| Objective The aim of this study was to explore whether IL-23 was involved in the thyroid redox balance differ, and whether the accumulation of ROS was associated with the impaired autophagy acitivity in HT patients.Methods1. IL-23 and IL-23 R expression in thyroid tissues from HT patients(n = 10) and euthyroid nodular goiter tissues(n = 10) were detected by immunohistochemistry.2. The expressions of IL-23 and IL-23 R in TFCs of Nthy-ori 3-1 with various concentration of IFN-γ(250, 500, 1000 U/mL) or LPS(0.1, 0.5, 1 μg/mL) or TNF-α(10,20, 50 ng/mL) treatment were measured by immunofluorescence.3. The relative expression levels of ROS in Nthy-ori 3-1 cells were detected by immunofluorescence and flow cytometry after the treatment with different concentrations of IL-23(0, 10, 50 ng/m L) only or with 50 ng/m L IL-23 after pretreatment with the anti-IL-23(1 μg/m L).4. The levels of autophagy-related proteins LC3 B and Atg12 expression in thyroid tissues from HT patients and euthyroid nodular goiter tissues were detected by immunohistochemistry.5. The expression levels of LC3 B, Atg12, p62 in Nthy-ori 3-1 were detected after treatment with IL-23(50 ng/m L) for different times(8 min, 15 min, 30 min, 1 h, 3 h, 6h, 12 h) or with 50 ng/m L IL-23 after pretreatment with the anti-IL-23(1 μg/mL) by western blotting.6. The relative expression level of p-mTOR protein was detected by immunofluorescence after treatment with different concentrations of IL-23(0, 10, 50ng/m L) for 24 h.7. The expression levels of AKT, p-AKT, mTOR, p-mTOR in Nthy-ori 3-1 cells were detected by western blotting after treatment with IL-23(50 ng/m L) for different times(8min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h).8. The expressions of p-mTOR and mTOR were detected by western blotting after treatment with IL-23(50 ng/m L) with or without the pretreatment of 1 μg/mL anti-IL-23.9. The relative expression level of p-mTOR in Nthy-ori 3-1 cells was detected by western blotting after the treatment with different concentrations of rapamycin(10, 20,50 nM) for different time( 12, 24 h).10. The expressions of LC3B-II, Atg12, p62 were detected by western blotting in the presence of IL-23(50 ng/m L) and/or rapamycin(10 nM).11. Effect of rapamycin on intracellular ROS levels in Nthy-ori 3-1 cells was detected by immunofluorescence and flow cytometry.12. The levels of p-AKT, AKT and p-mTOR, mTOR expression in thyroid tissues from HT patients and euthyroid nodular goiter tissues were detected by immunohistochemistry.Results1. Compared with the control thyroid tissues, IL-23 and IL-23 R expressions were markedly upregulated in TFCs of HT patients.2. In vitro, the expressions of IL-23 R in TFCs of Nthy-ori 3-1 with various concentration of IFN-γ( 250, 500, 1000 U/mL) or LPS in the concentrations of 0.1 and0.5 μg/mL were significantly increased, but there was no significant difference in the TNF-α treatment groups in comparison with control.3. Immunofluorescence and flow cytometry results showed the level of ROS expression in Nthy-ori 3-1 cells was sharply increased by IL-23 stimulation in a dose-dependent manner, and a neutralizing antibody against IL-23 abolished the effect of IL-23-induced ROS accumulation in Nthy-ori 3-1 cells.4. Immunohistochemistry results showed autophagy-related proteins LC3 B and Atg12 protein expressions in TFCs were sharply decreased in HT tissues as compared with that in healthy control. In vitro, IL-23 treatment significantly inhibited the expression ofautophagy-related proteins LC3 B and Atg12 in Nthy-ori 3-1 cells; whereas promoted the expression of p62(a negative indicator of autophagy)(P < 0.01). Furthermore, the effect that IL-23-induced alteration of autophagy-related proteins was blocked by the addition of neutralizing antibody against IL-23 that was the expressions of autophagy-related proteins LC3 B and Atg12 in Nthy-ori 3-1 cells were significantly increased, whereas the expression of p62 was inhibited.5. Immunofluorescence results showed IL-23 treatment resulted in a significant increase of phosphorylation of mTOR in a dose-dependent manner. Western blotting analysis verified that phosphorylation of AKT(P < 0.05) and mTOR(P < 0.05) was significantly increased after IL-23 stimulation in different times. Moreover, the addition of a neutralizing antibody against IL-23 reduced the expression of p-mTOR in Nthy-ori3-1 cells.6. Western blot results showed that there were significant decreases in the levels of both phosphorylated and total mTOR with the rapamycin treatment at 10 nM for 24 h.7. Western blot results showed that rapamycin treatment significantly increased the expression of autophagy proteins LC3 B and Atg12 in Nthy-ori 3-1 cells, whereas inhibited the expression of p62(P < 0.01) in comparison with the group of IL-23 treatment only; Immunofluorescence and flow cytometry results showed that mTOR inhibition by rapamycin significantly reduced IL-23-induced ROS accumulation in comparison with the group of IL-23 treatment only.8. Immunohistochemistry results showed that phosphorylated AKT and mTOR expression were significantly up-regulated in TFCs of HT tissues as compared with healthy control, whereas total AKT and mTOR expressions in TFCs were at the similar levels between healthy control and HT patients.ConclusionTaken together, our results indicated that significantly elevated IL-23 expression in the TFCs of HT patients, and IL-23 inhibited the autophagy activation through the activation of AKT/mTOR signaling pathway, leading to the ROS accumulation in TFCs and exacerbation of HT pathology. Therefore, inhibition of either IL-23 by its specific antibody or mTOR by rapamycin significantly reversed the autophagy phenotype inhuman TFCs, and the changes of these related-proteins in vitro were relatively consistent with the expression of thyriod tissues in HT patients, suggesting that IL-23,as a critical pathogenic factor, was closely related to the development of HT and might be a potential therapeutic strategy aimed at preventing cellular oxidative damage to thyroid cells of HT. |
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