| Objective Large numbers of lymphocytes infiltrating in the thyroid tissue is the important pathological characteristics of Hashimoto’s thyroiditis disease.The aim of this study was to investigate the effect and mechanism of β-catenin on the chemokines expression which contribute to lymphocytes migration into thyroid tissues of HT,which might provide a new insight into the pathogenesis of Hashimoto’s thyroiditis.Methods 1.The quantity of lymphocytes infiltrating in thyroid tissues from HT patients and euthyroid nodular goiter tissues were detected by HE staining.2.β-catenin and IFN-γ expression in thyroid tissues from HT patients and euthyroid nodular goiter tissues were detected by immunohistochemistry staining.3.The protein expression of β-catenin from the whole cell or the nucleus in TFCs of Nthy-ori 3-1 with various concentrations of IFN-γ(250,500,1000 U/mL)treatment were measured by Western blot.And the mRNA levels of β-catenin were measured by quantitative real time polymerase chain reaction(qRT-PCR).4.The expression levels of Wnt signaling proteins p-GSK-3β,GSK-3β and β-catenin in Nthy-ori 3-1 were detected after treatment with IFN-γ(500 U/mL)for different times(1.5,3,6,12,24 h)by Western blot.5.The protein expression of 38 chemokines in the culture supernatant which collected from TFCs of Nthy-ori 3-1 with IFN-γ(500 U/m L)for 24 h or with 500 U/m L IFN-γ after pretreatment with the LiCl(10 mM)were detected by protein microarray.And the mRNA levels of chemokines were measured by qRT-PCR.6.The application of si RNA down regulated the expression of GSK-3β in Nthy-ori 3-1,then the change of the chemokines expression with IFN-γ treatment was analyzed by qRT-PCR.7.The application of siRNA down regulated the expression of β-catenin in Nthy-ori 3-1,then the change of the chemokines expression with IFN-γ treatment was analyzed by qRT-PCR.Results 1.Compared with the control thyroid tissues,more lymphocytes infiltration andhigher levels of IFN-γ,but lower levels of β-catenin expression distributed in thetissues of HT patients.2.Compared with the control group,the protein levels of β-catenin from the wholecell and the nucleus of Nthy-ori 3-1were significantly down-regulated in IFN-γtreatment group,especially in 500 U/mL IFN-γ treatment group(P < 0.05),whilemRNA level of β-catenin was not changed obviously.3.Compared with the control group,p-GSK-3β expression level in Nthy-ori 3-1 wassignificantly increased after treatment with IFN-γ(500 U/mL)at different times(1.5,3,6,12,24 h),as well as β-catenin expression was reduced beginning at 6h(P<0.01).4.The chemokine levels of CCL2,CCL5,CXCL10,CXCL16 and GRO in Nthy-ori3-1were significantly increased in IFN-γ treatment group compared with thecontrol(P<0.05),whereas the levels of CCL5,CXCL16 and GRO in Nthy-ori 3-1were significantly reduced in the IFN-γ after pretreatment with the LiCl group,butnot CCL2 or CXCL10(P < 0.05).5.Down-regulation of GSK-3β by siRNA in Nthy-ori 3-1 weakened the induction ofIFN-γ on the chemokine expression of CCL5,CXCL16,GRO-2 and GRO-3(P <0.05).6.Down-regulation of β-catenin by siRNA in Nthy-ori 3-1 enhanced the induction ofIFN-γ on the chemokine expression of CCL5,CXCL16,GRO-2and GRO-3(P <0.05).Conclusion Taken together,our results indicated that significantly reduced β-catenin expression and elevated IFN-γ expression distributed in the tissues of HT patients.And in vitro,IFN-γ reduced β-catenin expression through the activating GSK-3β of Wnt signaling pathway,leading to the production of chemokines in TFCs.Therefore,inhibition of GSK-3β by its inhibitor LiCl or siRNA significantly reversed the induction of IFN-γ on chemokines.And down-regulation of β-catenin by siRNA enhanced the induction of IFN-γ on chemokines,suggesting that reduction of β-catenin was closely related to lymphocytes infiltration in the thyroid tissues of HT patients. |
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