Effects Of Iodine Excess During Pregnancy And Lactation On Offspring Of Rats And The Effects Of Adjustment From High Iodide To Normal Iodide And 1,25（OH）₂D₃ Supplementation

Aims:The aim of the present study is to establish the iodide induced thyroid disease model in the offspring of rats,and to investigate the effects and mechanism of adjustment from high iodide to normal iodide and 1,25（OH）₂D₃ supplementation on iodide-induced thyroid diseases.Method:For maternal rats:6-week-old female rats were mated with male rats（1:1）after one week of adaptation.For the female rats,gestation was verified by positive finding of vaginal plug.The pregnant rats were randomly assigned to three treatment groups（N=6,each group）:normal iodide intake（NI）,10 times high iodide intake（10 HI）and100 times high iodide intake（100 HI）.For the offspring at 180 days:Their offspring were continuously exposed to potassium iodide（KI）with different concentrations（NI,10 HI,100 HI）from weaning to postnatal day 180（PN180）.At PN180,we measured urinary iodine concentration by As-Ce catalytic spectrophotometry.Serum FT4,FT3,TSH,TPOAb and TgAb were measured by enzyme-linked immunosorbent assay（ELISA）.Single-Photon Emission Computed Tomography（SPECT）imaging was performed to evaluate the Na^99mTcO₄%thyroid uptake.Morris Water Maze was performed to evaluate spatial learning and memory behavior of rats.Echocardiography was performed to evaluate cardiac function alterations.Changes in histological and ultrastructure of the thyroid were detected by hematoxylin and eosin（HE）staining and electron microscopy.TRα1 and TRβ1 expression in the thyroid was measured by Western blotting and Dio1 mRNA expression was measured in the thyroid by Real-time Quantitative PCR（qPCR）.For the offspring at 90 days before treatment:Their offspring were continuously exposed to KI with different concentrations（NI,100 HI）from weaning to postnatal day 90（PN90）.At PN90,the body weight of the rats was measured.The serum FT4,FT3,TSH,TPOAb,TgAb and VD₃（Vitamin D3）levels were measured by ELISA.For the offspring at 90 days after treatment:The NI group was considered as control group.The 100 HI group was divided into NI group（Adjustment from 100 HI to NI）,NI+1,25（OH）₂D₃ group（Adjustment from 100 HI to NI）,100 HI+1,25（OH）₂D₃ group（Continued to 100 HI）and 100 HI group（Continued to 100 HI）.NI+1,25（OH）₂D₃ and 100 HI+1,25（OH）₂D₃ groups were administered 1,25（OH）₂D₃every day by gavage in an amount of 5μg/kg.The treatment was carried out for 4weeks.Urinary iodine concentration（UIC）was measured by As-Ce catalytic spectrophotometry.SPECT imaging was performed to evaluate the Na^99mTcO4%thyroid uptake.We also measured body weight.Serum FT4,FT3,TSH,TPOAb and TgAb were measured by ELISA.Histological changes in the thyroid were observed by HE staining and immunofluorescence.The mRNA expression of IL-17,IFNγ,IL-10,Dnmt1,Dnmt3a,Dnmt3b in the thyroid and spleen were measured by qPCR.Spleen lymphocyte proliferation was measured by Flow Cytometry.The protein expression of TRα1 and TRβ1 in the thyroid was detected by Western blotting.Result:For the offspring at 180 days1.Urinary Iodine Concentrations（UIC）:Compared to the NI group,the median UIC increased significantly in 10 HI and 100 HI group（p<0.05）;compared with 10 HI group,the median UIC was increased significantly in 100 HI group（p<0.05）.2.Serum thyroid hormone and autoantibody levels:Compared with NI group,the serum FT3 and FT4 levels decreased significantly（p<0.05）,while the levels of serum TSH,TPOAb and TgAb increased significantly in 10 HI and 100 HI group（p<0.05）.Compared with 10 HI group,the FT4 decreased significantly and the TPOAb and TgAb increased significantly in the 100 HI（p<0.05）.3.SPECT:Compared with NI group,the%thyroid uptake was decreased in 10 HI and 100 HI groups（p<0.05）.4.Morris Water Maze:Compared with NI group,the average escape latency was significantly increased.However,crossovers in the target quadrant decreased in both10 HI and 100 HI groups（p<0.05）.5.Echocardiography:Compared with NI group,LA（left atrial）was significantly increased（p<0.05）,and the%FS（fractional shortening）and%EF（ejection fraction）decreased significantly in 10 HI and 100 HI（p<0.05）.6.The body weight:Compared with NI group,the body weight had no significant changes in the 10 HI and 100 HI groups（p>0.05）.7.HE staining:In the NI group,the thyroid follicles were intact.Lymphocytic infiltration in the thyroid was observed in both 10 HI and 100 HI groups.8.Ultrastructure of the thyroid:In the NI group,the thyroid follicles were morphologically intact.In the 10 HI or 100 HI group,swollen mitochondria and increased dilatation of the rough endoplasmic reticulum（RER）was observed.9.Western blotting:Compared with NI group,the decreased expression of TRα1 and TRβ1 in the thyroid was found in both 10 HI and 100 HI groups（p<0.05）.10.qPCR:Compared with NI group,the mRNA expression of Dio1 in the thyroid of the 10 HI and 100 HI was significantly decreased（p<0.05）.For the offspring at PN90 before treatmentCompared with the NI group,there was no significant changes in body weight in the100 HI group（p>0.05）;FT3 and FT4 levels had no significant changes（p>0.05）;serum TSH,TPOAb,and TgAb levels were significantly elevated（p<0.05）;VD₃levels decreased significantly（p<0.05）.In the NI group,thyroid follicles were intact;in the 100 HI group,infiltrated cells within the thyroid follicular was detected.For the offspring after one month treatment1.Urinary iodine concentration（UIC）in different treatment groups:Compared with the 100 HI group,the median UIC was significantly decreased in the NI and NI+1,25（OH）₂D₃ groups（p<0.05）.2.SPECT results in different treatment groups:Compared with the 100 HI group,%thyroid uptake in the NI,NI+1,25（OH）₂D₃ and 100 HI+1,25（OH）₂D₃ groups was significantly increased（p<0.05）.3.Body weight in different treatment groups:Compared with the 100 HI group,body weight in the NI group decreased significantly（p<0.05）;4.Thyroid hormone and autoantibody levels in different treatment groups:Compared with the 100 HI group,FT3 levels were significantly increased in the NI,NI+1,25（OH）₂D₃ and 100 HI+1,25（OH）₂D₃ groups（p<0.05）.5.HE staining in different treatment groups:In the NI+1,25（OH）₂D₃,NI,100HI+1,25（OH）₂D₃ groups,the thyroid follicles were relatively intact.In the 100 HI group,lymphocytic infiltration in the thyroid was observed.6.Expression of IL-17,IFNγ,IL-10,Dnmt1,Dnmt3a and Dnmt3b mRNA in the thyroid in different treatment groups:In the thyroid,compared to 100 HI group,mRNA expession of IL-17,IFNγ,Dnmt1,Dnmt3a and Dnmt3b were significantly decreased,mRNA expession of IL-10 was significantly increased in the NI+1,25（OH）₂D₃,NI,100 HI+1,25（OH）₂D₃ groups（p<0.05）.7.Expression of IL-17,IFNγand IL-10 mRNA in the spleen in different treatment groups:compared with the 100 HI group,IL-17 and IFNγmRNA expressions were decreased significantly（p<0.05）;the IL-10 mRNA expression significantly increased in the NI+1,25（OH）₂D₃,NI,100 HI+1,25（OH）₂D₃ groups（p<0.05）.8.Expression of Dnmt1,Dnmt3a and Dnmt3b mRNA in the spleen in different treatment groups:Compared with the 100 HI group,mRNA expressions of Dnmt1,Dnmt3a and Dnmt3b in the NI+1,25（OH）₂D₃,NI,100 HI+1,25（OH）₂D₃ had no significant changes（p<0.05）.9.Proliferation of CD4⁺T cells in the spleen in different treatment groups:Compared with the 100 HI group,CD4⁺Foxp3^-Ki67⁺T cells frequency decreased in the NI+1,25（OH）₂D₃,NI,100 HI+1,25（OH）₂D₃ groups（p<0.05）.10.Immunofluorescence staining:In the 100 HI group,there was a positive staining of CD4⁺（red）co-localized with TRβ1/VDR（green）and Hoechst（blue）in the infiltrated cells.11.Thyroid hormone receptor expression in the thyroid in different treatment groups:compared with 100 HI groups,TRα1 and TRβ1 expression was significantly increased in the NI+1,25（OH）₂D₃,NI,100 HI+1,25（OH）₂D₃ groups（p<0.05）.Conclusion:1.Iodide induced thyroid disease model in the offspring at PN180（hypothyroidism and thyroiditis）can be established by high iodide supplementation.2.SPECT,Morris water maze,and echocardiography were used to evaluate thyroid,brain,and heart function alterations respectively,in an iodide induced thyroid disease model.3.In the NI,NI+1,25（OH）₂D₃,100 HI+1,25（OH）₂D₃ groups,the expressions of IL-17,IFNγmRNA were significantly decreased and the expression of IL-10 mRNA was significantly increased in the thyroid and spleen;the expression of Dnmt1,Dnmt3a and Dnmt3b mRNA in the thyroid was significantly decreased.