Study On Maternal Fetal Tolerance Of Recurrent Spontaneous Abortion Model Mice Induced By Bushen Huoxue Recipe Regulating Th17/Treg Balance

Objective:The effect of Bushen Huoxue Recipe on the balance of Th17/Treg cells（related factors）and the pregnancy outcome of the model mice with recurrent abortion were observed experimentally,so as to explore the mechanism of action of Bushen Huoxue Prescription on recurrent abortion and provide basis for clinical treatment.Methods:1.Female CBA/J mice were cage copulated with male DBA/2 mice at a ratio of 2:1 to establish RSA model,and were randomly divided into dydrogestrel group,model group,and Bushen Huoxue Recipe low-dose,medium-dose and high-dose groups.In addition,female CBA/J mice and male BALB/C mice were cage copulated at a ratio of2:1 as normal pregnancy group.On day 1 of gestation,normal pregnancy group and model group were given equal doses of normal saline,didrogesterone group was given 6.18mg/kg didrogesterone,Bushen Huoxue Recipe low-dose,medium-dose and high-dose groups were given 7.83g/kg、15.67g/kg、31.34g/kg,respectively,once a day,for consecutive 14 days.At the end of intragastric administration,peripheral blood was collected from pregnant mice to detect the levels of interleukin-17（IL-17）and transforming growth factor β（TGF-β）in serum and calculate the IL-17/TGF-β ratio.The pregnant mice were sacrificed after peripheral blood was collected,the uterus and spleen were separated,the embryo absorption rate was calculated,Th17 and Treg cell levels in splenic lymphocytes were detected,IL-17/TGF-β ratio was calculated,and Foxp3 protein expression level in uterine decidual tissue was detected.Results:1.Compared with model group,the embryo absorption rate of mice in Bushen Huoxue Recipe low-dose,medium-dose and high-dose groups and didrogesterone group were significantly reduced（P<0.05）,suggesting that the embryo absorption rate of mice in Bushen Huoxue Recipe and didrogesterone group could be reduced;Compared with dydrogesterone group,there was no statistical difference in the medium-dose Bushen Huoxue Recipe group（P>0.05）.2.Compared with normal pregnancy group,IL-17 expression and IL-17/TGF-β ratio were increased and TGF-β expression was decreased in model group（P<0.05）;Compared with model group,IL-17 expression and IL-17/TGF-β ratio were decreased and TGF-βexpression was increased in low,medium and high dose Bushen Huoxue Recipe groups and dydrogesterone group（P<0.05）.Compared with dydrogesterone group,there were no significant differences in the expression of IL-17 and TGF-β and the ratio of IL-17/TGF-β in the medium-dose Bushen Huoxue Recipe group（P>0.05）.3.Compared with normal pregnancy group,the proportion of Th17 cells and Th17/Treg ratio of pregnant mice in model group increased,but the proportion of Treg cells decreased（P<0.05）;Compared with model group,the proportion of Th17 cells and Th17/Treg ratio of pregnant mice in low,medium and high dose Bushen Huoxue Recipe groups,dydrogesterone group were decreased,while the proportion of Treg cells were increased（P<0.05）.Compared with dydrogesterone group,the proportion of Th17 cells,Th17/Treg ratio and Treg cell ratio in middle dose Bushen Huoxue Recipe were not significantly different（P>0.05）.4.Compared with normal pregnancy group,Foxp3 protein expression in uterine decidua of pregnant mice in model group was significantly decreased（P<0.05）;Compared with model group,the expression of Foxp3 protein in uterine decidua of pregnant mice in low,medium and high dose Bushen Huoxue Recipe groups,dydrogesterone group were increased（P<0.05）;Compared with dydrogesterone group,Foxp3 protein expression in uterine deciduous tissue of pregnant mice in medium dose Bushen Huoxue Recipe group had no significant difference（P>0.05）.Conclusion:（1）Low,medium and high dose Bushen Huoxue Recipe groups can significantly reduce the absorption rate of mouse embryos and improve the pregnancy outcome.（2）Bushen Huoxue Recipe can increase TGF-β level and reduce IL-17 level,which may be part of the mechanism for the treatment of recurrent abortion.（3）Bushen Huoxue Recipe increased the proportion of Treg cells in spleen lymphocytes of pregnant mice and reduced the proportion of Th17 cells,so that the balance of Th17/Treg tended to the normal pregnancy model,which may be part of the mechanism for the treatment of recurrent abortion.（4）Bushen Huoxue Recipe increased Foxp3 protein level in decidua of pregnant mice,which may be part of the mechanism of treating recurrent abortion.