Effects Of Thyroid Stimulating Hormone On The Secretion Of Proinflammatory Factors In Adipocytes

Objective:In recent years, it is gradually recognized that adipose tissue is not just a energy storage place, it is also an important endocrine organ. Adipose cells could secrete multiple bioactive factors through paracrine, autocrine and telecrine form, for example, tumor necrosis factor-α, interleukin-6, leptin and resistin those can cause the metabolism and inflammatory response and are associated with metabolic disease, insulin resistance. Insulin resistance is the key of various disease and closely correlated to impaired glucose tolerance, type2diabetes mellitus, hypertension, dyslipidemia, atherosclerosis and many kinds of cancer. So the exploration of the initiating factor of adipose cells to secrete proinflammatory factors has become a hot key in the etiology of insulin resistance. Nowadays,effects of thyroid stimulating hormone on the secretion of proinflammatory factors in adipocytes focus on subclinical hypothyroidism and microvascular disease, We find TSHR exists on other organs and cells except thyroid.So this study intends to explore whether TSH can promote the secretion of tumor necrosis factor-a through binding with TSH receptor, and participate in the occurrence and development of adipocytes insulin resistance.Methods:Mouse3T3-L1preadipocytes and differentiated adipocyte cell cultures were studied, and the mature adipocyte is the main study object. Use small interfering RNA to design plasmids which are applied to silence TSHR, transfectin these plamids into mature adipocytes, then adopt western blotting to assess TSHR protein expression, finally validate the interfering effectiveness and choose the best plasmids those could successfully silent TSHR. Use ELISA to assess0.1mIU/ml TSH on TNF-a protein release into the medium of the normal and TSHR-silented mature adipocytes over0to24hours to determine the best effect time. Then, the effect of0.01mIU/ml,0.1mIU/ml,1mIU/ml TSH on TNF-a protein release into the medium of the normal and TSHR-silented mature adipocytes was assessed by ELISA.Results:1.3T3-L1preadipocytes were induced in the inducer containing culture fluid to10days, induced to mature cells.2.Use siRNA to design4groups of plasmids. Compare to the blank controller, shRNAl, shRNA2and shRNA3groups are down-regulated(0.07±0.03,0.04±0.01,0.19±0.04), there is statistical significance difference(P<0.01). Compare to shRNA3group, shRNAl and shRNA2group are down-regulated,there is statistical significance difference(P<0.01),And there is no statistical significance difference between shRNA1group and shRNA2group(.P>0.05).Compare to the blank controller, TSHR protein of2ug-24h and4ug-24h group are down-regulated(0.32±0.10,0.31±0.05),there is statistical significance difference(P<0.05),and4ug-48h group protein is down-regulated (0.17±0.04),there is statistical significance difference(P<0.01). There is no statistical significance difference among2ug-12h group,2ug-48h group,4ug-12h group and6ug group(P>0.05).3.Use0.1mIU/ml bovine TSH to stimulate mature cells Oh to24h in culture fluid, the TNF-a levels increased with time.The TNF-a level of4h achieved peak position(537.07±45.19ng/L), and there is no statistical significance differences with the TNF-a level of24h(547.94±32.32ng/L,P>0.05).Use0.1mIU/ml bovine TSH to stimulate TSHR-silented mature cells, the TNF-a levels slowly increased with time, but compared with the controller, there is no statistical significance differences(P>0.05).4. When compare to controller,the TNF-a level of4h blank groups culture fluid stimulated by different concentration of Bovine TSH (0.01mIU/ml,0.1mIU/ml,1mIU/ml) are up regulated(522.67±36.22vs431.51±30.10、411.08±12.03、341.85±12.00ng/L, P<0.05或P<0.01).5.Compare to the controller, Use1.0mlU/ml bovine TSH to stimulate normal mature cells and TSHR-silented mature cells, the TNF-a level of4h blank and negative groups culture fluid are up-regulated(522.67±36.22vs341.85±12.00ng/L,521.26±34.23vs340.54±12.53, P<0.05).But there is no statistical significance difference in the TNF-a level of4h TSHR-silented group(368.50±26.11vs339.62±14.39,P>0.05).Conclusion:TSH can dose-dependently stimulate the3T3-L1adipocytes to promote the secretion of TNF-a through binding with TSHR. Closely monitoring TSH level and prevent subclinical thyroidism and its relevant complications, may help decrease the levels of inflammatory factors, improve insulin resistance and prevent or delay the related disease development and progression.