Comparison Of BMP-15and Smads Signal Pathway Between Bushen Treatment And Shugan Treatment In Improving Oocyte Quality Of The Mice

Objective: The research results of former experiments have confirmedthat Bushen treatment and Shugan treatment can improve pregnancy rates ofpatients with IVF-ET, and increase the protein content and mRNA expressionof BMP-15. It indicates that Bushen Tiaojing recipe and Xiaoyao Pill maypromote follicular development, improve oocyte quality by increasing theprotein content and mRNA expression of BMP-15. BMP-15and its Smadssignaling pathway plays an important role in the development of follicles, theexpansion of cumulus and the mature of oocytes. Bushen Tiaojing Recipe isused as the representative recipe to enhance the function of the kidney;Xiaoyao Pill is used as the traditional prescription to regulate the liverfunction.On this basis, we further explore the relationship between increasingoocyte quality of the mice by Bushen treatment and Shugan treatment andregulating BMP-15and Smads signal pathway through animal experiment,and then investigate the difference of the action mechanisms and links of thetwo methods. Then we can provide the experimental basis for the traditionalChinese medicine as adjuvant of IVF-ET, certain the role of Chinese medicinein the treatment of infertility, and promote the development of Chinesemedicine of gynecology.Methods:900healthy female KunMing mice of6-7weeks old were usedfor six times.Every time150mice were used for experiment.They wererandomly divided into6groups: control group, COH group, Shugan low dosegroup, Shugan high dose group, Bushen low dose group, Bushen high dosegroup (N=25). Xiaoyao Pill was producted by Henan Wan Xi pharmaceuticalcompany. We had200and100pills dissolved in125ml of distilled water intothe suspension, high dose of crude drug was0.6g/ml, low dose of crude drug was0.3g/ml. Bushen Tiaojing Recipe was purchased from Shijiazhuang LeRentang drugstore, and was made into fine oral liquid by Department ofChinese Materia Medica, high dose of crude drug5.4g/ml，low dose of crudedrug2.7g/ml.They were stored at4℃(high, low dose equivalent to20,10times of clinical dosage).The COH group were drenched with distilled water,Shugan groups were drenched with Xiaoyao Pill, Bushen groups weredrenched with Bushen Tiaojing Recipe once a day,1ml/100g weight.Treatments continued for10days. The treatment groups and the COH groupwere dealt with intraperitoneal injection of PMSG10IU/only on the eleventhday, after48hours intraperitoneal injection of HCG10IU/only. The smears ofvaginal epithelial cells in the control group were made to observe sexual cycle.The first experiment:16hours after intraperitoneal Injection of HCG,15mice of COH group,15mice of Shugan low dose group,15mice of Shuganhigh dose group,15mice of Bushen low dose group,15mice of Bushen highdose group were killed, and15mice of the control group whose vaginalepithelial cells showed cutinization were killed the next day. Then the bilateralfallopian tube were removed and placed in the dish. The ampulla of fallopiantube were pierced with1ml syringe needle by stereo microscope,and cumulusoocyte complexes (COCs) were collected,observed the follicles number andthe high-quality follicle rate quickly.Then cumulus granulosa cells weredigested by the hyaluronidase, washed repeatedly by PBS. Finally M IIoocytes were obtained and put in the ice for real-time fluorescent quantitativePCR. The MII oocytes of the rest mice were got as above,and preserved at-80℃for Western blot.The second time: After intraperitoneal injection of HCG,20mice ofCOH group,20mice of Shugan low dose group,20mice of Shugan high dosegroup,20mice of Bushen low dose group,20mice of Bushen high dose group,and20mice of the control group whose vaginal epithelial cells showedcutinization were mated with male mice of8-9weeks old to observe thepregnancy rate (the ratio of1:1). The MII oocytes of the rest mice were got asabove, and fixed in4%paraformaldehyde solution for immunohistochemistry. The MII oocytes of the rest mice were got as above.Some were fixed in4%paraformaldehyde solution for immunohistochemistry,and the other werepreserved at-80℃for Western blot.The protein content of BMP-15, BMPR II,ALK6, Smad1, Smad5and Smad8were detected by Western blot andimmunohistochemistry. The mRNA expressions of them were analyzed byreal-time fluorescent quantitative PCR.Results:1Comparison of the follicles number, the high-quality follicle rate andthe pregnancy rate Comparison of the follicles number: Compared withthe control group, the follicles number of the COH group, Bushen groups andShugan groups were increased significantly (P<0.05). Compared with theCOH group, the follicles number of Shugan groups and Bushen groups wereno statistical significance (P>0.05). Comparison of the high-quality folliclerate and the pregnancy rate: Compared with the control group, the high qualityfollicle rate and the pregnancy rate in COH group were discreasedsignificantly (P<0.05), Shugan low group and Bushen low group were nostatistical significance (P>0.05), Shugan high group and Bushen high groupwere increased significantly (P<0.05). Compared with the COH group, thehigh-quality follicle rate and the pregnancy rate of Shugan groups and Bushengroups were increased significantly (P<0.05).2The protein and mRNA expression of BMP-15, BMPR II,ALK6,Smad1, Smad5and Smad8in Shugan groups Compared with the COHgroup and the control group, the protein and mRNA expression of BMP-15were increased significantly in Shugan groups (P<0.05),while there were nostatistical significance between low dose group and high dose group (P>0.05);the protein and mRNA expression of BMPR II in low dose group were nostatistical significance (P>0.05);the protein and mRNA expression of BMPRII in high dose group were higher; the protein and mRNA expression of ALK6,Smad1, Smad5and Smad8were increased significantly in Shugan groups(P<0.05),and they in high dose group were higher than in low dose group(P<0.05). 3The protein and mRNA expression of BMP-15, BMPRII, ALK6,Smad1, Smad5and Smad8in Bushen groups Compared with the COHgroup and the control group, the protein and mRNA expression of BMP-15,ALK6, Smad1, Smad5and Smad8were increased significantly in Bushengroups (P<0.05).And the expressions of BMP-15, ALK6, Smad1, Smad5andSmad8in low dose group were lower than that in high dose group (P<0.05).Compared with the COH group and the control group, the protein and mRNAexpression of BMPR II in Bushen groups were no statistical significance(P>0.05).4The protein and mRNA expression of BMP-15, BMPRII,ALK6,Smad1,Smad5and Smad8in Shugan groups and Bushen groups Compared withShugan low group, the protein and mRNA expression of BMP-15, ALK6,Smad1, Smad5and Smad8were increased significantly in Bushen groups(P<0.05),while the protein and mRNA expression of BMPRII were nostatistical significance (P>0.05). Compared with Shugan high group, theprotein and mRNA expression of BMP-15, ALK6, Smad1were increasedsignificantly in Bushen low group (P<0.05), the protein and mRNAexpression of BMPRII, Smad5and Smad8were decreased significantly(P<0.05); the protein and mRNA expression of BMP-15, ALK6, Smad1,Smad5were increased significantly in Bushen high group (P<0.05), theprotein and mRNA expression of BMPRII were decreased significantly(P<0.05), the protein and mRNA expression of Smad8were no statisticalsignificance (P>0.05).Conclusions:1Without reducing the follicles number, Xiaoyao pill and BushenTiaojing Recipe can increase the high-quality follicle rate and the pregnancyrate.It shows that both Xiaoyao pill and Bushen Tiaojing Recipe couldimprove occytes quality.2Xiaoyao Pill can improve the expression of BMP-15in mouse oocytes,and may raise a second type of receptor BMPRⅡ, active receptor ALK6, andstart the signal transduction of Smads pathway. And the effect of the high dose group is better than the low dose group.3Bushen Tiaojing Recipe can improve the expression of BMP-15inmouse oocytes, and may activate receptor ALK6trough other type2receptor,and then start the signal transduction of Smads pathway. And the effect of thehigh dose group is better than the low dose group.4Both Xiaoyao pill and Bushen Tiaojing Recipe can improve oocytequality of the mice, but the mechanism between them is different.