Activation of the Nrf2-Keap 1 Pathway in Short-Term Iodide Excess in Thyroid in Rats

Abstract

Wistar rats were randomly divided into groups of varying iodide intake: normal iodide; 10 times high iodide; and 100 times high iodide on Days 7, 14, and 28. Insignificant changes were observed in thyroid hormone levels (p > 0.05). Urinary iodine concentration and iodine content in the thyroid glands increased after high consumption of iodide from NI to 100 HI (p < 0.05). The urinary iodine concentration of the 100 HI group on Days 7, 14, and 28 was 60-80 times that of the NI group. The mitochondrial superoxide production and expressions of Nrf2, Srx, and Prx 3 all significantly increased, while Keap 1 significantly decreased in the 100 HI group when compared to the NI or 10 HI group on Days 7, 14, and 28 (p < 0.05). Immunofluorescence staining results showed that Nrf2 was localized in the cytoplasm in NI group. Although Nrf2 was detected in both cytoplasm and nucleus in 10 HI and 100 HI groups, a stronger positive staining was found in the nucleus. We conclude that the activation of the Nrf2-Keap 1 antioxidative defense mechanism may play a crucial role in protecting thyroid function from short-term iodide excess in rats.

Figure 1

Effects of the iodide intake (NI, 10 HI, and 100 HI) on (a) the body weight; (b) the thyroid weight; and (c) the ratio of the thyroid weight/body weight on Days 7, 14, and 28. All data is presented as mean ± SD (N = 6 for each group). Statistical analyses were performed by one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test.

Figure 2

Effects of the iodide intake (NI, 10 HI, and 100 HI) on the changes of mitochondrial superoxide production on Days 7, 14, and 28. (a) There was a significant increase in the mitochondrial superoxide production in the 100 HI group on Days 7, 14, and 28 (p < 0.05). The difference was more significant on Day 28 when compared to Day 7 or Day 14. (b) Histogram analysis was performed on the mean fluorescence intensity of MitoSOX Red. All data is presented as the mean ± SD (N = 6 for each group). Statistical analyses were performed by one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test. ^∗ p < 0.05 versus the NI group on Days 7, 14, and 28, respectively. ^# p < 0.05 versus the 10 HI group on Days 7, 14, and 28, respectively. ^▲ p < 0.05 versus the Day 7 group in the 100 HI group. ^△ p < 0.05 versus the Day 14 in the 100 HI group. Experiments were repeated 3 times with similar results. (c) There was a significant increase in the mitochondrial superoxide production in the 100 HI group on Day 28 and was observed by confocal microscopy. Scale bar: 20 µm.

Figure 3

Effects of the iodide intake (NI, 10 HI, and 100 HI) on the changes of (a) Nrf2; (b) Keap 1; (c) Srx; and (d) Prx 3 expressions on Days 7, 14, and 28. Representative western blot and histograms of densitometric analyses normalized for the relative β-actin content. All data is presented as the mean ± SD (N = 6 for each group). Statistical analyses were performed by one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test. ^∗ p < 0.05 versus the NI group on Days 7, 14, and 28, respectively. ^# p < 0.05 versus the 10 HI group on Days 7, 14, and 28, respectively. Experiments were repeated 3 times with similar results.

Figure 4

Effect of the iodide intake (NI, 10 HI, and 100 HI) on the changes of immunofluorescence staining on Days 7, 14, and 28. (a) In the NI group, Nrf2 (red) was localized in the cytoplasm on Days 7, 14, and 28. In the 10 HI group, the positive staining of Nrf2 can be observed in both the nucleus and the cytoplasm. Moreover, in the 100 HI group, a stronger positive staining of Nrf2 can be detected in the nucleus on Days 7, 14, and 28; the nucleus was dyed with Hoechst (blue). (b) Srx (red) positive staining was located in the cytoplasm; the nucleus was dyed with Hoechst (blue). Scale bar: 20 μm.

Figure 5

Proposed mechanisms in the present study. (a) The urinary iodine concentration and thyroid function of Wistar rats were detected. (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid. (c) The balance between oxidative stress and antioxidative defense under physiological conditions and excessive iodide intake.