| Experiment PurposeBased on previous research,by establishing a chronic stress rat model,intervention treatment with"Jieyuyubao Prescription",further verifies of the effect of Jieyuyubao Prescription on endometrial receptivity in chronic stress rats.The mechanism of improving endometrial receptivity in chronic stress rats was preliminarily explored,which provided a reliable experimental basis for the application of Jieyuyubao Prescription in clinical practice.Experiment MethodChronic mild unpredictable stress modeling was used to establish a chronic stress rat model.Rats with successful modeling were randomly divided into five groups:Model control group,western medicine control group,Jieyuyubao Prescription of the low-dose group,Jieyuyubao Prescription of the medium-dose group and Jieyuyubao Prescription of the high-dose group,another normal control group.The administration methods of each group are as follows:Normal Control Group:Fill an equal volume of purified water 11.2ml/kg,once a day for 14 consecutive days;Model Control Group:Fill an equal volume of purified water 11.2ml/kg,once a day for 14 consecutive days;Western Medicine Control Group:Aspirin was administered at a dose of 6.75mg/kg,once a day for 14 consecutive days;Jieyuyubao Prescription of the Low-dose Group:The dosage of Jieyuyubao Prescription is 5.6g/kg,once a day for 14 consecutive days;Jieyuyubao Prescription of the Medium-dose Group:The dosage of Jieyuyubao Prescription is 11.2g/kg,once a day for 14 consecutive days;Jieyuyubao Prescription of the Medium-dose Group:The dosage of Jieyuyubao Prescription is 22.4g/kg,once a day for 14 consecutive days;The experiment is divided into five parts.The first part of the experiment is:the establishment and evaluation of chronic stress rat model.Establishing a chronic stress rat model by administering chronic mild unpredictable stress for21 consecutive days,and the behaviors of rats before and after modeling?including general behavior and saccharide water uptake rate,forced swimming rest time?and serum corticosterone?CORT?were observed to evaluate the model.The second part of the experiment is:the effect of Jieyuyubao Prescription on Estrogen and Progesterone Levels,Endometrial Thickness,Tissue Morphology and Receptivity in Rats with Chronic Stress.After the 14th day of administration,the rats in each group were selected to be erected in a 2:1 cage,and the female was examined at 9:00 in the morning.The vaginal smear was examined in the absence of vaginal suppository.Recorded as the first day of pregnancy.On the 4th day of pregnancy,6 rats in each group were sacrificed and the remaining 10 rats in each group were used for subsequent experiments.The histomorphological and thickness changes of rat endometrium were observed by HE staining.The serum estrogen and progesterone levels were detected by enzyme-linked immunosorbent assay?ELISA?.The expression of LIF in endometrium was detected by immunohistochemistry.The third part of the experiment is:Effect of Jieyuyubao Prescription on Wnt7/?-catenin signaling pathway in chronic stress rats.The rat uterus specimens collected in Experiment 2 were used to detect the expression levels of Wnt7a,HOXA10,?-catenin and p-?-catenin in Wnt7/?-catenin signaling pathways in rat endometrium by Western blot,the mRNA expression levels of Wnt7/?-catenin signaling pathway-related genes Wnt7a and?-catenin in rat endometrium were detected by qRT-PCR.The location and expression level of p-?-catenin protein in rat endometrium were detected by immunofluorescence.The fourth part of the experiment is:Effect of Jieyuyubao Prescription on mRNA expression of LPAR3 and COX-2 during peri-implantation in chronic stress rats.The rat uterus specimens collected in Experiment 2 were used to detect the expression levels of LPAR3 and COX-2 mRNA in rat endometrium by qRT-PCR.The fifth part of the experiment is:Effect of Jieyuyubao Prescription on Pregnancy Function in Chronic Stress Rats.The remaining 10 rats in each group of experiment 2 were sacrificed by cervical dislocation at 16:00 on the 10th day of pregnancy,and the uterus was dissected to observe the pregnancy and count the number of embryos.HE staining,immunohistochemical staining,and immunofluorescence sections were taken using a microscope to acquire images.The cell morphology and protein expression of the endometrium were directly observed.The image was analyzed by Image-pro plus 6.0 image analysis system.The software was used to measure the thickness of the endometrium and calculate the average optical density of specific proteins?average optical density=Cumulative optical density IOD/area area?.Western blot was used to obtain experimental images by scanning after film exposure,and BandScan was used to analyze the gray value of the film.The ratio of the target protein to the gray value of the internal reference protein was used as a statistical analysis of the relative expression level of the protein.qRT-PCR used ABI QuantStudio?6 Flex real-time PCR system to detect the expression level of target gene mRNA.Based on the normal control group,the relative expression of mRNA of each target gene was analyzed by 2-??Ct??Ct analysis.All data were processed with SPSS19.0 software package.The data were expressed as?x±s.The comparison between groups was performed by independent test T-test statistical method.The multiple comparisons were compared by one-way ANOVA.P<0.05 The difference was statistically significant.Experiment Result1.Establishment and evaluation of a rat model of chronic stressChronic stress causes behavioral changes in rats,such as decreased behaviors such as lifting,scratching,and adding feet,decreased sugar water intake,increased resting time for forced swimming,and serum corticosterone?CORT?in rats.The content is increased.In the evaluation of the chronic stress rat model,the saccharide water uptake rate can be reduced by 20%,the forced swimming rest time can be increased by 20%,and the serum corticosterone content is significantly increased to a quantitative evaluation index.2.Effects of Jieyuyubao Prescription on the levels of female?E2?,progesterone?Pg?,endometrial tissue and receptivity in chronic stress rats.Jieyuyubao Prescription can increase serum estradiol?E2?and progesterone?Pg?levels,increase endometrial thickness and LIF expression levels in chronically stressed rats in a dose-dependent manner.There were significant differences between the traditional Chinese medicine doses and the model control group?P<0.05?.There was significant difference between the high-dose Chinese medicine group and the western medicine control group?P<0.05?.3.Effect of Jieyuyubao Prescription on Wnt7/?-catenin Signaling Pathway in Chronic Stress RatsThe expression of Wnt7a,HOXA10,?-catenin,P-?-catenin,Wnt7a and?-catenin in chronic stress rats was increased in a dose-dependent manner by Jieyuyubao Prescription.There were significant differences between the traditional Chinese medicine dose group and the model control group?P<0.05?.There was significant difference between the high-and medium-dose Chinese medicine group and the western medicine control group?P<0.05?.4.Effect of Jieyuyubao Prescription on mRNA expression of LPAR3 and COX-2 during peri-implantation in chronic stress rats.Jieyuyubao Prescription can increase the mRNA expression levels of COX-2 and LPAR3 in chronically stressed rats in a dose-dependent manner.There was significant difference between the traditional Chinese medicine dose group and the model control group?P<0.05?.There was significant difference between the high-and medium-dose Chinese medicine group and the western medicine control group?P<0.05?.5.Effect of Jieyuyubao Prescription on Pregnancy Function in Chronic Stress RatsThe average number of embryos in medium and high dose group of Jieyuyubao Prescription and western medicine control groups was higher than that in the model control group,and the difference was statistically significant?P<0.05?.The average number of embryos ratsin the low-dose group of Jieyuyubao Prescription was also higher than that in the model control group,but the difference was not statistically significant?P>0.05?.Experiment ConclusionJieyuyubao Prescription can regulate serum estrogen?E2?and progesterone?Pg?levels in chronically stressed rats,increase the expression of endometrial thickness and LIF in chronically stressed rats,it also promotes the expression of key proteins and genes in the Wnt/?-catenin signaling pathway,enhance the mRNA expression of LPAR3 and COX-2;thereby improve the endometrial receptivity of chronically stressed rats,improve the number of embryo implantation in rats and improve their pregnancy function. |
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