| Objective:Assisted reproduction1prescription, a clinical topical prescription as adjuvant therapy for our mentor to improve infertility patients with kidney and blood stasis type and revised on the classical prescription wuziyanzong pills by a small quantity, has the effects of consolidating the kidney and supplementing essence and promoting blood circulation for removing blood stasis, which improves the endometrial receptive of patients, thus increasing the rate of embryo implantation. In order to evaluate the safety of assisted reproduction1prescription clarify its efficacy and further reveal its mechanism of action, the research aims at toxicology experiment of assisted reproduction1prescription, at the same time, and observing the influence on the body weight, the uterus viscera index, the endometrial tissue morphology, the endometrial tissue protease metal matrix MMP-9and the content of its inhibitor of TIMP-1of model rats with kidney deficience and blood stasis syndrome. Preliminary this paper discusses on its safety and the mechanism of improving uterine receptivity, which provides a theoretical basis for clinical use and lays a foundation for the further development of the experiment.Methods:1Experimen taltoxicologyAccording to the maximum concentration, maximum delivery capacity of enema, once per2hours,8times/day, observing by the assisted reproduction1prescription acute toxicity in mice.2Effect of assisted reproduction1prescription on model rat with kidney deficiency and blood stasisThe adult female SD mice of proestrus were randomly divided into five groups, namely blank group, model group, assisted reproduction1prescription by high and low dose group,wuzi group,the wuzi group as a positive drug control group. Each group lavage by hydroxyurea450mg/(kg·d) once per day,8days in a row due to kidney deficiency model rats, than close cage after the completion of the building,continues to give medicine until the fourth day of gestation. And one hour after the last dose, dosing the10%polymer dextran5ml/kg from the intravenous back quickly except blank group and wuzi group, by which the kidney and blood stasis model is established. Putting to death the rats after30min, taking out the rats and rapidly weighing the uterus tissue, fixed it in the FAA liquid, then calculating the index of uterine viscera, observing the pathological morphology change of the uterus endometrial tissue of the rats in each group under the microscope, and detecting the expression level of MMP-9and TIMP-1, those of values were determined by immunohistochemistry from endometrium.Results:1Acute toxicity test:During the observation period after the administration of enemas, mice in the control group and the medication group were active and had good spirit,their diets and stool and urine were quiet normal.Their eyes,noses and mouths were clean without any abnormal secretion or any abnormal reaction.There was no statistically significant difference in mice’s weight growth in this two groups (P>0.05). Not even one mouse died.Dissect the dead mice’s body which were killed by taking off the necks,the results of observation showed that there were abnormal changes of the main internal organs.In the experiment, the maximum dosage in mice which was determined within1days was77.38g/kg,which was106times equivalent to the daily doses of adult. At this dose,the enema was safe for mice.2Effect of assisted reproduction1prescription on model rat with kidney deficiency and blood stasis (l)The effect of assisted reproduction1prescription on the growth of body weight of model rats with kidney deficiency and blood stasisCompared to the blank group,the rats’weight of the model group is lower,and the differences had its statistical significant (P<0.05),which illustrated that the model may have inhibitory effect on the growth of rats’ weight;Compared to the model group, the rats’weight of assisted reproduction1prescription by high, and low dose group were higher,and and the differences had its statistical significant (P<0.05or P<0.01).The rats’ weight of assisted reproduction1prescription by high dose group was higher than that of the assisted reproduction1prescription by low dose group,but the difference was not remarkable (P>0.05),which illustrated that the assisted reproduction1prescription can promote the model rats’ weight growth.(2)The effect of assisted reproduction1prescription on the growth of the uterus viscera index of model rats with kidney deficiency and blood stasisThe uterus viscera index of model group was lower than that of the control group,and the differences had its statistical significant(P<0.05),which proved that model may inhibit the growth of rats’ uterus;The uterus viscera indexes of assisted reproduction1prescription by high, and low dose group were higher than that of the model group,and the differences had its statistical significant (P<0.05),which indicated that assisted reproduction1prescription had the effect of promoting the growth of rats’ uterus. The uterus viscera index of assisted reproduction1prescription by high dose group was higher than that of assisted reproduction1prescription by low dose group,but the differences between these groups had no statistical significant (P>0.05).(3)The effect of assisted reproduction1prescription on the endometrial tissue morphology of model rats with kidney deficiency and blood stasis.The Intima development of model group was poor, the glands are small and straight with interstitial density,the cells were spindle shaped and had few spiral artery which was located in the basement layer and observed in growth period;Intima which was in estrous period, part of the mesenchyme was dense,part of the mesenchyme was loose and dropsical,and the Interstitial growth is not synchronous. The blank group and the treatment group has the many endometrial gland which distributes evenly with gland bending.There were bright cells in glandular epithelium and a few had apocrine secretion.Glandular cavity had secreta and the mesenchyme was loose and dropsical.The spiral arterioles hyperplasia was close to the function layer. In the mordern group, there has34%of endometrial growth developed and17%of delyed developed, as well as50%of un-unnified developed.In the high-dose treatment group, it has similar with blank group of endometrial condition, there has83%of endometrial growth developed and17%of delyed developed. In the low-dose treatment group, there has64%of endometrial growth developed and34%of delyed developed. Every group has no observation of endometrial developed un-unnified except the modern group.(4)The effect of assisted reproduction1prescription on the endometrial tissue MMP-9of model rats with kidney deficiency and blood stasis.The expression level of MMP-9of the total area,optical density of the total,mean of the average black in endometrial epithelium and stroma of the model group is lower than the blank group, the differences had its statistical significant(P<0.01); Compared with model group, the expression level of MMP-9of the assisted reproduction1prescription by high and low dose group is higher than the model group(P<0.05or P<0.01); The expression level of MMP-9of the assisted reproduction1prescription by high is higher than the low dose group, the differences all values in endometrial epithelium had its statistical significant(P<0.05or P<0.01), the differences of the expression level of MMP-9of the total area,optical density of the total in endometrial stroma had its statistical significant(P<0.05).(5)The effect of assisted reproduction1prescription on the endometrial tissue TIMP-1of model rats with kidney deficiency and blood stasis.The expression level of TIMP-1of the total area,optical density of the total,mean of the average black in endometrial epithelium and stroma of the model group is lower than the blank group, the differences had its statistical significant(P<0.01); The expression level of TIMP-1of the assisted reproduction1prescription by high and low dose group is higher than the model group(P<0.05or P<0.01);The expression level of TIMP-1of the assisted reproduction1prescription by high is higher than the low dose group, the differences all values in endometrial epithelium had its statistical significant(P<0.05or P<0.01).(6)The effect of assisted reproduction1prescription on the endometrial tissue MMP-9/TIMP-1of model rats with kidney deficiency and blood stasis.The expression level of MMP-9/TIMP-1of the optical density of the total, mean of the average black in endometrial epithelium of the model group is lower than the blank group, the differences had its statistical significant(P<0.01); The expression level of MMP-9/TIMP-1of the assisted reproduction1prescription group is higher than the model group, but the differences between these groups had no statistical significant(P<0.05);Compared with treatment group, the differences between these groups had no statistical significant (P>0.05)The expression level of MMP-9/TIMP-1of the total area, mean of the average black in endometrial stroma of the model group is lower than the blank group, the differences had its statistical significant(P<0.01);The expression level of MMP-9/TIMP-1of the total area of treatment group is higher than the model group, the differences had its statistical significant(P<0.01).Conclusion: 1. Toxicology results show that the maximum dose in mice1day amount77.38g/kg, the equivalent of the clinical employing106times the daily amount, without an animal was killed. Indicate feeding on mice this dose is safe.2. The assisted reproduction1prescription can increase the body weight, the uterus viscera index of the model rats,and improve the endometrial tissue morphology, Synchronize the endometrium growth and embryonic development so as to favor embryo implantation.3. Assisted reproduction1prescription can significantly improve the expression of MMP-9and TMP-1in the endometrial epithelial and stromal of the model rats.It can also adjust the dynamic equilibrium of specific value of MMP-9/TIMP-l,then it moderately promote endometrium the degradation of extracellular matrix, involve in maintaining normal pregnancy, this is probably one of the important mechanism of assisted reproduction1prescription in improving the endometrium receptivity. |
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