The Effect And Mechanism Of Estradiol On Isolated Maternal Hypothyroxinemia

ObjectiveTo investigate the effects of different estradiol concentrations during pregnancy on deiodinase and thyroid hormone transporter in brain astrocytes and pituitary tissues from cellular and animal levels,and to preliminarily explore the mechanism of isolated maternal hypothyroxinemia.1.To investigate the correlation between serum estradiol and thyroid hormone levels in patients with IMH in late pregnancy.2.To investigate the changes of serum levels of E2 in rats during different pregnancy.3.To investigate the correlation between serum E2 and thyroid hormone changes in rats during different pregnancy.4.To investigate the effects of 17β-estradiol on deiodinase and thyroid hormone transporter in brain astrocytes.5.To investigate the effects of 17β-estradiol on pituitary deiodinase and thyroid hormone transporter in ovariectomized rats.MethodsThe thyroid function and estradiol were collected retrospectively in patients with isolated maternal hypothyroxinemia and pregnant women with normal thyroid function in late pregnancy.The correlation between estradiol and thyroid function was analyzed by spearman correlation.Female Wistar rats aged 6-8 weeks were selected and randomly divided into 4groups according to the process of pregnancy: first trimester group(7th day of pregnancy),second trimester group(14th day of pregnancy),third trimester group(20th day of pregnancy)and non-pregnant control group,with 15 rats in each group.Except the control group,the other groups were caged with male rats,and heart blood was collected on the 7th,14 th and 20 th day of gestation,respectively.Serum of Wistar rats in each group was collected,and the values of FT4,FT3,TSH and E2 were measured by electrochemical luminescence immunoassay.Spearman correlation was used to analyze the correlation between estradiol and thyroid function in rats.Human normal brain astrocytes(HA1800)were cultured in vitro,and the cells entered the logarithmic growth phase.After trypsin digestion,they were uniformly inoculated in six-well plates.When cells in the six-well plates contact each other and alignment reached 80%,with different concentrations of 17 beta estradiol and estrogen receptor inhibitor intervention for 48 hours,using CCK8 testing estradiol and estrogen receptor inhibitor activity of astrocytes,using Real Time PCR detection cells D2 mRNA,D3 mRNA,MCT8 mRNA,OATP1C1 mRNA expression level,the expression level of D2、D3、MCT8、OATP1C1 protein was determined by Western-blot.Thirty female Wistar rats aged 6-8 weeks were randomly divided into 5 groups with 6 rats in each group:Sham operation group(Sham),ovarian removal group(OVX),ovarian removal + cornoil group(OVX+V),ovarian removal + estrogen treatment group 1(OVX+E),ovarian removal + estrogen group 2,except for the sham operation group,only the same amount of adiposed tissue around the ovary was removed.The other groups ovarian surgery.Intraperitoneal injection was started three days after recovery from ovariectomies,ovarian removal + estrogen treatment is divided into two groups,give 0.1 ml contains 17 beta estradiol corn oil,dose per 100 g body weight of rats given 0.7ug or 2.1ug 17 beta estradiol(0.7 ug / 100 g bw or 2.1 ug / 100 g bw),and the ovariectomized corn oil group was replaced by 0.1 ml corn oil.The rats were weighed every other day,and the dosage was adjusted according to the weight of the rats.The rats were administered continuously for 21 days.On day 21,heart blood was collected and serum of Wistar rats in each group was collected.Serum FT3,FT4 and E2 in each group were determined by electrochemiluminescence analysis.The pituitary gland of Wistar rats in each group was retained,and the mRNA expression levels of TSHβ,D2,D3,MCT8 and OATP1C1 were detected by real-time PCR.Results1.There was no correlation between serum E2 and FT4,FT3 and TSH in normal women in late pregnancy(P > 0.05).2.There was a negative correlation between E2 and FT4 levels in women with isolated maternal hypothyroxinemia in late pregnancy(spearman correlation coefficient r=-0.575,P < 0.05).There was no correlation between serum E2 and levels of FT3 and TSH(P > 0.05).3.The estradiol in rats increased gradually with the progress of pregnancy,but only the estradiol in the late pregnancy were significantly higher than those in the control group(P < 0.05).4.There was a negative correlation between serum E2 and FT4 levels in rats with different pregnancy(spearman correlation coefficient r=-0.634,P < 0.05).There was a negative correlation between serum E2 and FT3(correlation coefficient r=-0.668).There was no correlation between serum E2 and TSH levels(P > 0.05).5.Different concentrations of 17β-estradiol and estrogen receptor inhibitors had no effect on the activity of brain astrocytes.6.The expression of D2 mRNA in brain astrocytes increased with the increase of estradiol concentration,and the difference was statistically significant when the estradiol concentration was 15000pg/ml compared with the control group(P<0.05).There was no significant difference in D3 mRNA among all groups.When estradiol was 15000pg/ml,the expression of MCT8 mRNA was significantly higher than that of the control group(P < 0.0001),and the expression of OATP1C1 mRNA was the highest(compared with the control group,P < 0.0001).7.The expression of D2 protein in brain astrocytes increased gradually with estradiol concentration,and there was a statistical difference when estradiol was15000pg/ml compared with the control group(P< 0.05).There was no statistical difference in the amount of D3 protein among all groups.The expression of MCT8 protein increased with the increase of estradiol concentration,but the difference was not statistically significant compared with the control group(P > 0.05).When estradiol concentration was 15000pg/ml,OATP1C1 protein expression was the highest,and the difference was statistically significant compared with the control group(P < 0.05).8.Estrogen receptor inhibitors reduced the effect of estrogen on enhancing the expression of D2(P < 0.05,VS E2),but did not change the expression of OATP1C1(P > 0.05,VS E2).9.Compared with sham operation group,serum E2 in OVX group was significantly decreased(P < 0.05).With the increase of E2 dose,serum FT4 in OVX+2.1 group(OVX+ 2.1ug/100 g bw E2)was lower than that in OVX+0.7 group(OVX+0.7ug/100 g bw E2)(P < 0.05).There was no significant difference in serum FT3 among all groups.10.There was no difference in the expression of TSHβmRNA in pituitary gland.Compared with OVX group,D2 mRNA expression in OVX+2.1 group was significantly increased(P<0.05),OATP1C1 mRNA expression was increased in OVX+2.1 group(P<0.0001).There was no significant difference in D3 mRNA and MCT8 mRNA expression among all groups.Conclusions1.There was a negative correlation between E2 and FT4 levels in women with isolated maternal hypothyroxinemia in the third trimester.2.The estradiol in Wistar rats showed an upward trend in different stages of pregnancy,which was basically the same as that of human gestational estradiol,and there was a negative correlation between serum E2 and FT4 and FT3 during pregnancy3.Estradiol during pregnancy can up-regulate the D2 and OATP1C1 transporters in astrocytes by binding to estrogen receptors(ER)on astrocytes.4.With the increase of estradiol concentration,the expression of D2 mRNA and OATP1C1 mRNA in pituitary tissue of the ovarian removal rats were significantly upregulated,serum FT4 was down-regulated,but TSHβmRNA in pituitary tissue had no change.