Effect Of Yiqi Huayu Decoction On Improving The Endometrial Receptivity In Endometriosis Model Rats

ObjectiveThe endometriosis model was established by the method of autotransplantation and the traditional Chinese medicine Yiqi Huayu Decoction was used to intervene.To observe the effect of Yiqi Huayu Decoction on pregnancy function in rats,the influence on the endometrial receptivity and the impact on the growth of ectopic endometrium of the rats.To investigate the mechanism of Yiqi Huayu Decoction in the treatment of endometriosis,advance endometrial receptivity and improve the pregnancy function,providing a theoretical basis for further clinical application.MethodsWe cut the rat's right uterus by sugical operation and the clipping endometrial tissue was transplanted to the mesenteric to establish a rat model of endometriosis.The successful model rats were randomly divided into three groups: Yiqi Huayu Decoction group,Gestrinone group,model group and sham operation group.The rats in Yiqi Huayu Decoction group were treated with Yiqi Huayu Decoction suspension(crude drug at a dosage of 3.55g/ml),according to 4ml/(Kg·d)by gavage for 6 weeks;The rats in Gestrinone group were treated with Gestrinone suspension(0.1mg/ml),according to 4ml/(Kg·d)by gavage for 4 weeks and sequentially given distilled water for 2weeks;The rats in the model group and sham operation group were given distilled water,according to 4ml/(Kg·d)by gavage for 6 weeks.The experimental research was composed of three experiments.The first part was to observe the effect of Yiqi Huayu Decoction on the pregnancy function of endometriosis model rats.After the drug intervention,male and female rats were mated with a ratio of 2:1 during the estrous period.The day that vaginal or vaginal smears were found was recorded as the first day of pregnancy.In the 16 th day of pregnancy,the female rats were killed.The number of pregnancy rats in each group was recorded.The number of embryos in each uterine was counted.The weight of each embryo and planceta was measured by electronic scales.The corpus luteum number of each left ovarian was counted.Consequently,pregnancy rate and implantation rate were calculated.The second part of the experiment was to investigate the mechanism of the effect of Yiqi Huayu Decoction on improving the endometrial receptivity and promoting embryo implantation.After the drug intervention,the male and female rats were mated with 2:1 ratio in the estrous period.In the 4th day of pregnancy,the female rats were killed and samples were obtained.ELISA was used to detect the estradiol(E2)and progesterone(P)level in serum.SP immunohistochemical method and Western blotting were used to detect the estrogen receptor(ER),progesterone receptor(PR),leukemia inhibitory factor(LIF),integrin ???3(???3)and matrix metalloproteinase-9(MMP-9)in endometrium of the rats.The third part was to study the mechanism of the effect of Yiqi Huayu Decoction on inhibiting ectopic endometrium growth.The diameter of each ectopic cyst was respectly measured by vernier caliperbefore and after the intervention.SP immunohistochemical method was used to detect the ER,PR,???3,MMP-9,metallopeptidase inhibitor-1(TIMP-1)and Casepase-9 in ectopic endometrium of the rats.Immunohistochemical sections were amplified 400 times and Image Pro Plus 6.0 software was used to analize the image.We measured the integral optica density(IOD)and the area of the specific proteins.The average optical density(IOD/area)was used to represent the intensity of protein expression.The amount of target protein expression was detected by Western blot with ?-actin as control.SPSS 19.0 software was used for data analysis,the measurement data was describe by mean and standard deviation.After parameter test,measurement data comparison among multi groups were analysed by one-way ANOVA or rank-sum test,data between two groups were analysed by LSD or Kruskal-Wallis test,data variation within group were analysed by paired samples T-test or Wilcoxon Signed-Rank test.Enumeration data comparison among multi groups were analysed by by ?2 test or Fisher exact test.P<0.05 showed significant difference.Results1.Effect on pregnancy function in rats with endometriosisThe ovulation number,implantation number,implantation rate,embryo weight and placental weight of the rats in model group were lower than that in sham operation group.The ovulation number,implantation number,implantation rate,embryo weight and placental weight of the rats in Yiqi Huayu Decoction group were higher than that in model group or Gestrinone group.Meanwhile,there was no significant difference between the Gestrinone group and the model group.2.Effect on serum E2 and P level in peri-implantation period in rats withendometriosisThe serum E2 level of the rats in Yiqi Huayu Decoction group was higher than that in model group or in Gestrinone group.Meanwile the E2 level between the rats in model group and that in Gestrinone group were no significant difference.The serum P level of the rats in model group was lower than that in sham operation group and that in Yiqi Huayu Decoction group was higher than that in model group.While the serum P level between Gestrinone group and the model group was no significant difference.3.Effect on the endometrial receptivity in endometriosis model ratsThe expression of ER in model group was higher than that in sham operation group,the expression of ER in Yiqi Huayu Decoction group and Gestrinone group was lower than that in model group,the expression of ER in Yiqi Huayu Decoction group was significantly higher than that in the pregnant group,the difference was significant.The expression of PR in model group was higher than that in sham operation group,and the expression of PR in Yiqi Huayu Decoction group and Gestrinone group was lower than that in model group,and the expression of PR was lower than that in sham operation group,the difference was significant.The expression of LIF,???3,MMP-9 in the model group were lower than that of sham operation group.The expression of LIF,???3,MMP-9 in Yiqi Huayu Decoction group were higher than that in model group.But there was no significant difference in LIF,???3and MMP-9 expression between Gestrinone group and the model group.4.Effect on ectopic endometrium of endometriosis model ratsThe diameter of ectopic cyst of the rats decreased significantly after the intervention in Yiqi Huayu Decoction group and Gestrinone group.But that change in model group was not significant.The expression of PR,ER in ectopic endometrium in Yiqi Huayu Decoction group and Gestrinone groupwere lower than that in model group,while there was no significant difference in that between the Yiqi Huayu Decoction group and Gestrinone group.The expression of ???3,MMP-9 in ectopic endometrium in Yiqi Huayu Decoction group and Gestrinone group were lower than that in model group,while there was no significant difference in that between the Yiqi Huayu Decoction group and Gestrinone group.The expression of TIMP-1,Casepase-9 in ectopic endometrium in Yiqi Huayu Decoction group and Gestrinone group were higher than that in model group,while there was no significant difference in that between the Yiqi Huayu Decoction group and Gestrinone group.Conclusions1.The pregnancy function of endometriosis rats model is lower.Yiqi Huayu Decoction can effectively increase the number of ovulation and implantation rate of the endometriosis rats,meanwile it can promote the development of embryos.2.Yiqi Huayu Decoction can effectively improve the serum progesterone level in peri-implantation period of the endometriosis model rats.In the meantime,it can downregulate the expression of PR and upregulate the expression of LIF,???3,MMP-9 in endometrium,suggesting that it may regulate endometrial receptivity and promote embryo implantation.3.Yiqi Huayu Decoction can effectively inhibit the growth of ectopic endometrium.On the one hand,it can effectively decrease the expression of ER,PR,???3,MMP-9 and enhance the expression of TIMP-1 in ectopic endometrium to inhibit ectopic endometrial invasion;on the other hand,it can upregulate the expression of Casepase-9 and promote apoptosis of ectopic endometrial cells.