| Background: Recurrent Spontaneous Abortion(RSA)refers to recurrent spontaneous abortion for two or more consecutive times with the same partner before 28 weeks of gestation,including biochemical pregnancy.The etiology of RSA is very complex,including chromosome or gene abnormalities,anatomical abnormalities,endocrine factors,infectious factors,coagulation factors,autoimmune diseases,male factors and psychological factors.The pathogenesis of immune factors in RSA is a hot topic in reproductive immunology.After systematic etiological screening,there are still about 50% of patients with unclear etiology,which is called Unexplained Recurrent Spontaneous Abortion(URSA),also known as isoimmune recurrent abortion.In order to achieve a successful pregnancy,the mother needs to receive a semi-allogenous fetus carrying the father’s antigens and allow invasion of trophoblast cells,implantation and placenta formation to provide oxygen and nutrients to the fetus.However,the mechanisms that regulate this particular immune behavior and how mother and fetal cells "talk to each other" are not well understood.The imbalanced immunity at the mother-to-fetus interface is closely related to URSA,especially the imbalanced immunity of CD4+T cells.Th17 cells mainly secrete cytokine IL17 to promote inflammation and participate in immune tolerance.Studies have found that the concentration of IL17 in URSA women in early pregnancy is higher than that in normal early pregnancy checkers.Autophagy is involved in immune escape during pregnancy,regulating trophoblast apoptosis and placental vascular remodeling.However,the effect of autophagy on recurrent abortion has been controversial.The interaction between cytokines such as IL17 and autophagy is involved in the coordination of innate and adaptive immune system activities.We hypothesized that IL17 in peripheral blood and decidua could affect trophoblast autophagy and participate in recurrent abortion.IL17 interacts with PI3K/AKT/m TOR signaling pathways in a variety of diseases.IL17 can be signaled through the nuclear factor NF-κB,mitogen activated protein kinase(MAPK),and phosphoinositol 3 kinase(PI3K)signaling pathways.m TOR is the master regulator of cell metabolism and plays a crucial role in regulating autophagy.The mechanism regulating the expression of IL17 in Th17 cells remains unclear.Aryl Hydrocarbon Receptor(Ah R)is a ligand-activated transcription factor that plays a varietyof biological roles,such as participating in normal endometrial development,reproductive immunity and maintenance of normal pregnancy.It was found that Ah R protein and m RNA levels in decidua and placenta in URSA patients were significantly higher than those in normal controls.Resveratrol is a polyphenol compound with Ah R antagonistic activity and has anti-inflammatory,antioxidant and anticancer properties.High affinity ligand activation of Ah R during Th17 cell development increases the percentage of Th17 cells.Therefore,we speculated that Ah R may be involved in regulating the secretion of IL17 by peripheral blood and decidua Th17 cells in early pregnancy,acting on trophoblast,influencing autophagy of trophoblast cells through PI3K/AKT/m TOR signaling pathway,and ultimately affecting the occurrence of URSA.Objective: This study was divided into three parts: The first part was to compare the difference of IL17 concentration in peripheral blood between women with unexplained recurrent spontaneous abortion and normal women before pregnancy and in early pregnancy,and the difference of Th17 percentage in peripheral blood CD4+T lymphocytes and Ah R positive expression percentage in Th17 cells between women with unexplained recurrent spontaneous abortion and normal women during early pregnancy.To compare the differences of villus autophagy level and expression of proteins affecting autophagy pathway between women with unexplained recurrent spontaneous abortion and healthy women with early pregnancy selective termination.In the second part,the relevant signaling pathway of IL17 in the regulation of autophagy function of healthy early pregnancy trophoblast cell lines was discussed,and the molecular mechanism related to the regulation of autophagy function of human villus trophoblast cell by PI3K/AKT/m TOR signaling pathway was further revealed.In the third part,mice models of normal pregnancy and recurrent spontaneous abortion were established,and the expression of Ah R in Th17 cells at the maternal fetal interface and the regulation mechanism of trophoblast autophagy on pregnancy outcome were investigated in vivo.Methods: Part I: 1.Flow cytometry was used to detect the expression level of IL17 in peripheral blood of pregnant women(70 cases)and pregnant women(40 cases)with URSA.2.Flow cytometry was used to detect the expression level of IL17 in peripheral blood of URSA women(100 cases)and normal women(50 cases).3.Flow cytometry was used to detect the percentage of Th17 cells in peripheral blood of URSA early pregnantwomen(20 cases)and normal early pregnant women(20 cases)and the percentage of Ah R positive in Th17 cells.3.Ah R protein levels in decidua induced abortion and autophagy related protein and PI3K/AKT/m TOR pathway protein levels in villi of healthy women with URSA early pregnancy and selective termination of pregnancy were detected by Western-Blot method.4.The positive proportion of Ah R in Th17 cells in decidua and villi was detected by multiple immunofluorescence.Part Ⅱ: 1.Western-Blot detection of autophagy related proteins LC3 I,LC3II,Beclin1 and p62 after different concentrations of IL17 A were treated with HTR-8/SVneo.2.Western-Blot analysis showed changes in the expression levels of PI3K/AKT/m TOR signaling pathway molecules PI3 K,p-PI3 K,AKT,p-AKT,m TOR and p-m TOR after IL17 A was treated with HTR-8/SVneo at different concentrations.3.After combined intervention of IL17 A and m TOR inhibitor rapamycin,The expressions of autophagy related proteins LC3 I,LC3II,Beclin1 and p62 of HTR-8/SVneo and the expression levels of PI3K/AKT/m TOR signaling pathway molecules PI3 K,p-PI3 K,AKT,p-AKT,m TOR and p-m TOR were detected by Western-Blot.Part Ⅲ:1.We adopt the CBA/J ×DBA / 2 recurrent spontaneous abortion mice model,using the CBA/J ×BALB/c model to establish the normal pregnancy.The recurrent spontaneous abortion model and female mice were randomly divided into four groups according to the order of conception: model group,low,medium and high dose resveratrol group.The pregnant rats were sacrificed on the 14 th day of gestation.The uterus and placenta of pregnant rats were separated and the absorption rate of embryos was calculated.2.The expression levels of IL17 A protein and Ah R protein in the labyrinthian region of placenta were detected by immunohistochemistry.3.The expression levels of Ah R protein,autophagy related protein and PI3K/AKT/m TOR pathway protein in placenta were detected by Western-Blot.4.The positive proportion of Ah R in Th17 cells in mouse placental labyrinthian region was detected by multiple immunofluorescence.Results: Part I: 1.Analysis of clinical data showed that the general data of the subjects were comparable.2.The level of IL17 in peripheral blood of pregnant women with unexplained recurrent abortion is higher than that of normal pregnant women.3.The level of IL17 in peripheral blood of early pregnant women with unexplained recurrent abortion was higher than that of normal early pregnant women.4.The percentage of Th17 cells inthe peripheral blood of women with unexplained recurrent spontaneous abortion and the percentage of Ah R positive in Th17 cells were higher than those in normal women with early pregnancy.5.The expression level of Ah R protein in decidua tissue of induced abortion increased in women with unexplained recurrent spontaneous abortion compared with healthy women with selective termination of pregnancy in early pregnancy.6.The proportion of Ah R positive in Th17 cells in decidua and villi was significantly higher in women with unexplained recurrent spontaneous abortion than in healthy women with selective termination of pregnancy in early pregnancy.7.Compared with healthy women who had elective termination of pregnancy in early pregnancy,the expression of Beclin1,p62,p-PI3K/PI3 K,p-AKT/AKT and p-m TOR/m TOR ratios in the lint of induced abortion increased in women with unexplained recurrent spontaneous abortion.Part Ⅱ: 1.After 2.5ng/ml to 10ng/ml IL17 A stimulation,the relative expression of p62 increased,the relative expression of Beclin1 decreased,and the ratio of LC3II/LC3 I decreased.2.7.5ng/ml and 10ng/ml of IL17 A can increase p-PI3 K /PI3 K,p-AKT/AKT and p-m TOR/ m TOR ratios.3.Compared with IL17 A group,rapamycin combined with IL17 A increased the expression of autophagy proteins LC3II/LC3 I and Beclin1 in HTR-8/Svneo cells,while decreased the expression of p62.Part Ⅲ: 1.The mean embryo absorption rate of recurrent abortion mouse model was 29.17%,which was significantly higher than that of normal pregnancy control group(3.92%).The embryo absorption rate of mice model of recurrent spontaneous abortion in resveratrol medium and high dose groups was lower than that in recurrent spontaneous abortion group,and there was no significant difference compared with normal pregnancy group.There was no statistical difference between the resveratrol low-dose group and the recurrent abortion group.2.The expression of Ah R protein in the placenta of mice with recurrent spontaneous abortion was significantly higher than that of mice with normal pregnancy.The expression of placental Ah R in mice with recurrent spontaneous abortion was decreased in medium and high dose groups.There was no significant difference in the low-dose resveratrol group.3.Compared with normal pregnancy mice,the expression of IL17 A protein in the placental labyrinthian region was significantly increased in the recurrent spontaneous abortion mice model.Resveratrol decreased the expression of IL17 A in placenta of mice with recurrent spontaneous abortion in medium and high dose groups.There was no significant difference in the low-dose resveratrol group.4.The results of multiple immunofluorescence staining showed that Ah R+IL17A+ double positive cells were present in the decidua area,the junction area and the labyrinthian area of the mouse placenta,and the expression of labyrinthian area was obvious.Compared with the normal pregnancy group,Ah R+/IL17A+ in the labyrinthian area of the mouse model of recurrent abortion was significantly increased.After oral administration of resveratrol,the Ah R+/IL17A+ cell count in the high-dose and medium-dose groups was significantly lower than that in the recurrent spontaneous abortion group,while there was no significant difference in the low-dose resveratrol group.5.Compared with normal pregnancy mice,Western-Blot results showed that the expression of autophagy protein p62 was increased and Beclin1 expression was decreased in recurrent spontaneous abortion mice,and the expressions of p-PI3K/PI3 K,p-AKT/AKT and p-m TOR/m TOR were significantly increased.After oral resveratrol treatment in relapsing aborted mice,Beclin1 expression increased and p62 expression decreased in high-dose mice.The ratios of pPI3 K /PI3 K and p-m TOR/m TOR in the medium-dose and high-dose resveratrol groups were significantly lower than those in the recurrent spontaneous abortion model group,while the ratios of p-AKT/AKT were not significantly different between all resveratrol groups and the recurrent spontaneous abortion model group.Conclusion: Part I: Compared with normal women,the level of IL17 in peripheral blood of URSA patients was higher before and during early pregnancy.The percentage of Th17 cells in peripheral blood and the positive percentage of Ah R in Th17 cells in URSA patients during early pregnancy were higher than those in normal women.URSA patients had higher Ah R protein in decidual tissue than healthy women with elective pregnancy termination,and a higher percentage of Ah R positive in Th17 cells in decidual and villi.The decreased villus autophagy level in URSA patients may be related to the regulation of PI3K/AKT/m TOR pathway.Part Ⅱ: IL17 A inhibits autophagy of trophoblast cells by regulating the phosphorylation of PI3 K,AKT and m TOR proteins in the PI3K/AKT/m TOR pathway.It is an important regulatory factor at the maternal-fetal interface in early pregnancy.The inhibition of IL17 A on autophagy of trophoblast cell lines could be partially reversed by rapamycin.Part 3: Autophagy was impaired in the placenta of recurrent spontaneous abortion mice,which may be related to activation of the PI3K/AKT/m TOR pathway activation.Resveratrol can inhibit the expression of Ah R and IL17 A at the maternal fetal interface of recurrent spontaneous abortion mouse models,reduce the positive proportion of Ah R in Th17 cells,and further inhibit m TOR phosphorylation,up-regulate autophagy,repair autophagy flow,and reduce the absorption rate of mouse embryos. |
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