Crocin Inhibits Orbital Fibroblasts Fibrosis in Thyroid-Associated Ophthalmopathy

To investigate the role of Crocin on proliferation, fibrosis, and migration of orbital fibroblasts, as well as the possible signaling pathway.

Immunofluorescence assay was performed to detect the expression of fibroblast marker proteins vimentin cytokeratin, desmin, and S-100. The quantity of 5‑ethynyl‑2'‑deoxyuridine-positive cells in orbital fibroblast was analyzed. Quantitative real-time PCR and western blots were performed to evaluate the expression level of fibrosis-related marker including alpha-smooth muscle actin, connective-tissue growth factor, collagen 1A1, and fibronectin. Scratch wound assays were performed to assess wound widths of orbital fibroblast. The expression and phosphorylation of extracellular signal-regulated kinase/signal transducer and activator of transcription 3 were evaluated using western blots. The phosphorylation of smad2 and smad3 was evaluated using immunofluorescence assay.

Crocin treatment reduced 5‑ethynyl‑2'‑deoxyuridine-positive cells, indicating inhibitory effect on orbital fibroblast proliferation. The expression levels of alpha-smooth muscle actin, connective-tissue growth factor, collagen 1A1 and fibronectin were declined in Crocin treatment. Delayed wound closures were observed in Crocin treatment. Furthermore, Crocin did not affect the expression of extracellular signal-regulated kinase and signal transducer and activator of transcription 3, but weakened extracellular signal-regulated kinase and signal transducer and activator of transcription 3 phosphorylation in orbital fibroblast. The phosphorylation of smad2 and smad3 was attenuated by Crocin as well.

In conclusion, Crocin inhibits the phosphorylation of extracellular signal-regulated kinase and signal transducer and activator of transcription 3, contributing to the inhibitory effect on proliferation, fibrosis, and migration of orbital fibroblast, suggesting that Crocin has potential to be a novel therapeutic candidate for thyroid-associated ophthalmopathy treatment.