Effect Of Bushen And Shugan Methods On Endometrial Receptivity Through VEGFR-2 Regulating The MAPK Signaling Pathway During Peri-implantation Of Controlled Ovarian Hyperstimulation Mice

Objective: To observe the effect of Bushen and Shugan methods on endometrial receptivity through VEGFR-2 regulating the MAPK signaling pathway during peri-implantation of controlled ovarian hyperstimulation mice.In order to investigate the reduced mechanism to improve the receptivity of endometrium,and what are the similarities and differences.Methods: Totally 240 female mice with normal estrous period were randomly divided into control group,model group,Bushen group and Shugan group with 60 mice in each.Except the control group,all the mice were made GnRHa controlled ovarian hyperstimulation model(GnRHa+HMG+HCG).First day of molding,Bushen group mice were given Bushen Zhuyun recipe,Shugan group mice were given Xiaoyao Pill suspension,control group and model group mice were given the same dose of distilled water,consecutive for 11 days.The day of inject HCG,female mice were mated with males at 6 PM with the ratio of 2:1 and checked by the vaginal smear at 9 AM the next day.Presence of vaginal plug was considered as evidence of mating,and the day was designated as the pregnancy day 1.The pregnant mice in each groups were killed at the day 2?3?4?5?6 of pregnancy respectively.The content of estradiol(E2),progesterone(P)in serum was detected by radio immunoassay.The morphology of different stages of endometrial tissue was observed by HE staining.Pinopodes expression was observed by scanning electron microscope(SEM).Expression and localization of ER,PR,MVD,VEGF,VEGFR-2,PCNA,CyclinD 1,MMP-9 protein in Uterineendometrium was detected by immunohistochemistry.Expression of estrogen receptor(ER)andprogesterone receptor(PR),PCNA,CyclinD1,MMP-9,VEGF and VEGFR-2downstream angiogenesis related molecular protein in Uterine endometrium was detected by Western Blot method.Expression of VEGF,VEGFR-2,PCNA,CyclinD 1,MMP-9 mRNA in uterine endometrium was detected by real-time fluorescence quantitative PCR.Comparing groups of vaginal plug rate,pregnancy rate and embryo implantation in mice.Results:1 Comparison of vaginal plug rate,pregnancy rate and embryo implantation: compared with the control group,model group mice in the vaginal plug rate,pregnancy rate and embryo implantation were decreased(P<0.05);compared with the model group,the vaginal plug rate,pregnancy rate and embryo implantation of treatment group were increased(P <0.05),The vaginal plug rate,pregnancy rate and embryo implantation in the Bushen group were higher than those in the Shugan group,but there was no significant difference between the two groups(P>0.05).2 Comparison of estrogen,progesterone in serum and its receptor levels during peri-implantation.With the increase of the number of days of pregnancy,the serum E2 and P values of each group increased gradually.During peri-implantation compared with the control group,content of E2 and P was lower in model group;The content of E2 and P was obviously higher in Bushen group and Shugan group than in model group.On the third day and fifth day of pregnancy,the serum E2 value of the Bushen group was higher than that of the Shugan group,the difference was statistically significant(P < 0.05),and there was no statistically significant difference between the two groups in other days(P > 0.05).There was no significant difference between the treatment group and the liver group(P>0.05).Compared with the normal group,model group ER and PR protein expression decreased,with statistical significance(P<0.05);compared with the model group,Bushen group and Shugan group ER and PR protein expression increased(P<0.05),between the Bushen group and Shugan groupshowed no significant difference(P>0.05).3 Comparison of endometrial thickness in each group of mice during peri-implantation.With the increase of pregnant time,endometrial thickening gradually.Compared with the control group,the thickness of endometrium in the model group was significantly decreased On the day 2?3?4?5?6 of pregnancy(P<0.01);Compared with the model group,the thickness of endometrium in Bushen group and Shugan group were significantly increased On the day2?3?4?5?6 of pregnancy(P<0.01);There was no significant difference between the Bushen group and the Shugan group(P>0.05).4 The morphological changes of endometrium of mice during the peri-implantation period.Control group HE staining showed:endometrial thickening gradually,the number of glands increased,the glandular cavity became larger and curved,the glandular secretions increased,Stromal edema and stromal cells increased.After the fourth day of pregnant,the cavity gradually closed,and the stromal cells were differentiated into decidual cells.Model group HE staining showed: compared with the normal group,the endometrium was thin and poorly developed,the glands were few and small,the glandular cavity was not dilated,the glandular cavity secretion was few.In some of the mice endometrium,the glandular and stromal development were not synchronized,the stroma showed the secretory phase and the glandular development was still the proliferative phase.The development of endometrium in the Bushen group and Shugan group was close to that of the normal group,which was better than that of the model group,and there was no significant difference between the two groups.5 The expression of Pinopodes in each group of mice during peri-implantation.Pinopodes were mainly located in the opening of endometrial gland.The surface of some endometrial cells is formed with some membrane protrusions,which is covered with short microvilli on the third day of pregnancy ofcontrol group mice,as developing pinopodes.On the fourth day of pregnancy,there were a large number of uniformly distributed and clear membrane protrusions on the surface of endometrium,as fully developed pinopodes.On the fifth day of pregnancy,pinocytosis surface shrinkage,microvilli appeared again,as degradation of pinocytosis.on the third day of pregnancy of model group mice,pinocytosis raised more obviously and the microvilli were shorter than that in the normal group,the development of model group was earlier than the normal group.On the fourth day of pregnancy,the number of pinocytosis was decreased and the surface of pinocytosis was shrunken in the model group compared with the control group.There was no fully developed pinopodes of the endometrium in the model group.On the fifth day of pregnancy,the cell apoptosis was more obvious than that in the normal group,and the microvilli were less.On the third day of pregnancy,the surface of the endometrium of the Bushen group and Shugan group mice appeared membranous,which covered with microvilli.On the fourth day of pregnancy,the surface of the endometrium of Bushen group and Shugan group mice were fully developed pinopodes.On the fifth day of pregnancy,the development of the pinopodes were close to that of the normal group,and these were degenerated.6 Comparison of MVD was detected by immunohistochemical method in different groupsCD34 is a marker of vascular density,which is expressed in endometrial vascular endothelial cells and is used to determine MVD.Compared with the control group,the model group MVD decreased significantly On the day 2?3?4?5?6 of pregnancy(P<0.05).Compared with the model group,Bushen group and Shugan group were increased significantly.On the day2?3?4?5?6 of pregnancy(P<0.05);There was no significant difference between the Bushen group and the Shugan group(P>0.05).7 The position expression of VEGF?VEGFR-2?PCNA?CyclinD1?MMP-9 protein in mice endometrium by immunohistochemical method.The expression of VEGF and VEGFR-2 protein in the endometrium ofmice was highly consistent in space,which was mainly located in the cytoplasm of endometrial luminal epithelium,glandular epithelium,stromal cells and vascular endothelial cells.PCNA located in the endometrial cavity epithelium and stroma within the nucleus,a small gland epithelial cells express.CyclinD1 was mainly expressed in the luminal epithelium and glandular epithelium of the nucleus,weak expression of interstitial cells.MMP-9 located in endometrial luminal epithelium and glandular epithelial cells.8 The temporal expression of VEGF?VEGFR-2?PCNA?CyclinD1?MMP-9 protein and mRNA in mice endometrium by immunohistochemical method.The expression of VEGF?VEGFR-2?PCNA?CyclinD1?MMP-9 protein and mRNA in mice endometriumis is low on the day 2 of pregnancy,it rised after pregnancy,topped on on the day 4 of pregnancy with statistical significance contrast with other time points(P < 0.05)and decreased after that.9 Comparison of the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD 1 protein and mRNA in mice endometrium among each group.On the day2?3?4?5?6 of pregnancy:Compared with control group,the expression of VEGF and VEGFR-2 protein and mRNA in model group decreased significantly(P < 0.05);Compared with the model group,the expression of VEGF and VEGFR-2 protein and mRNA in Bushen group and Shugan group was increased significantly(P<0.05).Compared with control group,the expression of VEGF protein and mRNA in Bushen group decreased significantly on the day 2?4?5?6 of pregnancy(P<0.05);the expression of VEGFR-2 protein and mRNA in Shugan group decreased significantly on the day 3?4?5?6 of pregnancy(P<0.05).On the day 6 of pregnancy,the expression of VEGF and VEGFR-2 protein and mRNA in Bushen group is higher than that in Shugan group,and the difference was statistically significant(P<0.05).On the day2?3?4?5?6 of pregnancy:Compared with control group,theexpression of PCNA protein and mRNA in model group decreased significantly(P < 0.05);Compared with the model group,the expression of PCNA protein and mRNA in Bushen group and Shugan group was increased significantly(P < 0.05).Compared with control group,the expression of PCNA protein and mRNA in Bushen group decreased significantly on the day4 and5 of pregnancy(P<0.05).On the day 2?3?4?5 of pregnancy,the expression of PCNA protein and mRNA in Bushen group is higher than that in Shugan group,and the difference was statistically significant(P<0.05).On the day2?3?4?5?6 of pregnancy:Compared with control group,the expression of CyclinD1 protein and mRNA in model group decreased significantly(P<0.05);Compared with the model group,the expression of CyclinD1 protein and mRNA in Bushen group and Shugan group was increased significantly(P < 0.05);Compared with control group,the expression of CyclinD1 protein and mRNA in Bushen group and Shugan group decreased significantly(P<0.05).There was no significant difference between the Bushen group and the Shugan group(P>0.05).On the day 2?3?4?5?6 of pregnancy:Compared with control group,the expression of MMP-9 protein and mRNA in model group decreased significantly(P<0.05);Compared with the model group,the expression of MMP-9 protein and mRNA in Bushen group and Shugan group was increased significantly(P < 0.05).Compared with control group,the expression of MMP-9 protein and mRNA in Bushen group decreased significantly on the day 2?3?4 of pregnancy(P<0.05);the expression of MMP-9 protein and mRNA in Shugan group decreased significantly on the day 2 and 4 of pregnancy(P<0.05).On the day 2?3?4?5 of pregnancy,the expression of PCNA protein and mRNA in Shugan group is higher than that in Bushen group,and the difference was statistically significant(P<0.05).10 Comparison of the expression of MAPK signaling pathway protein in mice endometrium among each group.On the day 2?3?4?5?6 of pregnancy: Compared with control group,the expression of p-ERK ? p-P38 ? p-JNK protein in model group decreasedsignificantly(P<0.05).Compared with the model group,the expression of p-ERK?p-P38?p-JNK protein in Bushen group was increased significantly(P< 0.05);the expression of p-P38 ? p-JNK protein in Shugan group was increased significantly(P<0.05);the expression of p-ERK protein in Shugan group was not increased significantly(P > 0.05).Compared with Bushen group,the expression of p-ERK protein in Shugan group decreased significantly(P<0.05).Conclusion:1 Expression of endometrial VEGF during peri-implantation have high concordance in time and it can used as one marker of the endometrial reception.2 COH may have some bad effect on the endometrial receptivity:lower serum E2,P levels in mice during iperi-implantation,decrease the expression of ER,PR in endometrial tissue,the endometrial glands and stromal development are not synchronized,pinopode expression in advance,reduce the expression of VEGF,VEGFR-2,PCNA,CycinD1,MMP-9,reduced the activity of the MAPK family 3 signaling pathways and the expression of phosphorylation of ERK,P38,JNK,decrease endometrial angiogenesis.3 Bushen and Shugan method may have some good effect on the endometrial receptivity:increase serum E2,P levels in mice during iperi-implantation,improve the expression of ER,PR in endometrial tissue.Promote the formation of pinopodes and development,promote the synchronization of the endometrial stroma and glands.Improve the expression of VEGF,VEGFR-2,PCNA,CycinD1,MMP-9.Increase the activity of the MAPK family 3 signaling pathways and the expression of phosphorylation of ERK,P38,JNK,promote endometrial angiogenesis.In the end,the endometrium during iperi-implantation reach the best receptive state.4 Bushen method in promoting p-ERK expression,endothelial cell proliferation and angiogenesis is better than that of Shugan method.Shugan method in degrading ECM and promoting cell migration is better than that of Bushen method.