Study For The Role Of IRE1Pathway In Fluoride Induced Damage In Thyroid Cells

Endemic fluorosis that seriously impairs human health is prevalent in the world. High fluoride can affect the thyroid function by altering the thyroid structure and disturbing thyroid hormone secretion, so that the thyroid may be a target organ of fluoride toxicity. Many research results suggested that the toxic effects caused by fluoride on the target organ may be related to the induction of apoptosis. In recent years the endoplasmic reticulum stress (ERS) pathway got focused in a variety of ways to induce apoptosis, and the IRE1pathway of the ERS pathway is the oldest one of the pathways in the process of biological evolution.ObjectiveThe purpose of this study was to determine the effects of different concentrations of fluoride on human thyroid cells in vitro models (Nthy-ori3-1cells) and to investigate whether the fluoride-induced thyroid cells apoptosis is via the IRE1signaling pathway of the ERS, and to provide a theoretical basis for the prevention of the toxic effects of high fluoride on thyroid.Methods1The cytotoxic effects of different concentrations of fluoride on the Nthy-ori3-1cellsThe Nthy-ori3-1cells were exposed to0,0.1,1,3mmol/L of sodium fluoride (NaF) in vitro. MTT assay and lactate dehydrogenase (LDH) assay were used to measure cell viability and the LDH leakage rate. ROS level, constituent ratio of the cell cycle, and apoptosis rate were measured by flow cytometry2The impact of different concentrations of fluoride on the IRE1signaling pathway of ERS in Nthy-ori3-1cellsThe Nthy-ori3-1cells were exposed to0,0.1,1,3mmol/L of NaF in vitro. After24hours incubation, total RNA was extracted and RT-PCR was used for GRP78, IRE1, XBP-1(S) and CHOP mRNA detection.Total protein was extracted and GRP78, IRE1and CHOP protein were detected by Western blot.Results 1Compared to control group and0.1mmol/L fluoride-treated group, the cell viability of1,3mmol/L NaF-treated groups decreased significantly (P<0.05); The cell viability of the3mmol/L NaF-treated group was lower than that of1mmol/L group (P<0.05).2Compared to control group,0.1and lmmol/L NaF-treated groups, LDH leakage rate of3mmol/L NaF-treated group increased significantly (P<0.05).3Compared to control group,0.1and lmmol/L NaF-treated groups, ROS level of the3mmol/L NaF-treated group were significantly increased (P<0.05).4After treatment with different concentrations of NaF for24h, The Go/Gi phase cells of the1mmol/L NaF-treated group were lower (P<0.05) than that in the other groups but the percentage of cells in S phase were higher (P<0.05) than that of the other groups. Comparing to the other groups, the precentage of apoptosis notablely increased in3mmol/L NaF-treated group (P<0.05).5The expressions of GRP78, IRE1, XBP-1(S) and CHOP mRNA in1mmol/L NaF-treated group were higher than those in the control group (P<0.05); The expressions of XBP-1(S)、CHOP mRNA in3mmol/L NaF-treated group were higher than those in the control group (P<0.05).6The expressions of GRP78, IRE1and CHOP protein in1mmol/L NaF-treated group were higher than those in the control group (P<0.05); The expressions of IRE1and CHOP protein of the3mmol/L NaF-treated group were higher than those in the control group (P<0.05).Conclusions1Fluoride can cause cytotoxicity to thyroid cells via decreasing cell viability and increasing LDH leakage.2Fluoride can increase level of ROS and block the cells in S phase, and induce thyroid cells apoptosis.3Fluoride can activate IRE1signaling pathway, which may be one of the ways to induce thyroid cells apoptosis. This work was supported by grants from National Natural Science Foundation of China (NO.30972555).