Chitosan inhibits inflammation and adipogenesis of orbital fibroblasts in Graves ophthalmopathy

Purpose: The aim of this study was to investigate the roles of chitosan in inflammation and adipogenesis of primary cultured orbital fibroblasts in Graves ophthalmopathy (GO). Methods: Cell viability, apoptosis, and cell cycle were determined with the Cell Counting Kit-8 (CCK-8), the Annexin V-FITC/PI kit, and flow cytometry, respectively. Inflammation of orbital fibroblasts was stimulated by interleukin-1 beta (IL-1 beta). The levels of IL-6 and prostaglandin E-2 (PGE-2) were measured using an enzyme-linked immunosorbent assay (ELISA). The expression of cyclooxygenase-2 (COX-2) was measured with real-time PCR and western blot assay. Phosphorylation of c-Jun N-terminal kinase (JNK) was evaluated with western blot assay. An inhibitor of JNK was used to investigate the signal transduction pathway of cytokine production. Orbital fibroblasts differentiated to adipose cells in differentiation medium Adipose cells were dyed with Oil Red O. FABP4, adiponectin, C/EBP alpha, PPAR-gamma, and phosphorylation of AKT were evaluated with western blot assay. Results: The results showed that IL-1 beta statistically significantly increased the expression of IL-6, PGE-2, and COX-2 in orbital fibroblasts Phosphorylation of JNK was promoted by IL-1 beta. IL-6 and PGE-2 were modulated by the JNK signaling pathway as determined with the inhibition experiments. Chitosan downregulated expression of IL-1 beta-stimulated IL-6, COX-2, and PGE-2 and downregulated phosphorylation of JNK. Chitosan inhibited the production of adipose cells dyed by Oil Red O. Chitosan statistically significantly decreased the protein levels of FABP4, adiponectIN, C/EBP alpha, and PPAR-gamma with downregulation of AKT phosphorylation during adipocyte differentiation. Conclusions: Chitosan statistically significantly inhibits inflammation and adipogenesis, as well as related signaling pathways, of orbital fibroblasts in GO. This indicates a possible therapeutic effect of chitosan on Graves ophthalmopathy.