Endocrine Abstracts

Background: Lymphocytic infiltration and inflammation in autoimmune thyroid diseases (AITDs) results in accumulation of HA, contributing to the pathogenesis of both thyroidal and extra-thyroidal (ophthalmopathy, pretibial dermopathy and mixedema) manifestations of AITDs. HA fragments, originating from native HA during tissue inflammation and injury, in turn promote the expression of different mediators of oxidative stress and inflammation, by interacting with the Toll-like receptor 2 (TLR-2) and 4 (TLR-4) and CD44, via nuclear factor kappa-B (NF-kB). Curcumin (diferuloylmethane) is a phytochemical with anti-inflammatory properties. It has been reported to have suppressive effect on NF-kB signaling pathway in various cell types. This study was aimed at investigating the effects of curcumin treatment in cultured primary human thyrocytes and fibroblasts after exposure to 6-mer HA oligosaccharides (6-mer HA).

Methods: Cultured cells were treated with increasing concentrations of curcumin (5 and 10 μg/ml), with and without 6-mer HA (50 μg/ml). mRNA and proteins expression for TLR-2, TLR-4, inducible nitric oxide synthases (iNOS), interleukin-1beta (IL-1beta), IL-6, matrix metalloproteinase 9 (MMP-9), and thyroid-specific genes [thyroglobulin (Tg) and sodium iodide symporter (NIS)] were evaluated by real-time PCR and Western Blot, respectively. Protein quantification was assessed by densitometry analysis. NF-kB (p65) activation was determined in nuclear extracts by DNA binding activity assay. The pro-inflammatory cytokines IL-1 beta and IL-6 levels were measured by ELISA. Levels of NO were measured in culture medium by a fluorometric assay.

Results: In both cell lines 6-mer HA treatment induced the increase in mRNA and protein of TLR-2, TLR-4, CD44, as well as the activation of NF-kB, that in turn increased iNOS, IL-1beta, IL-6 and MMP-9 expression and NO levels. The addition of curcumin at increasing concentrations (5 and 10 μg/ml) decreased NF-κB activation and significantly reduced, iNOS, IL-1beta, IL-6, MMP-9, and NO levels in a dose-dependent manner (P<0.01 and P<0.001 respectively). Furthermore, in thyrocytes curcumin significantly restored the mRNA expression of Tg and NIS, decreased after exposure to 6-mer HA. Curcumin only slightly reduced CD44 expression (P<0.05) and did not change TLRs levels, suggesting that its anti-inflammatory effect mainlly depends on the inhibitory effect on NF-kB activation.

Conclusions: Curcumin attenuates the pro-inflammatory effects of HA oligosaccharides in both thyrocytes and fibroblasts. Since HA fragments might contribute to inflammation in both thyroidal and extra-thyroidal (i.e. dermal and orbital) tissues in the course of AITDs, curcumin could be beneficial in these disorders as a suitable adjunct to conventional pharmaceutical therapy.