The Immunoregulatory Mechanism Of Daphnetin On Th17/Treg Imbalance In RSA Model Mice

Objective:To investigate the immune regulation mechanism of recurrent spontaneous abortion(RSA)model mice with Th17/Treg imbalance induced by daphnetin,and to further provide theoretical and experimental basises for the drug development of daphnetin(E003)and the treatment of RSA.Methods:1.RSA mouse model: the female CBA/J and male BALB/c mice were cage-mated in a 2:1 ratio to establish a normal pregnant mouse model,and the female CBA/J mice and male DBA/2 mice were cage-mated in a 2:1 ratio to establish an RSA mouse model.2.Experimental groups:(1)Normal pregnancy group;(2)RSA model group;(3)E003 low-dose group(1 mg/kg?day);(4)E003 high-dose group(4 mg/kg?day).3.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression of cytokines Il-2 and Il-6 in peripheral blood and Il-2 and Il-6 in the homogenate of decidual tissues.Flow cytometry was used to determine the Th17/Treg ratio of peripheral blood mononuclear cells(PBMCs).The expressions of Foxp3,ROR?t,STAT3 and STAT5 in PBMCs from decidual tissues were detected by real-time fluorescence quantitative PCR.The expression of STAT3 and STAT5 proteins in decidual tissues was detected by Western blot.HE staining was used to observe the pathological changes of the decidual tissue.Transmission electron microscopy(TEM)was used to observe the ultrastructure changes of the decidual membrane.Results:1.RSA mouse model was established by a classical method.The uterine embryo diagram showed that the number of embryos in the normal pregnancy group was higher than those in the RSA model group.Furthermore,the number of embryos in the low-dose E003 group was significantly higher than those in the RSA model group,and the number of embryos in the high-dose E003 group was more higher2.ELISA results showed that the levels of Il-2 in peripheral blood and decidual tissues in the RSA model group were significantly higher than those in the normal pregnancy group,and the levels of Il-6 were significantly lower than those in the control group.After treatment with E003,the levels of Il-2 in serum and decidua tissues of RSA model mice were significantly reduced,while the levels of Il-6 were significantly increased in a dose-dependent manner.These results suggesting that the effect of the high-dose group was better than that of the low-dose group on regulating IL-2 and IL-6(P < 0.05).3.Flow cytometry results showed that compared with normal pregnancy group,the ratio of Th17 in RSA model group was significantly higher and the ratio of Treg cells was significantly lower(P < 0.05).After gavage treatment with E003,the Th17 ratio decreased and the Treg cell ratio increased.The proportion of Th17/Treg cells in the RSA model group was significantly higher than that in the normal pregnancy group,and the proportion of cells decreased significantly after E003 treatment.4.RT-PCR results showed that the expressions of Foxp3,ROR?t,STAT3 and STAT5 mRNA in the decidual tissues were significantly decreased in the E003 treatment group compared with the RSA model group,while the expressions of the Treg-related cytokines ROR?t and STAT5 were significantly increased.5.Western blot results further showed that after E003 treatment,the expression of STAT3 protein significantly decreased,and the expression of STAT5 protein significantly increased.All the above results indicated that E003 may improve the immune balance of Th17/Treg by regulating related cytokines in RSA model mice.6.HE staining results showed that the number of decidual cells in the RSA model group was significantly reduced compared with that in the normal pregnancy group.The number of decidual cells in both the low-dose and high-dose E003 groups were increased.7.Transmission electron microscopy showed that the volume of decidual cells in the RSA model group was decreased,the intercellular space was increased remarkably,the nuclear curvature was increased,heterochromatin was increased and was close to the nuclear membrane,the cytoplasm was dense,and the rough endoplasmic reticulum and a few vacuol-like mitochondria were observed.Vascular wall was incomplete,vascular endothelial cell structure was incomplete,and the nucleus was fusiform.The volume of decidual cells in the low-dose E003 group was larger than that in the model group,and the intercellular space was larger than that in the model group.The vascular wall was more complete than that of the model group,and the vascular endothelial cells were in a long spindle shape.The volume and intercellular space of decidual cells in the high-dose E003 group were larger than that in the model group.The vascular wall was relatively complete,which was formed by a single or multiple vascular endothelial cells.The vascular endothelial cells showed a relatively complete structure,the cell membrane protrudes towards the lumen,and the nuclear membrane was relatively completed.Conclusion:Daphnetin can improve Th17/Treg cell imbalance in peripheral blood mononuclear cells of RSA model mice by regulating Th17 and Treg cell-related cytokines,repair the damaged cell structure in the decidual tissue of RSA model mice,improve angiogenesis and organelle integrity,and thus have a certain therapeutic effect on RSA model mice.