| ObjectiveThyroid hormone plays an important role in the regulation of fetal brain development in uterus.Mild thyroid insufficiency during pregnancy can impair the cognitive function of offspring.Most of the models of hypothyroidism in rodents reported in the literature at present were given antithyroid drugs from embryonic to lactating stage,and brain development abnormalities were found in offspring of young and adult rats.However,there is no report of mild thyroid dysfunction only in embryonic stage and its effect on brain development of offspring.This experiment was only exposed to the antithyroid drug propylthiouracil(PTU)during pregnancy.It simulated that human beings suffered from thyroidism deficiency only in embryonic stage.The effect of thyroidism deficiency during pregnancy,especially mild damage,on the brain development of offspring was discussed,which provided experimental basis for the appropriate level of TH during pregnancy.MethodsExperiment 1: 18 pregnant Wistar rats were randomly divided into hypothyroidism group and control group according to their body weight.Pregnant mice were fed tap water containing 0,20 ppm of propylthiouracil(PTU)from 4 days of gestation to 7days of postnatal period(PND7)to replicate the hypothyroidism model of pregnant mice.The differentially expressed genes of the gene family related to substance transport were screened out by microarray analysis of brain tissue of offspring 1 day after birth(PND1).The expression of thyroxine transporters in brain tissues of PND1 and PND7 offspring was confirmed to be significantly regulated by hypothyroidism.Experiment 2: 56 Wistar pregnant rats were randomly divided into control group,3hypothyroidism group and T4 group according to body weight.From the 4th day of gestation to the birth of offspring(PND0),three groups of neonatal rats were fed with tap water of 0,1,3,5,0 ppm propylthiouracil(PTU)respectively.The control group and T4 group drank tap water during pregnancy.From postnatal 1 day(PND1)to 28days(PND28),T4 group was intraperitoneally injected with 0.4 ug/100 g body weight perday T4,and the control group and three hypothyroidism groups wereintraperitoneally injected with the same dose of normal saline(NS).The growth and development indexes,thyroid function and thyroid morphology of offspring of 1 day(PND1),7 days(PND7),14 days(PND14),21 days(PND21),28 days(PND28)and70 days(PND70)were observed.The learning and memory abilities of adult rats were measured by water maze,and the expressions of MCT8,DCX,SHH,ARC/ARG3.1,GFAP,MBP and PVALB in the hippocampus or cortex were detected by Western Blot or immunohistochemistry.Results Experiment 1:Compared with the control group,the serum TSH level of hypothyroidism pregnant rats increased significantly,the levels of TT4,FT4,TT3 and FT3 decreased significantly(P < 0.01);the body weight of PND1 and PND7 offspring decreased significantly;the blood TT4 of PND1 offspring decreased significantly;the levels of TT4,FT4,TT3 and FT3 of PND7 offspring decreased significantly.The results of gene microarray in brain tissue of neonatal 1-day-old hypothyroidism mice showed that 21 up-regulated genes and 32 down-regulated genes were differentially expressed more than twice in 369 SLC gene family genes related to substance transport,and 19 genes were selected for validation,11 of which were successfully validated.Slc16a2,Slco4a1 and Slco4c1,which can mediate thyroid hormone transport,were significantly up-regulated in the brain tissues of 1-day and 7-day-old offspring,but the expression of Slco1c1 was not significantly down-regulated in the brain of2-day-old offspring with hypothyroidism.The level of MCT8(Slc16a2)protein of T3 transporter increased significantly in two-day-old offspring,but the expression of OATP1C1(Slco1c1)protein of T4 transporter did not change significantly.Experiment 2:1.Thyroid function,morphological changes and brain neuron development in pregnant and neonatal rats.Serum TH level of pregnant rats at 16 days of gestation: TSH level gradually increased with the increase of PTU exposure dose,while FT4 level decreased with the increase of PTU exposure dose.It showed that drinking 1,3 and 5 ppm PTU water in pregnant rats could lead to different degrees of hypothyroidism from pregnancy tohypothyroidism.Total TT4 level in whole blood of PND1 offspring decreased significantly in PTU 3 ppm and 5 ppm groups.In PND7 offspring,TSH increased significantly and FT4 decreased significantly in 5 ppm group.The thyroid follicle number density,mean volume,surface area and cross-sectional area of neonatal rats in two-day-old PTU group were significantly higher than those in the control group.In cortex and hippocampus,MCT8 protein increased with the increase of PTU exposure dose in two-day-old offspring,and increased in PND1 5 ppm group with statistical significance;SHH protein expression in cortex of PND7 offspring decreased with the increase of PTU exposure dose;DCX protein expression in cortex of two-day-old offspring increased with the increase of PTU exposure dose.The expression of ARC/ARG3.1 protein in hippocampal CA3 region of offspring rats increased significantly only in the 1ppm group of PND 1.2.Growth and development,thyroid function and brain neuron development of young rats.In the embryonic stage,the weight of immature mice with insufficient thyroidism decreased and the hanging time of forelimbs shortened.From 14 to 28 days after birth,the thyroidism of immature mice gradually recovered,FT3 of PND14 offspring decreased significantly in 3ppm group,FT4 and FT3 of 5ppm group,FT3 of PND21 offspring decreased significantly in 3ppm and 5ppm group,and TSH of PND28 offspring increased significantly,which still showed subclinical hypothyroidism.Compared with the control group at PND14,the TSH,FT3/FT4 levels of offspring in T4 group(hyperthyroidism group)decreased significantly,while FT4 and FT3 levels increased significantly.The expression pattern of neuronal development marker proteins in three PTU exposed groups was similar to that in T4 group.3.Learning and memory abilities of adult offspringAt the age of 70 days,the expression of PVALB and MBP positive products in hippocampus of three PTU groups was still significantly higher than that of the control group.Water maze test showed that the latency,swimming distance and the first time to enter the target quadrant of PTU exposure group were longer than those of the control group,and the expression of PVALB and MBP positive products in 3and 5 ppm groups was more obvious,but the gender difference was not significant.Conclusion1.Slc16a2,Slco4a1 and Slco4c1,which have the function of transporting thyroid hormone,are increased in the brain of hypothyroidism offspring rats to compensate for the deficiency of thyroid hormone in brain tissue.2.Inadequate thyroid function in embryonic stage delayed the growth and development of neonatal rats and young rats,and caused irreversible damage to the development of brain neurons in young rats and learning and memory in adult rats.3.The damage of embryonic thyroidism deficiency to offspring’s brain development is the result of overlapping the inhibition of pregnant thyroidism deficiency on brain neuron development and the hyperthyroidism-like expression pattern of neuron development during the recovery of postnatal TH. |
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