| Background: Natural abortion occurs 2 times or above is defined as recurrent spontaneous abortion(RSA), and the morbidity is about 1%-5% in fertile woman. Because of the complex etiology, the unexplained recurrent spontaneous abortion(URSA) accounts for approximately 80% with the exception of chromosome abnormality, endocrine disorders, infection, and abnormal anatomy, etc. URSA is harmful to the reproductive health of fertile woman severely, moreover, the probability of patients with recurrence of abortion will be increased with the increase of the number of abortion. With the in-depth reproductive immunology research, the role of maternal-fetal immune tolerance mechanism on the pathogenesis of URSA is highly concerned.Normal pregnancy is similar with successful allograft transplantation and its establishment and maintain largely depends on the formation of maternal-fetal immune tolerance, which is affected by the immune cells significantly. Both URSA patients and model mice exist T lymphocyte subsets imbalance according to the research. In addition, our previous study also confirmed that pregnancy outcome is closely related to the migration status of T subsets. However, it is rare to known the factors resulting in T subsets imbalance when abortion occurs. Dendritic cells(DCs) are the unique antigen-presenting cells(APCs) to activate the na?ve T cell in vivo and they are a crucial link relating the process of starting and regulating adaptive immune responses. A new study suggests that DCs play an important role in regulating immune tolerance and maintaining homeostasis apart from triggering immune response against microbial infection. With the in-depth research of the reproductive immunology, the effect of DCs in maternal-fetal immune tolerance and pregnancy-related diseases receives increasingly attention. JAK/STAT signal pathway can be one of the main signal pathways during DC differentiation and development, what's more, STAT proteins plays a vital roles in regulating DC's function and its differentiation and development. At present, researches on JAK/STAT signal pathway regulating the differentiation and development of DC and its function in URSA have not been reported. Furthermore, It is lack of the research of the differentiation and function of DC subsets regulated by drug targeting in curing URSA.Currently, treating URSA with western medicine mainly adopts hormone replacement therapy. However, there are unstable efficacy, side-effect and other shortcoming with this method. Traditional Chinese medicine(TCM) have a long history in treating abortion and should be claimed to be safe and quite effective. In traditional Chinese medicine, Scutellaria baicalensis is commonly used to prevent miscarriage and is regarded as fetus effective medicine by Dan Brook Heart Law. But its mechanism of action and efficacy of tocolysis material basis is not yet clear. Baicalin is the main monomer composition of Scutellaria baicalensis. Our previous study found that DCs surface molecular MHC class II and co-stimulatory molecules CD80 expressed abnormal during maternal-fetal interface of URSA mice, and Baicalin could regulate the m RNA expression of DCs MHC class II and co-stimulatory molecules CD80. As a result, the change of DC function may play an important role in URSA and it is more likely to become the new targets for drug intervention.Objective: To observe the change of DCs in URSA patients and model mice and clear the role of DCs subsets and function change in URSA from levels of cells, molecular and gene by clinic and mice experiments; Furthermore, to discuss the main mechanism of Baicalin regulating the differentiation and function of DCs subsets in treating URSA.Methods:Clinical research(1) Clinical specimen collection and preservation: draw peripheral venous blood of normal pregnancy group and URSA patients with 6 m L, respectively. EDTA-2 Na anticoagulation and separate samples serum and peripheral blood mononuclear cells.(2)The expression levels of IL-12, IL-6, IL-10, TGF-? and IL-23 of DCs in serum were measured by ELISA;(3)The change of DC subsets and the expression of DCs surface's HLA-DR molecules, costimulatory molecules(CD80/CD86) and functional molecules(CD274/CD275/CD205/33D1/Tim3) were detected by flow cytometry staining.(4)The DCs surface's HLA-DR molecules, costimulatory molecules(CD80/CD86) molecules and functional molecules(CD274/CD275/CD205/33D1/Tim3) were measured by RT-PCR. Animal experiment(1)Methods of mice model establishment and Baicalin intervention: CBA/J? were taken as non-pregnancy group, CBA/J ?mating with DBA/2?came to establish URSA model group, and CBA/J?mating with Balb/c?were used to establish thenormal pregnancy model group; URSA model mice were treated with different doses of Baicalin, which were divided into four groups: Saline control group, Baicalin low-dose group, Baicalin middle-dose group and Baicalin high-dose group;(2)The change of DC subsets and the expression of DCs surface's MHC-II molecules, costimulatory molecules(CD80/CD86) molecules and functional molecules(CD274/CD275/CD205/33D1/Tim3) in mice spleen and maternal-fatel interface were detected by flow cytometry staining.(3)The m RNA expression levels of DC MHC-II molecule, costimulatory molecules and key molecules of JAK/STAT signaling pathway(STAT3, E2-2, STAT5, ID2) were measured by q RT-PCR.(4) The correlation analysis between embryo resorption rate and DC subsets as well as function by Person correlation analysis.Results:(1)The changes of DC subsets and function in peripheral blood of URSA patients. Compared with normal pregnancy women, the proportion of c DC(CD11c~+HLA-DR~+ of URSA patients in peripheral blood was increased significantly(P<0.05), while the proportion of p DC(CD11c-CD123~+)and the ratio of p DC/c DC were both decreased markedly(P<0.05); Moreover, compared with normal pregnancy women, the proportion of CD11c~+CD80~+, CD11c~+CD86~+, CD11c~+CD275~+, CD11c~+CD205~+, CD11c~+Tim3~+ cells and the m RNA expression of HLA-DR, CD80, CD86, CD275, CD205,Tim3 of URSA patients in peripheral blood were both increased significantly(P<0.05), while the proportion of CD11c~+CD274~+, CD11c~+33D1~+ and the m RNA expression of 33D1 and CD274 were decreased markedly(P<0.05). Additionally, the level of IL-12, IL-23 and IL-6 in serum of URSA women were significantly increased(P<0.05), while the level of IL-10 and TGF-? were decreased markedly(P<0.05). Those results suggest that the subsets and function of DC of URSA patients in peripheral blood may be changed.(2)The changes of DC subsets and function in URSA mice. Compared with normal pregnancy group, the proportion of c DC(CD11c~+B220-) of URSA mice in spleen and maternal-fatel interface were both increased significantly(P<0.05), while the proportion of p DC(CD11c~+B220~+)and the ratio of p DC/c DC were both decreased markedly(P<0.05); The proportion of CD11c~+MHC-II~+, CD11c~+CD80~+, CD11c~+CD86~+, CD11c~+CD205~+, CD11c~+CD275~+, CD11c~+Tim3~+cells of non-pregnancy group, normal pregnancy group and URSA group in spleen and maternal-fatel interface were all increased successively, moreover, URSA group was increased significantly(P<0.05) than normal pregnancy group, while the proportion of CD11c~+33D1~+cells and CD11c~+CD274~+ cells were decreased markedly(P<0.05). As a result, the change of subsets and function of DC participate in the URSA.(3)The correlationship of between embryo resorption rate and DC subsets as well as function.The embryo resorption rate were negatively related both to the proportion of CD11c~+33D1~+DCs, CD11c~+CD274~+ DCs, p DC and the ratio of p DC/c DC, whereas the embryo resorption rate were positively related to the proportion of CD11c~+MHC-II~+DCs, CD11c~+CD80~+DCs, CD11c~+CD86~+DCs, CD11c~+CD275~+DCs, CD11c~+CD205~+DCs, CD11c~+Tim3~+ DCs and c DC in spleen and maternal-fatel interface.(4)The protective effects of Baicalin on pregnancy.Compared with URSA control group, the embryo resorption rate of URSA mice were does-dependent decreased significantly(P<0.05) after Baicalin administration, which indicated that Baicalin could decrease the embryo resorption rate of URSA mice.(5)The regulatory effect of Baicalin on DC subsets and function. Compared with normal pregnancy group, the proportion of c DC of URSA mice was increased(P<0.05), while the proportion of p DC and the ratio of p DC/c DC were both decreased(P<0.05), both the CD11c~+MHC-II~+, CD11c~+CD80~+, CD11c~+CD86~+, CD11c~+ Tim3~+, CD11c~+CD205~+, CD11c~+CD275~+ cells propation and the m RNA expression of DC moleculars MHC-II, CD80, CD86, CD275, CD205, Tim3 were increased significantly(P<0.05); After Baicalin administration, the proportion of c DC of URSA mice was decreased, while the proportion of p DC and the ratio of p DC/c DC were increased. These findings suggest that Baicalin induce maternal-fetal immune tolerance by regulating the change of DC function and subsets of URSA mice in spleen and maternal-fatel interface to treat URSA.(6)The regulatory effect of Baicalin on JAK/STAT signaling pathway. Compared with normal pregnancy group, the m RNA expression of E2-2 of URSA mice in spleen and maternal-fatel interface were both decreased significantly(P<0.05), while the m RNA expression of STAT5 and ID2 were both increased markedly(P<0.05); the m RNA expression of STAT3 and E2-2 of URSA mice were both increased(P<0.05), while the m RNA expression of STAT5 and ID2 were both decreased(P<0.05) after Baicalin administration and all of them were dose-dependent. Those results suggested that JAK/STAT signal pathway may be one of the main mechanisms of Baicalin regulating the differentiation and function of DCs subsets to treat URSA.Conclusions :(1)The change of subsets and function of DCs participate in the URSA. During the process, the proportion of c DC and the expression of DC surface markers MHC-II and co-stimulation moleculars(CD80, CD86, CD275, CD205, Tim3)were all increased, while the proportion of p DC, the ratio of p DC/c DC and the expression of CD274 and 33D1 were all increased; additionally, the level of IL-12, IL-6 and IL-23 in serum were increased while IL-10 and TGF-? were decreased.(2)Baicalin could effectively reduce the embryo resorption rate and provide protective effects for gestation.(3)Baicalin could induce maternal-fetal immune tolerance by regulating DCs shifting to p DC and decreasing the expression of DC surface markers MHC-II and co-stimulation moleculars(CD80, CD86, CD275, CD205, Tim3) and increasing the expression of CD274 and 33D1. Thereby to induce the maternal-fetal immune tolerance and treat URSA.(4)The expression of E2-2 of Maternal-fetal interface and mice spleen was increased while the expression of STAT5 and ID2 were decreased after Baicalin administration, which indicated that JAK/STAT signal pathway may be one of the main mechanism of Baicalin regulating the differentiation and function of DCs subsets to treat URSA. |
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