| ObjectiveHashimoto’s thyroiditis(HT)is a common autoimmune disease.The main pathological is the damage of thyroid follicular epithelial cells and thyroid follicular cavity,resulting in the production of thyroid auto-antibody,deficency of thyroid hormones synthesis,further clinical manifestations for multiple organ function damage and long-term chronic damage to the human body.There are many causes for HT etiology,one of the most important is the excessive iodine intake,but the specific mechanism of excessive iodine leading to the pathogenesis of HT is not clear.The purpose of this study is to explore the specific mechanism of HT caused by excessive iodine damage to thyroid follicular epithelial cells,and provide a new intervention target for the clinical treatment of HT.Methods1.The pyroptosis-related protein gasdermin D(GSDMD)protein levels in the thyroid tissue of HT patients and controls were analyzed by western blot(WB)or immunohistochemistry(IHC).“Control” indicates tissues from patients with nodular goiter of the thyroid.2.To evaluated pyroptosis degree,Nthy-ori 3-1 cells were harvested after treatment with a gradient of concentrations of sodium iodide(Na I;0,1,10,20,50 m M)for 24 h,the levels of GSDMD-FL and GSDMD-N was detected by WB.3.The cell viability of Nthy-ori 3-1 cells was assessed by CCK-8 assays after Na I(0,1,10,20,50 m M)treatment for 24 h.4.The ROS level of Nthy-ori 3-1 cells was detected by flow cytometry(FCM)and fluorescence analysis after Na I(0,20,50 m M)treatment for 24 h.5.To evaluated NF-κB signaling pathway activation and the relation between ROS,Nthy-ori 3-1 cells was treated with Na I(50 m M)at times(0,15,30,60 min)or in the presence of NAC(10 m M)at 24 h,the p65 and p-p65 expression levels were measured by WB.6.To sure the influence of ROS and NF-κB signaling pathway to pyroptosis,Nthy-ori 3-1 cells were treated with Na I and/or NAC(10 m M)and / or IKK-16(2 μM)for 24 h,and GSDMD expression levels were measured by WB.7.The NLRP3 expression levels were measured by WB when Nthy-ori 3-1 cells were treated with Na I with or without NAC(10 m M)or IKK-16(2 μM) for 24h.8.In order to explore the effect of NLRP3 inflammasome on cell pyroptosis,the protein levels of GSDMD were detected by WB after transfection of si NLRP3 in Na I(50 m M)treated Nthy-ori 3-1 cells at 24 h.9.IL-1β levels in the supernatants of Nthy-ori 3-1 cell cultures were detected by ELISA in the presence of Na I(50 m M)treatment,with or without NAC(10 m M),IKK-16(2 μM),and silencing of NLRP3 at 24 h.Results1.Compared with the control,abnormally increased pyroptosis and IL-1β were found in the thyroid tissue of HT.2.Na I promoted the upregulation of GSDMD-FL and GSDMD-N of Nthy-ori 3-1 cells in a dose-dependent manner,and the most significant effect was found at the concentration of 50 m M.3.The activity of Nthy-ori 3-1 cells induced by Na I was decreased gradually with the increase of concentration,and it was the most significant at the concentration of 50 m M.4.Compared with the untreated group,the intracellular ROS level of Nthy-ori 3-1 cells was increased significantly in the presence under the stimulation of Na I(50 m M).5.After Na I(50 m M)stimulation for 24 h,the phosphorylation level of p65 in Nthyori 3-1 cells was increased significantly,but there was no significant change in nonphosphorylation,and when intracellular ROS wasblocked by NAC(10 m M),the activation level of NF-κB was significantly inhibited.6.Na I(50 m M)induced the upregulation of GSDMD-FL and GSDMD-N protein levels in Nthy-ori 3-1 cells,which was significantly inhibited in the presence of ROS inhibitor NAC(10 m M)and NF-κB inhibitor IKK-16(2 μM).7.The expression of NLRP3 in Nthy-ori 3-1 cells was increased significantly after treatment with Na I(50 m M)for 24 h,and the expression of NLRP3 was significantly inhibited when co-treated with NAC(10 m M)or IKK-16(2 μM).8.The expression of GSDMD-FL and GSDMD-N protein induced by,Na I(50 m M)after NLRP3 knockout in Nthy-ori 3-1 cells was significantly inhibited by small interference technique.9.The concentration of IL-1β in the culture supernatant of Nthy-ori 3-1 cells was increased significantly after treatment with Na I(50 m M)for 24 h.In addition,the concentration of IL-1β was decreased significantly in the presence of NAC(10 m M)or IKK-16(2 μM)or si NLRP3.ConclusionThe data presented in the current study demonstrated that increased pyroptosis occurred in the thyroid tissues of HT patients,excessive iodine induced ROS accumulation,followed by NF-κB signaling,NLRP3 inflammasome activation,and subsequent abnormal pyroptosis under circumstance of HT.Finally,IL-1β was released into the extracellular matrix.Reducing ROS production,suppressing NF-κB signaling or silencing the NLRP3 inflammasome,all inhibited Na I-induced pyroptotic death of TFCs and IL-1β secretion. |
PDF Full Text Request