The Mechanism Of Thyroid Colloid Retention Developed By Excess Iodide Intake In Mice And The Processing Technology Of Its Functional Food

Background and objectThyroid colloid retention is a pathological phenomenon of the goiter mostly resultingfrom overproduced colloid which include I-,T3, T4or damaged transportation towardsthyroid cells. The pathogenesis is multifactorial, among the factors that have beensuggested to influence the development of thyroid colloid retention, and iodine intake maybe the most important one. But the mechanism is still unclear. In recent years, there is agreat attention over the issue that the increased incidence of goiter.Thyroid colloid retention is a very complex process that involves several aspects, suchas iodine uptake and translocation, iodide ion activation, iodization and condensation oftyrosine residues, synthesis and storage of thyroid colloid, release of thyroid colloid,thyroglobulin hydrolysis and thyroid hormone transport and other processes.The aboveprocess is orderly close contact, which regulate thyroid colloid synthesis, release andtransport. As long as one of the aspects is abnormal, thyroid colloid retention mayoccurred.And, the unbalanced regulation of hypothalamic-pituitary-thyroid axis may alsoaggravate thyroid colloid retention. However, which exactly are the key aspects and the keygenes response for abnormal thyroid colloid retention, and how the hypothalamic-pituitary-thyroid axis plays its regulatory mechanism during the occurrence of thyroid colloidretention. Currently, there are lacking research.Therefore, this research established a excess iodine mouse model of seven-month,studied the multiple aspects and genes involved in the processes of synthesis, release andtransport of thyroid colloid, combined with thyroid hormone feedback mechanism, toinvestigate the mechanism of thyroid colloid retention, which is the pathological features of thyroid goiter, develope by long-term excessive iodine through morphological methods andmolecular biological methods. Thus, thses data could provide insights into the mechanismof iodide goiter.Meanwhile, besides study the mechanism of thyroid colloid retention developed byexcess iodide intake in mice but also for the processing technology of its functional food.Studies have shown that excess iodine can reduce thyroid and blood antioxidant levels,causing oxidative stress, resulting in thyroid dysfunction, which is likely to the mechanismsof the occurrence of thyroid colloid retention. By supplementing selenium, vitamin C,vitamin E and other antioxidants may improve antioxidant levels.Therefore, with theperspective of food treatment, the development of antioxidant-rich functional food factorswill play a positive role in preventing thyroid diseases, especially for high iodine goiter. Inthis study, a higher antioxidant capacity of the extract from pine needles and skim milkyogurt were chosen as raw materials to evaluate the intervention effect of masson pineneedles extract, to develop masson pine needles yoghourt with antioxidant function, toprovide a theoretical basis for the development of functional yogurt and play a positive rolein prevention of chronic disease caused by antioxidant reduce from the perspective ofprevention.Methods1. Experimental animal feeding and grouping1)300female BALB/c mice,3-4weeks old, weighing15-17g, were randomly divided into5groups by body weight. Experimental groups B, C, D, and E were fed differentconcentrations of high-iodine water (1500μg/L,3000μg/L,6000μg/L and12000μg/Lof potassiumiodate, respectively). The control group, A, was fed on tap water. Theanimals used in the experiment were approved by the Guangdong Province MedicalLaboratory Animal Center, China, and housed in temperature-(22±2°C) andlight-controlled (12±3h) conditions and had free access to food and water. The studyperiod was seven months. KIO3formulated into water to a concentration of iodine600mg/Lof iodine standard solution was standby, various concentrations of iodine excesswater were prepared weekly.For all the animal feed were approved by the Experimental Animal Center of Guangdong Province, feed contains iodine were365μg/kg. Tap watercontains iodine were8μg/L.2) Using same experimental animal and feed environment,28female BALB/c mice, wererandomly divided into4groups, including blank group (tap water of free access), thehigh iodine group (3000μg/L of free access) and the control group (0.2ml/gadministration of masson pine needles extract+tap water of free access) andintervention group (0.2ml/g administration of masson pine needles extract+3000μg/Lof free access). To do serum GSH-Px level detection and assessment of masson pineneedles extract intervention effect after three months.2. Mice thyroid colloid retention producing testing After seven months,determination of thyroid organ index, observe changes in thyroid tissue underpathological and analysis of the expression of thyroglobulin by immunohistochemical,detect blood level of TSH, FT4, FT3levels by using radioimmunoassay kit.3. The study of the mechanism of thyroid colloid retention developed by excessiodide intake in mice1) Determination of urinary iodine: After seven months, collected the mice morning urine,measured mice urine iodine levels by using urinary iodine kits.2) Thyroid iodine uptake rate determination: In the morning, after0.5Mci I125administration for20min and2hours,all mice were killed.The removed thyroids wereweighed and the radioactive count examined at both points in time with γ calculator.3) Thyroid genetic testing: the removed thyroids and livers tissue were frozen quickly byliquid nitrogen and stored in-80℃. Detect the mRNA expression of Tshr, Slc5a5(NIS),Slc26a4(pendrin), Tpo, Duox2, Iyd, Ctsb, Ctsd, Slc16a2(Mct8) by qPCR assay.4) Determination of serum GSH-Px: After seven months, collected the mice serum,measured GSH-Px levels by using GSH-Px kits.4. Masson pine needles extract intervention effect assessment and ProcessingTechnology of intervention functional food Through the test of serum GSH-Px levelfor3months in mice, evaluated the intervention effect on excessive iodine of massonpine needles extract; the optimum formula and processing technology of the yoghourt weredetermined by single factor test, orthogonal experiment and sensory evaluation.. Results1. Excessive iodine-induced thyroid colloid retention1) The effect of excess iodine on thyroid gland organ/body mass indexFrom Group C (3000μg/L) to Group E (12000μg/L), excess iodine can cause thyroidorgan/body mass index significantly increased compared with the normal group(P<0.05), by Spearman correlation test, mice thyroid organ/body mass index wassignificantly positively correlated with iodine concentrations (r=0.655, P=0.01).2) Thyroid tissue morphology observationUnder light-microscopy examination, in the normal control Group A, thyroid follicularmedium size, round or oval, and mostly of follicular epithelial cells were singlecuboidal or columnar. In Each excess iodine group,the thyroid follicular significantlyincreased,and were filled with red gum, epithelial cells were flat. High-dose groups hadphenomenons of follicular fusion. The phenomenon of thyroid colloid retention becamemore and more obvious with the iodine concentration increasing.3) Immunohistochemical detection of thyroid Tg protein expressionImmunohistochemical analysis showed that the normal control Group A has the of Tgbasal expression, compared with the control group, the scope and intensity of thyroidfollicular lumen of Tg expression significantly increased in Group D, E.4) The effect of excess iodine on serum thyroid hormone levels in miceFrom Group C (3000μg/L) to Group E (12000μg/L), serum TSH levels weresignificantly elevated compared with the control group (P<0.05); comparison with thenormal group, in all experimental groups, serum FT4elevated and FT3levels reducedwhich have significant differences (P<0.05); through Spearman correlation test, theiodine concentration and mice serum TSH showed a significant positive correlation (r=0.439, P=0.001), the iodine concentration and serum FT4showed a significantpositive correlation (r=0.649, P=0.000), the iodine concentration and serum FT3showed a significant negative correlation (r=-0.320, P=0.03).2. The study of the mechanism of thyroid colloid retention developed by excessiodide intake in mice 1) The effect of excess iodine on the thyroid iodine uptake in miceFom Group B (1500μg/L) to Group E (12000μg/L), mice urinary iodine levels weresignificantly increased compared with the normal group, the difference was statisticallysignificant (P<0.05). By Spearman correlation test, urine iodine levels wassignificantly positively correlated with iodine concentrations (r=0.878, P=0.000).2) The effect of excess iodine on the thyroid iodine uptake in miceAfter20min, compared with the normal control group, From Group C (3000μg/L) toGroup E (12000μg/L), thyroid iodine uptake rate was significantly lower (P <0.05).2hlater, compared with the normal control group A, From Group B (1500μg/L) to GroupE (12000μg/L), thyroid iodine uptake rate was significantly lower (P <0.001).3) The effect of excess iodine on the mRNA expression levels of thyroid Tshr, Slc5a5(NIS), Slc26a4(pendrin), Tpo, Duox2, Iyd, Ctsb, Ctsd, Slc16a2(Mct8)Excessive iodine severely inhibited the mRNA expression of Tshr, NIS, Pendrin, Tpo,Duox2, the order of the severity of decreased: NIS, Pendrin, TPO, Tshr, Duox2, whoseexpression have significant differences (P<0.05); Although the mRNA expression ofIyd, Cstb, Cstd decreased, but showed no difference (P>0.05); for gene Mct8, themRNA expression significant was down-regulated in Group D (6000μg/L)(P<0.05).4) Thyroid cell ultrastructure was injured and the number of lysosomes reducedElectron microscopy showed that the normal control group set clearly thyroid follicularepithelial cell structure, cell nuclei were round or oval, uniform distribution oforganelles in the cytoplasm, rich in lysosomes. In Group E follicular were filled withhigh electron density of the material, visible colloid drops, thyroid follicular epitheliumflattened, oblong nucleus, organelles blurred, extreme expansion and degranulation ofthe endoplasmic reticulum, which some fused into irregular vesicles, the number ofmitochondria was reduced, vacuoles, structural fuzzy, lysosomes rare. Compared withthe normal control group, high dose iodine group can significantly make structuraldamage of the thyroid follicular epithelial cells and reduce the number of lysosomes.5) The effect of excess iodine on the serum GSH-Px in miceFom Group C(1500μg/L) to Group E (12000μg/L), mice serum GSH-Px levels weresignificantly decreased compared with the normal group, the difference was statistically significant (P <0.05).3. Masson pine needles extract intervention effect assessment and ProcessingTechnology of intervention functional food1) Compared with the high iodine group (3000μg/L), mice serum GSH-Px levels weresignificantly increased in the blank group, control group and intervention group, thedifference were statistically significant (P <0.05).2) The best extracting technology of masson pine needles extract was the solid-liquidratio of1:20(g/ml), extracting at90℃for120min and the optimum enzymolysis andclarification process of masson pine needles extract was0.12%of pectinaseconcentration, warming at65℃for2h.Meanwhile,it tasted best when dissolved14%ofnonfat dried milk,7%of sugar and0.1%of gelatin in masson pine needles extract.Theoptimal fermentation process indicated that fermentation works best when0.12%ofcompound starter culture was added in the yogurt after8h of fermentation in43℃.Thetotal antioxidant capacity of masson pine needles yoghourt was51.31(U/mL),whichshowed a strong antioxidant activities.Conclusion1. Long-term excessive iodine-induced thyroid colloid retention was mainly caused bythe abnormalities of thyroid colloid synthesis and release, the imbalance ofhypothalamic-pituitary-thyroid axis feedback and the reduction of antioxidant ofbody.5. Masson pine needles extract had intervention effects on excessive iodine, which can beused as raw material of function food for excessive iodine intervention.6. Under optimal formulation and process development, the pine needles antioxidantyogurt have antioxidant intrusive effect.