iodine is a key ingredient in the synthesis of thyroid hormones and also a major factor in the regulation of thyroid function [ 1 , 2 ] . at the organ level , small changes in the absorption of iodine can allow the thyroid to adjust its response to tsh stimulation and stabilize thyroid function . long - term severe iodine deficiency will cause diffuse goiter and thyroid dysfunction . on the other hand , large dose of iodine intake blocks iodine organification and inhibits the synthesis and release of thyroid hormones . this is called the  wolff - chaikoff  effect [ 4 , 5 ] . at the cellular level , iodine has been shown to inhibit multiple signaling pathways of thyroid cells , including camp and pip2 . iodine not only reduces the expression of thyroid - specific proteins such as nis but also has been shown to alter the tsh / tshr signaling pathway [ 68 ] . at the follicular level , iodine in follicular lumen , namely , iodinated thyroglobulin ( iodinated tg ) , is the main form of iodine reserve in the body . it has been demonstrated that iodinated tg in the follicular lumen can suppress thyroid function and that the suppression takes place through downregulation of tshr expression in thyroid follicular cells [ 911 ] . tshr belongs to a class of g - protein - coupled receptors and it is expressed on the surface of thyroid cells . it is regulated by iodine and also by the thyroid - specific transcription factors ttf-1 and pax8 [ 12 , 13 ] . tsh binds with tshr to deliver extracellular signal and regulates thyroid function and growth of thyroid cells [ 14 , 15 ] . overexpression of local follicular tshr may overstimulate this part of thyroid tissue to form hyperplasia , ultimately leading to the formation of nodules . the expression patterns of tshr , ttf-1 , and pax8 in the diseased tissues and the regulation of their protein expression by follicular lumen iodine have not yet been elucidated . to preliminarily study the pathogenesis of nodular goiter , the association of the follicular lumen iodine with tshr , ttf-1 , and pax8 expression in nodular goiter lesions was here investigated . after obtaining the approval of the institutional research ethics board , 30 resection specimens clinically histopathologically confirmed to have nodular goiter and 30 normal thyroid specimens from adjacent tissues of nodular goiter are used as the negative control were collected . all the patients with course of the disease 0.510 years and age of 40.5  14 years have euthyroid function and were not treated with medicine . all protein samples were prepared as follows : 0.5  g of tissue was frozen in liquid nitrogen , ground to yield tissue powder , and then suspended in ice - cold ripa lysis buffer ( abcom co. , ltd . , the suspension was then sonicated for 1  min at 08c and centrifuged at 8000  g for 30  min . the protein concentration was determined by bca ( sigma , shanghai , china ) assay . the lysates were then separated electrophoretically in 12% polyacrylamide gels and transferred onto pvdf membranes ( beijing biosynthesis biotechnology co. , ltd . , the membranes were blocked for 1.5  h at room temperature in 5% nonfat milk and incubated overnight at 48c with tshr , ttf-1 , and pax8 antibodies ( the same as those used in immunohistochemistry ) , respectively . after the triple wash in tris buffered saline with tween ( tbst ) for 30  min , the membranes were incubated with hrp - conjugated secondary antibodies for 45  min and then triple washed again in tbst . immune reactive bands were revealed using ecl detection system . in the negative group , 2% bsa the x - ray film was scanned , and the band density was calculated using imagej 1.45 software . sections ( 4  mm ) were taken from the tissues , affixed to 3-aminopropyl triethoxysilane - coated slides , and air - dried overnight at 37c . after dewaxing and antigen retrieval , endogenous peroxidase was quenched with 3% hydrogen peroxide for 5  min . after blocking with goat serum , the slides were incubated for 30  min at 37c with mouse anti - tshr ( at 1  :  100 dilution ) , rabbit anti - ttf-1 ( at 1  :  300 dilution ) , and rabbit anti - pax8 ( at 1  :  150 dilution ) polyclonal antibody ( beijing biosynthesis biotechnology co. , ltd . , all procedures were performed with a diaminobenzidine ( dab ) detection kit ( beijing biosynthesis biotechnology co. , ltd . , beijing , china ) following the standard protocol . after washing , all slides were counterstained with haematoxylin - eosin for histological evaluation . for the negative control , the sections were observed randomly in 10 high - power fields ( not follicular lumen area ) . the intensity of staining was scored as no staining ( 0 ) , weak staining ( 1 ) , moderate staining ( 2 ) , or strong staining ( 3 ) . the percentage of stained thyroid cells was scored as no stained cells ( 0 ) , staining in 25% of the cells ( 1 ) , staining in 2550% of the cells ( 2 ) , staining in 5075% of the cells ( 3 ) , or staining in more than 75% of the cells ( 4 ) . the final result of each section was evaluated by averaging the sum of the intensities and percentages of the stained thyroid cells . the average was then scored as 02 ( negative ) , 3 - 4 ( positive ) , and 57 ( strongly positive ) . each protein suspension was taken and the iodine concentration of suspension was then determined by arsenic - cerium catalytic spectrophotometry ( venter health science and technology co. , ltd . , student 's t - test was used to determine the differences of the western blot whereas the fisher exact test was used to determine the differences in positive - staining rate . in addition , the wilcoxon rank sum test was used for determining the differences in stain results and the spearman rank coefficient was used for determining the correlation between the parameters . western blot was performed to determine the expression of tshr , ttf-1 , and pax8 in 10 nodular goiter and 10 control thyroid samples . in addition to the increase in tshr expression , the expressions of ttf-1 and pax8 in nodular goiter lesions were also significantly higher than those in control tissues ( figures 1(a ) and 1(b ) ) . follicular lumen iodine concentration was also determined by arsenic - cerium catalytic spectrophotometry in 30 nodular goiter and 30 control thyroid samples . the iodine content ( g / mg ) in nodular goiter was significantly lower than those in control tissues ( figure 1(c ) ) . immunohistochemistry was also performed to determine tshr , ttf-1 , and pax8 expression in 30 nodular goiter and 30 control thyroid samples . tshr was localized in the basolateral membrane , and the immune reactivity of tshr in nodular goiter was significantly higher than those in the control ( figures 2(a ) and 2(b ) and table 1 ) . ttf-1 and pax8 were confined to the nucleus of the thyrocytes , and both types of immune reactivity in nodular goiter were significantly higher than those in the control ( figures 2(c ) , 2(d ) , 2(e ) , and 2(f ) and tables 2 and 3 ) . the spearman rank coefficient was performed to determine the correlation among the parameters of tshr , ttf-1 , pax8 , and iodine in 30 nodular goiter and control samples . the results showed that iodine content in follicular lumen of nodular goiter was less than that in control group ; moreover , the content of iodine in follicular lumen was found to be negatively correlated with tshr ( rs = 0.857 ; p < 0.01 ) , ttf-1 ( rs = 0.805 ; p < 0.01 ) , and pax8 ( rs = 0.652 ; p < 0.05 ) expression in nodular goiter . environmental factors include iodine nutrition , smoking , estrogen , obesity , age , and gender [ 17 , 18 ] . genetic factors are mainly related to the polymorphism or mutation of thyroid - specific genes , including tshr , tpo , tg , and nis [ 19 , 20 ] . epidemiological studies have suggested that iodine deficiency is the major risk factor of nodular goiter , which may be related to thyroid dysfunction caused by iodine deficiency   and subsequent chronic thyroid stimulation caused by enhanced tsh level and its binding to tshr . individual thyroids may differ considerably in response even when stimulated by the same concentration of tsh . some follicles respond normally to tsh , but others may form nodular hyperplasia due to excessive stimulation of tsh . it has been found that the heterogeneity of thyroid follicles was correlated with different levels of tshr expression on the surface of follicles . follicles rich in expression of tshr showed a strong response to tsh / tshr stimulation . on the contrary , the reaction was weak . western blot analysis showed tshr protein expression to be significantly greater in nodular goiter lesions than in the adjacent normal thyroid tissues . likewise , immunohistochemical staining also showed there to be more tshr staining was stronger in the basement membranes of cells in lesions than in normal tissues , indicating that tshr is highly expressed in nodular goiter lesions . this shows that tsh stimulation is stronger in nodular goiter lesions than in normal tissues . this leads to excessive proliferation of follicular cells and is one of the causes of nodule formation . tshr is g protein - coupled receptor , mainly located in the basement membrane of thyroid epithelial cells . tshr expression is mainly regulated by the thyroid - specific transcription factors ttf-1 and pax8 . ttf-1 and pax8 bind to the regulatory region of the tshr gene , promoting the expression of tshr and increasing tsh / tshr stimulation signals [ 25 , 26 ] . in addition , tshr is also regulated by iodine in the follicular lumen [ 9 , 27 ] . results showed that , in addition to the increase in tshr expression , nodular goiter lesions also expressed significantly more ttf-1 and pax8 than normal thyroid tissues . tshr expression in nodular goiter lesions may be associated with upregulated expression of transcription factors ttf-1 and pax8 . the thyroid is the body 's main storage site of iodine , and thyroid iodine is mainly stored in the thyroid follicles . in this way , iodinated tg represents iodine in the follicular lumen . iodinated tg in the follicular lumen reflects not only the size of the thyroid hormone reserve but also that of the iodine reserve . studies have shown that exogenous tg could inhibit the expression of thyroid - specific genes in monolayer cells in vitro [ 21 , 30 , 31 ] . in addition , results also showed that the iodinated tg in follicular lumen reduced the sensitivity of thyroid follicles to tsh from the pituitary by inhibiting the expression of tshr , eventually leading to downregulated expression of thyroid - specific genes nis , tpo , and tg [ 10 , 32 ] . the discovery also explained the heterogeneity of thyroid follicles and indicated that when all thyroid follicles in the body had the same concentrations of iodine and tsh , there were significant differences in the sizes and functions of individual follicles . negative feedback regulation of iodinated tg in the follicular lumen may be an important cause of thyroid follicular heterogeneity in the body . the present work showed nodular goiter lesions to contain significantly less iodine than normal thyroid tissue , and iodine content was found to be negatively correlated with tshr expression , suggesting that iodine in follicular lumen inhibits expression of tshr and low iodine status in the follicular lumen of nodular goiter promotes upregulation of tshr expression . it remains unclear whether low iodine in follicular lumen upregulated tshr expression directly or through induction of ttf-1 and pax8 . the current study showed that low iodine status in nodular goiter was associated with high levels of expression of ttf-1 and pax8 . ttf-1 and pax8 are positive transcription factors in the expression of thyroid - specific proteins tshr , tpo , tg , and nis . they not only strengthened the iodine absorption of thyroid epithelial cells , iodine organification , polyiodide , and other reactions , enhancing thyroid hormone synthesis and iodine accumulation , but also stimulated the hyperplasia of thyroid gland , which also plays a role in the formation of thyroid nodules . follicular iodinated tg is a macromolecular glycoprotein and can not freely penetrate the thyroid cells . the mechanism by which regulation of iodinated tg in turn regulates tshr , ttf-1 , and pax8 is still unclear . one previous report has suggested that tg binds to sialic acid receptors in the apical membrane of follicular lumen [ 3234 ] . further study is necessary to determine whether sialic acid receptor mediates the regulation of tg . the incidence of nodular goiter has increased significantly in recent years . this study demonstrated the negative feedback regulation of iodine and iodinated tg in the expression of tshr , ttf-1 , and pax8 in the follicular lumen , which further regulates the response heterogeneity of thyroid follicles to tsh stimulation . the current findings have important implications for further studies of the pathogenesis of these diseases .
objective . it has been testified that iodine regulates thyroid function by controlling thyroid - restricted genes expression and is closely related to diffuse goiter and thyroid dysfunction . 
 however , the effects of follicular lumen iodine , the main form of iodine reserve in the body , on thyroid - restricted genes in nodular goiter are poorly understood . in this study , 
 correlations between follicular lumen iodine and the expressions of thyroid stimulating hormone receptor ( tshr ) , its transcription factors ttf-1 , and pax8 in nodular goiter were investigated . 
 patients . in this study , 30 resection specimens clinically histopathologically confirmed to have nodular goiter and 30 normal thyroid specimens from adjacent tissues of nodular goiter are used . 
 measurement . 
 western blot immunohistochemistry was performed to assay tshr , ttf-1 , and pax8 in thyrocytes of nodular goiter as well as in extranodular normal thyroid tissues 
 . meanwhile , follicular lumen iodine of both nodular goiter and extranodular normal thyroid tissues was detected as well . 
 results . 
 the tshr , ttf-1 , and pax8 in nodular goiter were significantly higher than those in the controls . 
 the iodine content in nodular goiter was significantly lower than those in control tissues . 
 conclusion . 
 upregulation of tshr , ttf-1 , and pax8 is associated with low follicular lumen iodine content in nodular goiter .