| Objective: This topic was discussed theoretically Jieyuyubao Prescription TCM intervention in assisted reproductive endometrial receptivity chronic stress in rats and its mechanism. Aimed at improving the living environment because clinical infertility itself, IVF-ET and other psychological stress caused by low receptivity, adjuvant therapy of traditional Chinese medicine treatment of infertility and assisted reproductive technology license for the clinical application of liver Liver method provides a theoretical basis, combined with progesterone increase the success rate of conception by Chinese and Western medicine.Methods: In this study, Sprague-Dawley(SD) rats system as the research object, through the establishment of chronic stress model, the use of Jieyuyubao Prescription experiments. Select the normal estrous cycle 8-week-old female SD rats 80 included in the experiment, stratified according to body weight were random Ly divided into 5 groups, each 16. A group including normal control group(control group): Do not give any stimulation, no drenching; group B, chronic stress model(model group): Daily given chronic stimulation, no drenching; group C + Chronic Stress Relieving Jieyuyubao Prescription of the low-dose group(Xie low group); D group chronic stress + middle group(Xie middle group); E group chronic stress + high dose group(Xie high group). In addition to the A group, and the remaining four groups of rats received 14 d accept different stressor stimuli, including limited mobility(2hrs), clip the tail(1min, 3 times / day), ice swimming(4 ℃, 5mins), noise(75d B, 1hrs), fasting(24hrs), water deprivation(24hrs), electric shock foot(voltage 50 m V,every 50 s stimulate a times, each lasting 10 s, a total of 30 times). These stressors in several different applications throughout the experiment, two kinds of random daily stimulation, the rats were not expected stimulation occurs, to avoid adaptation. Modeling rats 14 days after emergence and looked tired, slow weight gain, decreased interest in eating, grooming(lifting, scratching, licking, etc.) reduction, suggesting that successful model. In Group D solution: to stress the 15 th day, daily 9:00 am to give Jieyuyubao Prescription solution extracellular side gavage, according to body surface area conversion clinically effective dose, a dose of 11.8g crude drug / kg body weight, into a suspension liquid 11.8m L / kg body weight, once a day, to death day. Low Group C solution: to stress the 15 th day, daily 9:00 am to give Jieyuyubao Prescription solution extracellular side gavage solution in accordance with the clinically effective dose group converted half a dose of 5.9g crude drug / kg body weight, into a mixed liquid suspension 5.6m L / kg body weight, once daily, to death day. High Group E solution: to stress the 15 th day, daily 9:00 am to give Jieyuyubao Prescription solution extracellular side gavage, according to the solution of the group converted clinically effective dose two times a dose of 23.6g crude drug / kg body weight, into a suspended liquid 23.6m L / kg body weight, once a day, to death day. A normal group, B model group were fed daily 9:00 am according to body surface area conversion volume and other clinically effective dose of distilled water once a day until the date of death. Five groups of rats at 16:00 pm Day 28 male and female 2: 1 in the cage, 9:00 am the next morning when checking vaginal suppository, and after vaginal smears found that sperm or vaginal suppository is listed as day 1 of pregnancy. Each group reach the first four days of pregnancy eight rats eyeball blood 3-5m L, within 30 mins at 1000 * g centrifugation 15 mins, the supernatant wasdetected by manual detection of β- endorphin receptor content. After the blood of rats were sacrificed by cervical dislocation, dissected and isolated uterus, the endometrium cut along the longitudinal axis, flattening, repeatedly washed with saline after taking unilateral uterus into the 4% glutaraldehyde, embedded in paraffin, sliced, HE staining, made TEM specimens. Take the other side of the uterus cut into upper and lower two sections, the upper section in 10% paraformaldehyde fixed to be tested, using immunohistochemical SP method to detect VEGF in rats endometrial tissue expression levels. After removing the lower segment of the uterus immediately placed in-80 ℃refrigerator, real-time quantitative PCR(Real-Time PCR) test HOXa-10 m RNA content. The remaining eight rats in each group were caged on the 14 th day all were killed by cervical dislocation, dissected uterus, uterine embryo view about the number, and for the record, and the average number of embryos pregnancy statistics.The Result: 1. The general observation of the rats Modeling 14 d, the situation observed in rats and other growth state, eating and activity, appear in the model group rats demeanor reconciliation low burnout, eyes glazed, get together and lazy move, when not actively fight modeling, dull coat, grooming(lifting, scratching, licking, etc.) to reduce, reduce food intake, with stool changes(dry or thin pond) performance. Description stress on mood and behavior in rats caused some impact. Normal group, the group solution, the solution set high Mao Faguang clean and tidy, lively eyes of God, feeding properly, more rapid weight gain. Until the end of the experiment, no significant adverse reactions during treatment in each group of rats, the rats after stress-free death. 2. estrous cycleChronic variable stress for 14 days, beginning vaginal smears, continuous observation of changes in rat estrous cycle of 14 days. Model group showed prolonged estrous cycle, mainly for prolongation, a large number of white blood cells under a microscope and a small amount of time to nuclear degradation of epithelial cells and mucus appear longer. Other groups of prolonged stress, irregular estrous cycle. From the time of the estrous cycle, the model group reconciliation estrous cycle low group was significantly longer compared with the normal group(P<0.05). Solution in groups, solve the high group compared with the model group, estrous cycle decreased significantly(P<0.05); no significant difference(P with the normal grou P>0.05). Xie Xie low group and the high group compared reconciliation, there are significant differences in the estrous cycle(P<0.05). 3. The serum levels of β- endorphin(β-EP) Compare Rats serum β-EP group than the control group significantly increased, and by statistical calculations P<0.05 statistically significant, proved successful modeling; compared with the normal group, the solution of the low group β-EP was significantly increased, and the difference was statistically significant(P<0.05); and the solution of the group, de high group and normal group, the difference was not statistically significant(P>0.05). Compared with the model group,β-EP content Jieyuyubao Prescription incubated in the experimental group in each experimental group were lower than the model group, but were higher than the normal group, there was a significant difference between the solution and the solution in the high group with model group, and the solution low group compared with the model group, β-EP was no significant difference(P>0.05); the solution in the group, β-EP content of the solution in the high group was significantly lower than the solution low group(P<0.05). Solution in the group, no significant difference(P>0.05) between the solution of the high group. 4. TEM rats endometrial expression pinopodesCompared with the normal group, model group, low group solution, the solution in the group pinopodes score decreased, which in the model group, the solution has a low group difference was statistically significant(P<0.05), no significant difference in the solution group(P > 0.05); and the solution pinopodes score high group and the normal group increased, the difference was not statistically significant(P>0.05). Compared with the model group, the extracellular side Jieyuyubao Prescription fertility experiments pinopodes score higher than those in the untreated group in each group, the group solution, the solution set pinopodes high score was significantly higher than the model group(P<0.05), the solution low group pinocytosis no sudden score significant difference compared with the model group(P>0.05); the solution of the group, the group of high solution pinopodes score was significantly higher than the solution of the low group(P<0.05). Solution in the group, no significant difference(P>0.05) between the solution of the high group. 5. Each group of rats expressing endometrial HOXa-10’s HOXa-10 m RNA content by calculating the ratio of specimens HOXa-10 m RNA and β-Actin m RNA measurement, HOXa-10 m RNA were expressed in the three groups endometrium and decidua in normal value of 0.965 ± 0.102, model group was 0.6583 ± 0.031, the solution is low group 0.7983 ± 0.017, the solution in the group is 0.8663 ± 0.125, Xie high group is 0.8246 ± 0.317. HOXa-10 m RNA relative content in the endometrium of rats under different conditions showed that, compared with the normal group, the expression level of the model group reconciliation HOXa-10 m RNA low group was significantly decreased(P<0.05), and the solution in the group with high solution no group compared with the control group significant difference(P>0.05). Compared with the model group, the solution of the low group, the solution of the group, significantly increased the expression level of the solution in the high group HOXa-10 m RNA, there are statistically significant(P<0.05); compared with the solution of the low group, the solution of thegroup, the group of high solution no significant difference(P>0.05) HOXa-10 m RNA expression levels. Solution in the group, no significant difference(P>0.05) between groups HOXa-10 m RNA solution high expression levels. 6. Comparison of rats in each group of endometrial VEGF VEGF expression mainly located in the cytoplasm and some stromal vascular endothelial cell cytoplasm of endometrial glandular epithelial cells, stromal cells have a weak part of the coloring strength of each test group was mixed. Compared with the normal group, model group, the expression of VEGF decreased, there was statistically significant(P<0.05), description of Chronic Stress on endometrial angiogenesis inhibition.Jieyuyubao Prescription in the experimental group(low, medium, and high group) compared to the difference between the expression of VEGF was no control group was statistically significant(P>0.05), but compared with the model group was statistically significant(P<0.05). No significant difference(P>0.05) between the expression of VEGF Jieyuyubao Prescription incubated in the experimental group(low, medium, and high groups). 7. Comparison of the number of rats in each group and the number of implanted pregnancy.After 14 days of each group mated, the normal group, the group solution, the solution of all pregnancies are high group, the model group reconciliation low group have not been a rat pregnancy. Compared with the normal group, the average number of points implantation rat model group and the low group solution significantly reduced, and the difference was statistically significant(P<0.05). Group reconciliation group average implantation high points with no significant difference between the control group(P>0.05) in the solution. Compared with the model group, the average number of points the solution low implantation group had no significant difference(P>0.05), the average number of points implantation reconciliation solution in the high group was significantly higher(P<0.05); compared withthe solution of the low group, the solution the average number of points in the group reconciliation implantation high group was significantly higher(P<0.05); the solution in group reconciliation between the average number of points higher implantation was no significant difference(P>0.05).Conclusions: 1. The model group behavior science, estrous cycle, blood β-EP and other indicators of change, successful modeling; 2. Chronic stress can have an impact on endometrial receptivity in rats related indicators; 3. Jieyuyubao Prescription can improve cellular stress(general state of the estrous cycle, serum β-EP, etc.) rats; 4. Jieyuyubao Prescription because of stress caused by intracellular side on endometrial receptivity index pinopodes, VEGF, HOXa-10 m RNA lowering effects have improved, and showed a dose-dependent characteristics, thus improving the uterus Chronic Stress receptivity membrane, thus increasing rat pregnancy, implantation points; 5. Because the pressure caused by the environment, infertility itself, IVF-ET, etc.,so that chronic stress on endometrial receptivity change, stagnation sterile cell side has a supporting role to improve the prognosis. |
PDF Full Text Request