Crocin Inhibits Orbital Fibroblasts Fibrosis in Thyroid-Associated Ophthalmopathy.

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

METHODS: 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.

RESULTS: 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.

CONCLUSION: 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.