Human Thyrocyte Dysfunction Induced By Polychlorinated Biphenyl 118 Through The Akt/FoxO3a/NIS Pathway

Objective:1. To investigate the effects of low-dose 2,3’,4,4’,5-pentachlorobiphenyl (PCB118) on proliferation and apoptosis of the human thyroid epithelial cells.2. To investigate the effects of low-dose PCB118 on the synthesis of Tg and T4 in human thyroid epithelial cells.3. To explore the possible molecular mechanism of thyrocyte dysfunction induced by low-dose PCB118 through the Akt/FoxO3a/NIS pathway.Methods:1. Thyroid follicular cells extracted from normal human thyroid tissue in patients who underwent nodular goitre surgery were cultured in vitro.2. Cells were seeded in 96-well culture plate and were treated with PCB118 at the doses of 0,0.025,0.25,2.5,25,250,2500,25000 nM/L, respectively. After 24 h,48 h and 72 h, Cell-Counting Kit-8 (CCK-8) assay was performed and cell viability were calculated by Adose group/A solvent control group x 100%.3. After the cells treated with PCB118 at the doses of 0,0.025,0.25,2.5,25 nM/L for 72 h, flow cytometry was performed to evaluate the cell apoptosis.4. After the cells treated with PCB118 at the doses of 0,0.025,0.25,2.5,25 nM/L for 48 h, the concentrations of Tg and T4 in the cell medium were determined by radioimmunoassay.5. After the cells treated with PCB118 at the doses of 0,0.025,0.25,2.5,25 nM/L for 48 h, quantitative real-time PCR was performed to measure the expression levels of Akt, FoxO3a and NIS mRNAs, and western blotting was performed to detetect the protein expression levels of Akt, p-Akt, FoxO3a, p-FoxO3a and NIS.6. Cells were pretreated with or without 25 μM/L LY294002 for 1 h and then stimulated with PCB118 at 25 nM/L. After the cells were treated with LY294002 for 6 h,12 h and 24 h, the protein expression levels of p-Akt, p-FoxO3a and NIS were meatured.7. Immunofluorescent staining was performed to detect the subcellular localization of FoxO3a after cells treated with or without 25 nM/L PCB118.Results:1. After PCB118 treatment 24 h, compared with the solvent control group, no significant difference was seen in cell viability in all the PCB118-treated groups (P> 0.05). After PCB118 treatment 48 h and 72 h, compared with the control group, cell viability were significantly decreased in 250,2500,25000 nM/L PCB118-treated groups (P< 0.05), whereas no significant difference was observed in 0.025,0.25,2.5, 25 nM/L PCB118-treated groups (P> 0.05).2. Compared with the solvent control group, no significant difference was seen in apoptosis rate in all the PCB118-treated groups (P> 0.05).3. In contrast to the solvent control group, the concentrations of Tg were significantly decreased in 2.5,25 nM/L PCB118-treated groups (P< 0.05), as well as T4 in 25 nM/L PCB118-treated group (P< 0.05).4. (1) Compared with the solvent controls, the expression level of Akt mRNA was significantly increased in 25 nM/L PCB118-treated group (P< 0.05), but not in the 0.025,0.25,2.5 nM/L PCB118-treated groups (P> 0.05). (2) Expression level of NIS mRNA was significantly decreased in 0.25,2.5,25nM/L PCB118-treated groups (P< 0.05), but not in 0.025 nM/L PCB118-treated group (P> 0.05), compared with the vehicle-treated controls. (3) There was no significant difference on expression level of FoxO3a mRNA among all PCB-treated groups and the vehicle-treated controls (P> 0.05).5. (1) Protein expression levels of Akt and p-FoxO3a were significantly increased in 0.25,2.5,25 nM/L PCB118-treated groups (P< 0.05), but not in 0.025 nM/L PCB118-treated group (P> 0.05), compared with the vehicle-treated controls. (2) Compared with the solvent controls, the protein expression levels of p-Akt were significantly increased in 2.5,25 nM/L PCB118-treated group (P< 0.05), but not in the 0.025,0.25 nM/L PCB118-treated groups (P> 0.05). (3) Protein expression levels of NIS were significantly decreased in the 2.5,25 nM/L PCB118-treated group (P< 0.05), but not in 0.025,0.25 nM/L PCB118-treated group (P> 0.05), compared with the vehicle-treated controls. (4) There was no significant difference on expression level of FoxO3a protein among all PCB-treated groups and the vehicle-treated controls (P> 0.05).6. Protein expression levels of p-Akt, p-FoxO3a were all significantly decreased (P< 0.05) whereas NIS was significantly increased in time-dependent manners (P< 0.05), compared with the vehicle-treated controls.7. Compared with the vehicle-treated controls, increased cytoplasmic shift of FoxO3a was observed in the PCB118-treated group.Conclusions:1. The human thyroid epithelial cells treated with low-dose PCB118 (≤ 25 nM/L) for 48 h and 72 h showed no significant difference in cell viability.2. Low-dose PCB118 treatment can inhibit the synthesis and secretion of Tg and T4 of the human thyroid cells.3. Low-dose PCB118 exposure can activate PI3K/Akt signal transduction pathway, increase the phosphorylation level and cytoplasmic shift of FoxO3a, and then inhibit the mRNA and protein expression of NIS.