Objective:Autoimmune thyroiditis(AIT)is one of the common endocrine diseases,and it is also a common thyroid disease in women of childbearing age.A large number of researches have shown that pregnant women with AIT have 2-3 times higher risk of miscarriage than normal pregnant women[1,2].Our previous study confirmed that the level of ENO1Ab in patients with AIT was significantly higher than that in patients without AIT.This confirms that ENO1Ab,a non-thyroid specific autoantibody,is produced in AIT,a classic organ autoimmune disease[3].Our team detected the expression ofα-enolase in many tissues such as brain,thyroid,vascular endothelium,ovary and so on by western-blot,which provides a certain basis for this research.α-enolase(ENO1)is a highly conserved key enzyme for glycolysis of cytoplasm[4],It plays an important role in cell energy metabolism and many physiological processes.ENO1 is also expressed in embryos and most adult tissues,and its antibody exists in many autoimmune diseases,inflammatory diseases and so on[5].However,currently,only a few studies have reported the relationship between the expression of ENO1 protein and abortion,and there are no reports on the effect of ENO1mediated autoimmune response on abortion.The objective of this study is to investigate whether the autoimmune response induced by ENO1 as an autoantigen in the mother increased the occurrence of abortion and the preliminary mechanism of abortion.Methods:The 5-week-old CBA/J female mice were divided into two groups:the experimental group(E group,n=8)and the control group(C group,n=8).CBA/J female mice in experimental group were given ENO1 recombinant immunoglobulin 100ug and complete Freund’s adjuvant subcutaneously,and CBA/J female in control group was immunized with equal dose PBS instead of ENO1.Two weeks later,the same dose of ENO1 or PBS was added to the same volume of incomplete Freund’s adjuvant for the final immunization.Four weeks after the last immunization.The two groups of CBA/J female mice were mated with10-week-old CBA/J mice(normal mice pregnancy model group)and DBA/2 male mice(abortion mice model group)respectively.The vaginal suppository was recorded as the first day of pregnancy and the mice were killed on the 13th day of pregnancy(G13).The levels of ENO1Ab in serum and amniotic fluid of G13 female mice were measured by ELISA method.Observe and compare the embryo absorptivity and number of embryo implantation of G13 female mice,Weighing and comparing the G13 female mice embryo and placenta.The serum levels of TT4,TSH and thyroglobulin antibody(TgAb)of G13 female mice were detected to evaluate thyroid function.Western-Blot was used to determine the expression of ENO1 protein in placenta of normal CBA/J female mice.The ENO1Ab of placenta of G13day was determined by ELISA method.The serum and placental tissue plasminase-antifibrinolytic enzyme complex(PAP)levels of G13 female mice were measured.The expression of angiogenic factor(VEGF)in placenta was observed by immunofluorescence method.The structure and morphology of G13 female mice placenta were observed by HE staining,and the area and cell density of placenta(decidual area,spongy layer area,labyrinth area)were calculated and compared.The apoptosis of G13 female mice placenta was observed by TUNEL staining.Similarly,the two groups of CBA/J female mice and 10-week-old CBA/J male mice were mated in 2:1 four weeks after the last immunization.After mating in the cage,the vaginal suppository was found to be the first day of pregnancy.Mice were killed on the day of birth(P0).The levels of ENO1Ab in serum and amniotic fluid of P0 mice were detected by ELISA method.Observe and compare the birth number of P0 mice.Results:1.Determination of ENO1Ab levels in serum and amniotic fluid of G13 and P0female mice:The serum and amniotic fluid of G13 and P0 female mice anti-ENO1autoantibodies were significantly increased after ENO1 immunization(P<0.01);2.The embryo absorptivity of G13 female mice ENO1 immunized group increased significantly(P<0.05),while the number of embryo implantation decreased,but there was no statistical difference.The birth number of P0 female mice ENO1 immunized group were significantly lower than that of control group(P<0.05);3.Placental weight of G13 female mice ENO1immunized group decreased significantly(P<0.05),but there was no significant difference in embryo weight between the two groups(P>0.05);4.The Serum TT4,TSH and TgAb of G13 female mice were determined:the serum levels of TT4,TSH and TgAb in the ENO1immunized group were not significantly different from those in the control group(P>0.05);5.The expression of ENO1 protein was found in placenta of normal CBA/J female by Western-Blot.6.The placenta ENO1Ab of G13 female mice after ENO1 immunization was significantly higher than that of the control group(*P<0.01);7.Determination of PAP in serum and placental tissue of G13 female mice:There was no significant difference in serum PAP level between ENO1 immunized group and control group.The level of placental tissue PAP in ENO1 immunized group was significantly lower than that in control group(P<0.05);8.G13 female mice placenta immunofluorescence showed:the VEGF protein in placenta of ENO1 immunized group was significantly lower than that of control group.9.The G13female mice placental tissue HE staining showed that the area of decidua in the ENO1immunized group was significantly lower than that in the control group(P<0.05),but there was no significant difference between the two groups in the area of the spongy layer area and the labyrinthine area.The cell density of decidua and labyrinth in ENO1 immunized group was significantly lower than that in control group(P<0.05),but there was no significant difference between the two groups.10.Tunel staining:The apoptosis of placenta in ENO1 immunized group was significantly higher than that in control group.Conclusion:The autoimmune response mediated by ENO1 can induce the increase of abortion in mice.ENO1Ab may be caused placental tissue function damage by inhibiting the fibrinolytic system activation,thus increasing the risk of abortion. |