| Objective Hashimoto's thyroiditis(HT)is characterized by lymphocytic infiltration of the thyroid parenchyma,which ultimately leads to tissue destruction and loss of function.In this study,we intend to further evaluate the relationships between peroxisome proliferator-activated receptor gamma(PPAR?)and Caveolin-1(Cav-1),and participate in regulating chemokine secretion in the thyroid follicular cells(TFCs)and thyroid tissue infiltration of lymphocytes in the process of disease.Additionally,the intervention effects of PPAR? agonist pioglitazone in the process,which may lead to a potential treatment for lymphocyte infiltration into the thyroid gland lesion and thereby prevent HT development.Methods 1.Cav-1 expressions in thyr oid tissu es fro m H T pat ients(n = 9)and euthyroid nodular goiter tissues(n = 9)were detected by immunohistochemistry staining.2.Knockdown(shRNA)and overexpression(OE)of Cav-1 were constructed by lentiviral transfection in TFCs of Nthy-ori 3-1 cells,and the effect was measured by quantitative real time polymerase chain reaction(qRT-PCR)and Western blot.3.The chemokines mRNA expression levels of CCL5,CXCL9,CXCL11,CXCL16,GRO-2 and GRO-3 in Cav-1 knockdown or overexpression of Nthy-ori 3-1 cells were determined by qRT-PCR.4.The expression levels of PPAR? in Cav-1 knockdown or overexpression of Nthy-ori 3-1 cells were determined by qRT-PCR and Western blot.5.The protein expression levels of PPAR? and Cav-1 in Nthy-ori 3-1 cells were detected after treatment with pioglitazone(0,4.2,8.4,16.8 and 33.6 ?M)for 24 h by Western blot.6.The mRNA expression levels of CCL5 in Cav-1 knockdown Nthy-ori 3-1 cells were detected after treatment with pioglitazone(0,16.8 and 33.6 ?M)for 24 h by qRT-PCR.7.Cav-1 shRNA and shRNA negative control cells were cultured with(0,16.8 and 33.6 ?M)or without pioglitazone for 24 h.The supernatant of cells were removed and were used to detect the migration ability of PBMCs,photograph under a microscope and calculate mobility by TranswellT M assay.Results 1.Compared with the control thyroid tissues,lower levels of Cav-1 expression and more lymphocytes infiltration in the tissues of HT patients.2.Compared with the negative control group,the lentiviral shRNA had a specific knockdown effect on the Cav-1 mRNA(P < 0.001)and protein(P < 0.001)in Nthy-ori 3-1 cells;Compared with the control group,the level of mRNA(P < 0.05)and protein(P < 0.001)of Cav-1 was effectively upregulated in the overexpression system in Nthy-ori 3-1 cells.3.The qRT-PCR results showed that,compared with the negative control group,the mRNA expression levels of CCL5 was dramatic increased in the Cav-1 shRNA group(P < 0.001),while CXCL9(P < 0.05),CXCL11(P < 0.05),CXCL16(P < 0.05),GRO-2(P < 0.05),and GRO-3(P < 0.05)were slight increased in Nthy-ori 3-1 cells.The qRT-PCR results showed that,compared with the control group,the mRNA expression levels of CCL5(P < 0.05)were slightly downregulated,CXCL11(P < 0.05),CXCL16(P < 0.05)and GRO-2(P < 0.05)were slightly upregulated,while CXCL9 and GRO-3 showed only a slight,but not significant,increase in the overexpression of Cav-1 group in Nthy-ori 3-1 cells.4.Compared with the negative control group,the mRNA levels of PPAR? exhibit a slightly increase(P = 0.06)and the level of PPAR? protein was significantly decreased in Cav-1-knockdown cells in Nthy-ori 3-1 cells(P < 0.05).Compared with the control group,overexpression of Cav-1 in Nthy-ori 3-1 cells resulted in an increase of mRNA(P = 0.07)and PPAR? protein(P < 0.05)levels.5.The Western blot results showed that,the protein levels of PPAR? and Cav-1 from the whole cell of Nthy-ori 3-1 were significantly increased(P < 0.05)in a dose-dependent manner after pretreatment with different concentration of pioglitazone(0,4.2,8.4,16.8,33.6 ?M).6.The qPCR results showed that,the different concentration of pioglitazone(0,16.8,33.6?M)treatment of Nthy-ori 3-1 cells significantly inhibited CCL5 expression in the Cav-1 shRNA group compared to the negative control group(P < 0.001).7.Treated with a gradient concentration of pioglitazone(0,16.8,33.6?M),the percentage of PBMCs migrating towards the supernatant from Cav-1 shRNA treated Nthy-ori 3-1 cells was significantly decreased compared to the control by the transwell assay(P < 0.001).Conclusion First of all,our studies demonstrated that Cav-1 expression was significantly reduced in thyrocytes from HT patients,especially where lymphocytes heavily infiltrated.Secondly,inhibition of Cav-1 downregulated the expression of PPAR? and upregulated the expression of CCL5,as well as increased PBMCs migration in thyroid follicular cells.Finally,pioglitazone,a PPAR? agonist,reversed the detrimental consequence,which might be a potential target for the treatment of lymphocyte infiltration into the thyroid gland lesion and HT development. |
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