of oestrus cycle by smear observation of vaginal exfoliated cells.But there are few individual differences.2.Effect of Cuyun Anyi Decoction on the regulation of reproductive endocrine in the early-aged model rats Cuyun Anyi Decoction can increase the levels of AMH,INH-B,E2 and decrease the levels of FSH and LH.The difference between the early-aged model group and the normal control group was statistically significant(P <0.05);and so does the difference between the early-aged model group and the early-aged drug group(P < 0.05).3.Effect of Cuyun Anyi Decoction on the ovarian histomorphology and the expression of ovarian stem cell factor(SCF)and growth differentiation factor-9(GDF-9)in the early-aged model rats Cuyun Anyi Decoction can increase the expression of SCF and GDF-9.And it can also improvethe microstructure of ovary.The difference between the early-aged model group and the normal control group was statistically significant(P<0.05);and so does the difference between the early-aged model group and the early-aged drug group(P<0.05).4.Effect of Cuyun Anyi Decoction on endometrial histomorphology and expression of LIF and HOXa-10 m RNA in the early-aged female rats The expression of LIF and hoxa-10 mrna in the uterus of the early-aged model rats can be increased by Cuyun Anyi Decoction.And this treatment can improve the endometrial microstructure and increase the number of pinocytes.The difference between the early-aged model group and the normal control group was statistically significant(P<0.05);and so does the difference between the early-aged model group and the early-aged drug group(P<0.05).Conclusion With the development of China’s economy,improvement of people’s living standards,the influence of higher education,and personal occupation development,women’s marriage and childbearing age has been postponed.So Late marriage and late childbirth have become a common social phenomenon.With the release of the two child policy,the elderly women not only have the need to prevent the occurrence of menopausal syndrome and delay aging,but also have the demand of more and more fertility.Cuyun Anyi Decoction is formulated by the tutor with the method of tonifying the kidney and strengthening the spleen and the method of nourishing the blood and regulating the liver.It is clinically used to treat the decline in fertility or even infertility of elderly women.It can improve ovarian reserve function and endometrial receptivity to increase fertility by treating congenital or acquired disease concurrently and regulating Chong Ren.This treatment can get the best result.The results suggest that Cuyun Anyi Decoction can regulate the levels of serum anti Mullerian hormone(AMH),inhibin B(INH-B),estradiol(E2),follicle stimulating hormone(FSH)and luteinizing hormone(LH)in the rats of the early-aged model.And it can raise the expression of ovarian stem cell factor(SCF)and growth differentiation factor-9(GDF-9),enhance the expression of endometrial leukemia inhibitory factor(LIF)and HOXa-10 m RNA.However it can improve the microstructure of ovary and uterus.So as to improve the ovarian reserve function and endometrial receptivity of the early-aged model rats and enhance the fertility of the rats. |
PDF Full Text Request