Study Of Cell Apoptosis In Iodine-induced Autoimmune Thyroiditis In NOD.H-2～(h4) Mice

ObjectiveThe relationship between iodine intake and thyroid diseases has been continuously paid much attention by endocrinologists in the recent years. It is well known that shortage of iodine intake would induce iodine deficiency disorders (IDD). In addition, excessive iodine may also be harmful to thyroid. Both population-based epidemiological survey and animal experiments have approved that iodine excess could increase the dangers of thyroid disorders, especially autoimmune thyroid disease (AITD). In animal models of BB/W rat and OS chicken, iodine ingestion could cause the development of autoimmune thyroiditis. Epidemiological sstudies have shown that the frequency and incidence of autoimmune throiditis and thyroid autoimmunity in population with iodine excess are higher than those with iodine deficiency.AITD is a group of thyroid diseases which specifically involves thyroid gland and manifests obvious genetic susceptibility, which includes Hashimoto's thyroiditis (HT), atrophy thyroiditis, Graves' disease (GD) and postpartum thyroiditis (PPT). Autoimmune thyroiditis (such as HT, atrophy thyroiditis and PPT) makes an important part of AITD. In thyroid gland of patients with AITD, lymphocytes infiltration, destruction or hyperplasia of thyroid follicular cells could be found. Abnormal thyroid function, which contains thyroid hyperfunction or hypofunction, is also a main feature of AITD.Some surveys have discovered that apoptosis may be involoved in the destruction or hyperplasia of thyrocytes in AITD. In human Hashimoto's disease, there are apoptosis thyrocytes at the site of thyroid folliculus destruction and lots of lymphocytes infiltrate aroud the location of thyrocytes apoptosis. At the same time, the expression of apoptosis related protein including apoptosis stimulating protein (such as Fas and its ligand FasL) and apoptosis inhibitory protein (such as Bcl-2 family) in thyrocytes and infiltrating lymphocytes altered in autoimmune thyroiditis. These findings demonstrated that cell apoptosis may play an important role in the mechanism of thyrocytes destruction in AITD. In addition,it is also found that iodine could induce thyroid cells apoptosis by specific mechanism. In cultured human thyrocytes in vitro, iodine treatment could make them apoptosis. The mechanism of this apoptosis was due to free radical injury induced by excessive iodine. Apoptosis induced by the interaction between Fas and FasL also was found involved the resolution of goiter in goiter model of Wistar rat.Although most of the studies demonstrate the close relationship between iodine ingestion and cell apoptosis, there are still lots of questions remained to be discussed. The animals used in the studies have not an autoimmune genetic background, there was no autoimmune thyroiditis and no lymphocytes infiltration, thus it is hard to illustrate the status of cell apoptosis and the expression of apoptosis related protein in autoimmune thyroiditis. The mechanism of the interaction between different iodine intake (especially iodine excess) and thyroid cells apoptosis, the role of cell apoptosis in the iodine induced autoimmune thyroiditis, both require an illustration by the endocrinologists.NOD.H-2^h4 mice is an animal model of spontaneous autoimmune thyroiditis (SAT). After 7-8 weeks year-old mice receive 0.05% NaI drinking for 8 weeks, almost 100 percent of them occurred autoimmune thyroiditis. We selected NOD.H-2^h4 mice as the objective of investigation, observing the degree of autoimmune thyroiditis, the status of apoptosis in thyroid gland and the expression of apoptosis related protein in thyroid cells and infiltrating lymphocytes. Thus, through combing the factors such as iodine, autoimmunity and cell apoptosis, we could discuss the relationship among them and investigate the effect of iodine on the apoptosis of thyroid cells and the role of apoptosis in autoimmune thyroid disease.Methods7-8 weeks year-old NOD.H-2^h4 mice received 0.05% NaI solution drinking (about 1000 times of normal daily iodine intake amount ), 0.005% NaI solution drinking (about 100 times of normal daily iodine intake amount ) and 0.001% NaI solution drinking (about 20 times of normal daily iodine intake amount ). Animals were sacrificed at the time point of 4 weeks, 8 weeks, 16 weeks and 32 weeks after drinking NaI solutions, thyroids were dissected and formalin fixed. After paraffin embedded and sectioned, incidence and degree of spontaneous autoimmune thyroiditis were evaluated by Haematoxylin&Eosin staining. The in situ status of thyroid cells and infiltrating lymphocytes apoptosis were detected by terminal-deoxynucleotidyl transferase (TUNEL), including different time point of iodine ingestion and different scores of thyroiditis. Immunohistochemistry staining in paraffin sections of thyroid was conducted to analyze the expression of apoptosis related proteins (including stimulating protein such as Fas and FasL, inhibitory protein FLIP) in thyroid follicular cells and infiltrating lymphocytes under different iodine ingestion, time point and scores of thyroidits.ResultsWhen the time of iodine ingestion processed, on the one hand, the incidence of AT elevated gradually; on the other hand, the extent of lymphocytes infiltration enhanced also and was related with the amount of iodine ingestion, showing that the severity of thyroiditis increased with time and iodine intake level. In some severe thyroiditis that the period of iodine ingestion lasted above 32 weeks, apparent thyroid follicullus decreasing could be discovered. There was no obvious apoptosis of thyroid follicular cells in normal thyroid. In thyroid with autoimmune thyroiditis, there were some apoptosis infiltrating lymphocytes and a few apoptosis thyroid follicular cells. In thyroid without autoimmune thyroiditis but with different iodine ingestion amount, no apoptosis thyroid follicular cell was found. The apoptosis stimulating protein Fas or FasL was not found in thyroid follicular cells in normal NOD.H-2^h4 thyroids. In autoimmune thyroiditis, Fas was mainly expressed in infiltrating lymphocytes and quite a few thyroid follicular cells expressed Fas in severe autoimmune thyroiditis. In thyroid from animals which received NaI but did not developed thyroiditis, the iodine ingestion did not induce the expression of Fas on thyroid follicular cells. FasL was mainly detected in infiltrating lymphocytes, the frequency and intensity of FasL staining was weaker than that of Fas. A few thyroid follicular cells expressed FasL in severe thyroiditis and the staining of FasL was not related with the iodine ingestion amount. The apoptosis inhibitory protein FLIP was mainly detected in almost all infiltrating lymphocytes in autoimmune thyroiditis. There was not apparent expression of FLIP in thyroid follicular cells. Western blotting detected obvious positive Fas and FLIP protein in thyroids with 3+thyroiditis but none in normal controls; positive FasL protein in thyroiditis but weak positive FasL in normal controls.Conclusions1. Excessive iodine ingestion induces thyroiditis in NOD.H-2^h4 mice, the frequency and severity of thyroiditis related to the amount of iodine ingestion and period of exposure in iodine excess.2. Excessive iodine ingestion could lead to destruction of thyroid follicles and ultrastructural damage of thyroid cells in NOD.H-2^h4 mice.3. Excessive iodine ingestion does not induce apoptosis in thyroid follicular cells of NOD.H-2^h4 mice in spite of whether autoimmune thyroiditis has developed.4. Long-term iodine excess may inhance Fas protein expression in thyrocytes under existed autoimmune thyroiditis in NOD.H-2^h4 mice.5. In autoimmune thyroiditis of NOD.H-2^h4 mice, the reason that infiltrating lymphocytes are not obviously apoptotic is probably due to their extensive FLIP expression.