To investigate the effect of tumor necrosis factor-α (TNF-α) on the differentiation of orbital fibroblasts (OF) in thyroid-associated ophthalmopathy (TAO) and its regulation mechanism.

Six patients (six eyes) diagnosed with TAO were collected in Tianjin Medical University Eye Hospital from December 2019 to August 2020.Adipose connective tissue was collected during the orbital decompression surgery.OF was isolated and cultured using the tissue block method and vimentin was identified by immunofluorescence.Lipogenic differentiation of OF was induced and identified by oil red O staining.Complete culture medium containing 0, 0.1, 1.0 and 10.0 μg/L TNF-α was used to induce the dedifferentiation of orbital mature adipocytes.Primary culturing cells, 14-day differentiation cells and 20-day dedifferentiation cells were collected.The relative mRNA expression levels of peroxisomal proliferation-activated receptor (PPARγ), extracellular regulatory protein kinase1 (ERK1), ERK2 and fat-coated protein1 (perilipin1) were detected by real-time fluorescent quantitative PCR.The relative protein expression levels of PPARγ, P-ERK1/2 and perilipin1 were detected by Western blot.

Human TAO-derived OF were successfully cultured in vitro, spindle-shaped or polygonal, tightly arranged in a vortex pattern, and immunofluorescence staining for vimentin was positive.After OF adipogenic differentiation, lipid droplet structures could be seen in the cytoplasm of some cells, and the stained lipid droplet structures in the cytoplasm could be seen by oil red O staining, which confirmed that the cells obtained after differentiation were adipocytes.Dedifferentiation of adipocytes was induced by 0.1, 1.0, and 10.0 μg/L TNF-α.With the extension of induction time, the volume of lipid droplets in the cytoplasm and the number of cells containing lipid droplets decreased.Lipid droplets disappeared in the cytoplasm on the 20th day of dedifferentiation, and the cells became long spindle-shaped and tightly arranged, dedifferentiated into fibroblast-like cells.Real-time fluorescence quantitative PCR detection results showed that the relative expression levels of PPARγ, ERK1, ERK2 and perilipin1 mRNA in 14-day differentiation group were 4.26±0.09, 2.01±0.09, 3.23±0.10 and 8.69±0.33, respectively, which were significantly higher than 1.00±0.09, 1.05±0.19, 1.00±0.10 and 1.05±0.07 in primary group, and 1.06±0.03, 1.15±0.11 and 6.27±0.09 in 20-day dedifferentiation group (all at P<0.05). Western blot analysis showed that the expression levels of PPARγ, ERK1/2 and perilipin1 proteins in 14-day differentiation group were 1.07±0.03, 1.00±0.03 and 1.13±0.02, respectively, which were significantly higher than 0.37±0.02, 0.29±0.02 and 0.00±0.00 in primary group, and 0.20±0.02, 0.38±0.06 and 0.00±0.00 in 20-day dedifferentiation group (all at P<0.001).

TNF-α has a dedifferentiation effect on TAO orbital adipocytes.The mechanism may be related to the downregulation of ERK1/2-PPARγ-perilipin1 signaling pathway.

Thyroid-associated ophthalmopathy; Orbital fibroblasts; Tumor necrosis factor-α; Dedifferentiation; PPARγ