Swainsonine Protects Human Thyrocytes from Fas-Induced Apoptosis: In vitro Study on Nthy-Ori 3-1 Cell Line.

Abstract

Background: The role of Fas is crucial for preserving immunotolerance. The mechanisms and roles of Fas/FasL signaling in the immune system are well understood, but the knowledge of how this process is regulated remains limited. Fas-mediated apoptosis is a way of thyrocyte elimination and thyroid destruction in Hashimoto's thyroiditis (HT). Proinflammatory cytokines, produced abundantly by immune cells in the thyroid in HT, stimulate the expression of Fas in thyrocytes, making them susceptible to apoptosis induced by FasL on the immune cell surface.

Purpose: The present study aimed to evaluate the impact of changes in Fas N-glycosylation on the death of human follicular thyroid cells of the Nthy-ori 3-1 line.

Methods: To induce thyrocyte apoptosis, an in vitro model was established. Cells were stimulated with IFNγ to express Fas and treated with the N-glycosylation inhibitors, kifunensine and swainsonine. Then apoptosis was induced by human recombinant FasL. MALDI-Tof mass spectrometry monitored kifunensine- and swainsonine-induced changes in N-glycosylation of Nthy-ori 3-1 thyrocytes, and cell death was analyzed using flow cytometry (annexin V staining, caspase 3/7 activity, TMRE mitochondrial membrane potential assay) and fluorescence microscopy (DAPI staining).

Results: We found that swainsonine reduces Nthy-ori 3-1 cell apoptosis, caspase 3 and 7 activity, and restores mitochondrial potential. DAPI staining showed a decreased rupture and fragmentation of Nthy-ori 3-1 cell nuclei in the presence of swainsonine. The protective effect of kifunensine was only shown in the TMRE assay and for the late apoptotic cells in annexin V⁺/PI⁺ staining.

Conclusion: Our study demonstrated for the first time the anti-apoptotic effect of swainsonine on follicular thyroid cell death through the Fas/FasL signaling pathway. This finding may have later applications in controlling Fas/FasL-induced thyrocyte apoptosis and preventing thyroid destruction in HT.