| ObjectiveExcess iodine intaken through drink and food maybe cause autoimmune thyroiddisease(AITD), but its pathogenesy is still unknown. Previous studies confirmed that virsusinfection was involved in the development of some autoimmune diseases and TLR3 mRNAexpression was found in the FRTL-5 cells, and high expression of TLR3 protein was detectedin the thyroids from Hashimoto's patients. We get the hypothesis that virus infection couldaffect the development of high iodine-induced AITD. NOD mice were administered with highiodine to reproduce murine models of lymphocyte infiltrating thyroiditis. In order toinvestigate the influence of poly(I:C) on pathogenensis of autoimmune thyroiditis, we usedvirus mimics(poly(I:C)) to observe the effects of virus infection on the alterations of thyroidfunction, apoptosis and inflammation-related gene expression induced by high iodine.MethodsIn vivo: Female, 24 7-8 week-old NOD mice were randomly divided into three groups i.e.①control(C);②high iodine(HI);③high iodine and poly(I:C) (I+P). C mice drank the waterwithout iodine added. Mice in HI and I+P groups drank 0.05% NaI water. Additionally, micein I+P group administered poly(I:C)(5.0μg/g body weight; at first, daily for 7 days and finallyltime/2days lasted 7 days). Mice in C and HI groups administered 0.9% sodium chloride.Then, 9 weeks later after administration, mice were sacrificed. The following parameters weredetermined, i.e., body weight, thyroid weight and anatomic form, thyroid hormones andanti-thyroid antibodies in serum by RIA, thyroid morphology observed through HE stain,apoptosis measured by TUNEL, the quantitive analysis of the level of NIS, TSH-R, TRAIL,TRAIL-sR1, CCL21, CXCL10, ICAM-1 mRNA expression by the method of real timeRT-PCR(SYBR GREEN), GAPDH was used as contrast gene.In vitro: Thyroids were excised from 7-8 week-old NOD mice, digested with double enzymes. Obtained thyroid follicular cells ch were cultured in Coon's F-12 mediumsupplemented with 5-10% FCS, thyrotropin(TSH), insulin, hydrocortisone, transferrin,L-Glutamic acid. Thyroid specific antigen thyroglobulin (Tg) expression were detected byimmunohistochemical staining. The expression of thyroid peroxidase(TPO), thyroglobulin(Tg), sodium/iodide symporter (NIS), and TSH- receptor as well as secretion of thyroidhormones by cultured cells were assessed by RT-PCR and electro-chemiluminescenceimmunoassay(ECLIA), respectively. After identification, the cultured thyroid cells wererandomly divided into four groups: control group(C), high iodine group(HI), poly(I:C)group(P), and high iodine combination with poly(I:C) group(I+P). Besides control group,three groups was supplemented with final concentration of 10-3M KI, 25.0μg/ml poly(I:C),10-3M KI and 25.0μg/ml poly(I:C), respectively. Stimulated for 24 hours, culture medium wasabandoned and cultured thyroid cells were used to extract total RNA with Trizol. Then,through RT-PCR to synthesize cDNAs, which were used to quantitively analyze the level ofNIS, TSH-R, TRAIL, TRAIL-sR1, CCL21, CXCL10, ICAM-1 mRNA expression by realtime RT-PCR(SYBR GREEN), GAPDH was used as contrast gene.ResultsIn vivo: Body weight varied in the extent of 25.0-27.0g, no significant changes amongthree groups. Thyroid abslute weight and relative weight of HI and I+P groups were higherthan that in control (P＜0.01). Compared in the different groups, the levels of total T4 in serumof HI and I+P groups were lower than that in C group (P＜0.05). Both anti-Tg Ab andanti-TPO Ab concentrations in serum had no significant changes in different groups. Result ofHE stain: no thyroiditis developed in C group, on the contrary, all mice in HI and I+P groupshad thyroiditis. Although the incidence of HI and I+P groups, pathological changes degreewere different, I+P group were more serious than that in HI group. TUNEL stain: More ceilswith positive staining were demonstrated in thyroid tissue of HI and I+P groups than that in Cgroup; AI of thyroid cells increased in HI and I+P groups than that in control. (P＜0.01). Highiodine could inhibit the expression of NIS and upregulate the expression of TSH-R, TRAIL,TRAIL-sR1, CCL21, CXCL10, ICAM-1. High iodine associated with poly(I:C) couldaggravate the inhibition of NIS mRNA expression and upregulate the expression of TRAIL,TRAIL-sR1, CCL21, CXCL10, ICAM-1. Additionally, a inhibitory tendency of TSH-R mRNA expression was found in the I+P group.In vitro: The cultured ceils showed the attached epithelial-like cell characteristics andstrong cytoplasmic staining for Tg. The cells expressed TPO, Tg, NIS and TSH-R at mRNAlevels and were able to release T3 and T4 for up to 14 days after seeding, although theexpressing and secreting gradually decreased with time. The expression of CCL21 could notbe detected in the cultured NOD mice thyroid cells. The effects of high iodine and high iodineassociated with poly(I:C) on the expression of NIS, TSH-R, TRAIL, TRAIL-sR1, CXCL10,ICAM-1 were similar with in vivo. The expression of NIS and TSH-R were inhibited bypoly(I:C) alone in the cultured NOD mice thyroid cells. The expressions of TRAIL,TRAIL-sR1, CXCL10, ICAM-1 were upregulated by poly(I:C) alone in cultured NOD micethyroid cells.Conclusion1. Iodine excess can induce autoimmune thyroiditis directly. The declination of serumtotal T4, the presentation of many thyroid cells with apoptosis, the infiltration oflymphocytes, the destruction of thyroid follicles in some regions and fibrosis ofmatrix were displayed in HI group. But the anti-thyroid specific antibodies were notdetected. Additionally, iodine excess can inhibit the expression of NIS mRNA, andupregulate the expressions of TSH-R, TRAIL, TRAIL-sR1, CCL21, CXCL10, ICAM-1mRNA.2. Poly(I:C) can facilitate the declination of serum total T4, the destruction of thyroidfollicles and fibrosis of matrix caused by iodine excess. Furthermore, poly(I:C) canaggravate the inhibition of NIS and TSH-R mRNA expression, and potentialize theupregulation of TRAIL, TRAIL-sR1, CCL21, CXCL10, ICAM-1 mRNA.3. Primary cultures of functional mouse thyrocytes were successfully established thatwere used to observe the effects of iodine excess, poly(I:C), combination of iodineand poly(I:C) on the expressions of function-related genes, apoptosis-related genesand inflammation-related genes. Besides the expression of CCL21 can not be found,the results had a similar tendency with the results in vivo.4. Poly (I:C) can aggravate autoimmune thyroiditis caused by iodine excess, but thesignal pathway of effects should be further explored.
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