Active Vitamin D Ameliorates Arsenite-Induced Thyroid Dysfunction in Sprague-Dawley Rats by Inhibiting the Toll-like Receptor 4/NF-KappaB-Mediated Inflammatory Response

Abstract

Arsenic, a well-known environmental endocrine disruptor, exerts interference on the body's endocrine system. Our previous investigations have demonstrated that chronic exposure to sodium arsenite (NaAsO₂) can induce thyroid damage and dysfunction in Sprague-Dawley (SD) rats. Vitamin D (VD) is an indispensable fat-soluble vitamin that plays a crucial role in maintaining thyroid health. In recent years, numerous studies have demonstrated the association between VD deficiency and the development of various thyroid disorders. However, the precise intervention roles and mechanisms of VD in arsenic-induced thyroid injury remain elusive. This study aimed to investigate the intervention effect of VD on NaAsO₂-induced thyroid dysfunction in SD rats. The results demonstrated that exposure to NaAsO₂ activates the TLR4/NF-κB signaling pathway in thyroid tissue of rats, leading to apoptosis of thyroid cells and subsequent inflammatory damage and disruption of serum thyroid hormone secretion. Supplementation with TAK-242 (a TLR4 inhibitor) and VD effectively inhibits the activation of the TLR4/NF-κB signaling pathway in rat thyroid tissue exposed to NaAsO₂, thereby reducing the inflammatory damage and dysfunction caused by arsenic exposure. In conclusion, the findings of this study offer innovative insights into the application of VD in the prevention and treatment of thyroid dysfunction caused by arsenic exposure.

Keywords: TLR4/NF-κB signaling pathway; sodium arsenite; thyroid hormone; thyrotoxicity; vitamin D.

Figure 1

The alterations in serum 25(OH)D₃ levels and VDR expression within the thyroid tissue of rats. (A) The serum 25(OH)D₃ levels were quantified using the ELISA method. (B) The VDR expression in rat thyroid tissue in each group was detected by IHC (scale = 50 μm). (C) The average optical density of VDR was analyzed using Image J software (Image J 2X, MD, USA) (n = 6); * p < 0.05.

Figure 2

The protein expression of the TLR4/NF-κB signaling axis in rat thyroid tissue was assessed. (A) The expression levels of the TLR4/NF-κB signaling pathway-related proteins TLR4, MyD88, p50, p-P50, p65, and p-P65 in thyroid tissue of rats were detected by IHC (bar = 50 μm). (B) Image J software (Image J 2X, MD, USA) was selected to analyze the average optical density of TLR4, MyD88, p50, p-P50, p65, and p-P65 proteins (n = 6); * p < 0.05.

Figure 3

The serum levels of the TLR4/NF-κB signaling-related inflammatory cytokines, including (A) IL-1β, (B) IL-6, (C) IL-10, and (D) TNF-α in SD rats were detected (n = 6); * p < 0.05.

Figure 4

TUNEL staining and positive cells in thyroid tissues of rats. (A) Representative TUNEL staining plots were observed in sections of rat thyroid tissue from each experimental group. Thyroid cells labeled with TUNEL exhibit a green fluorescence, while DAPI-labeled nuclei displayed a blue fluorescence (bar = 50 μm). (B) TUNEL positive cell rate in rat thyroid tissue (%); * p < 0.05.

Figure 5

The apoptosis-related protein expression levels in the thyroid tissue of rats. (A) The protein levels of Caspase-3, Caspase-9, Bax, and Bcl-2 in different groups of rat thyroid tissue were evaluated by IHC (bar = 50 μm). (B) Mean optical density analysis of the above proteins was performed using Image J software (Image J 2X, MD, USA). (n = 6); * p < 0.05.

Figure 6

The expression levels of THs synthesis-related proteins in rat thyroid tissue. (A) The protein levels of TSHR, NIS, TPO, and TG in each group were detected by IHC (bar = 50 μm). (B) The optical density values of the above proteins were quantitatively analyzed by Image J software (Image J 2X, MD, USA) (n = 6). * p < 0.05.

Figure 7

The histopathological staining of thyroid and serum THs secretion levels in rats. (A,B) The provided histopathological images depict rat thyroid tissue stained with HE as well as Masson’s trichrome (bar = 50 μm). (C) The percentage of Masson positive area in rat thyroid tissue. (D–I) The serum levels of TT₃, FT₃, TSH, TT₄, FT₄, and TG-Ab in each group (n = 6); * p < 0.05.