TSG-6 protects orbital fibroblasts via anti-inflammation and anti-fibrosis effects in thyroid eye disease

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Tumor necrosis factor-α-stimulated gene-6(TSG-6), a secreted protein with anti-inflammatory and tissue-protective properties, mediates a cascade of proinflammatory cytokines and ameliorates tissue fibrosis. Previous studies have found that TSG-6 can attenuate the degree of fibrosis, inhibit the inflammatory response, and reduce adipogenesis in orbital tissues in a Thyroid Eye Disease (TED) mouse model of thyroid-eye disease; however, the exact mechanism has not been elucidated. In the present study, we investigated the mechanism by which TSG-6 exerts its anti-inflammatory and antifibrotic effects in an in vitro cellular model of TED. Human orbital connective tissue was collected from primary and passaged cultures and 3-5 passages-cells were used in subsequent experiments. The expression of relevant inflammatory markers, including tumor necrosis factor-α(TNF-α), Interleukin-6(IL-6), monocyte chemoattractant protein-1(MCP-1), Cyclooxygenase-2(COX-2), and intercellular cell adhesion molecule-1(ICAM-1), was detected by western blotting with 5, 10, and 15 ng/ml TSG-6 pretreatment in the presence or absence of 10 ng/ml Interleukin-1beta (IL-1β) and H2DCFDA (DCFH-DA) fluorescent staining, and flow cytometry was used to detect reactive oxygen species(ROS) indices. Orbital fibroblasts (OF) were treated with TSG-6 in the presence or absence of transforming growth factor-beta 1(TGF-β1) agonist (SRI-011381:MCE, HY-100347) and the expression of TGF-β1/Smad pathway-associated fibrosis factors, including α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and collagen type 1 (COL1A1). TSG-6 inhibited IL-1β-induced production of the inflammatory mediators TNF-α, IL-6, MCP-1, COX-2, and ICAM-1, and the release of ROS in a dose-dependent manner in an in vitro cell model of TED. TSG-6 downregulated the expression of TGF-β1, Smad2/3, p-smad2/3, α-SMA, CTGF, and COL1A1 in a dose-dependent manner after TGF-β1 pretreatment and reduced the phosphorylation of Smad2/3, suggesting that TSG-6 inhibits the TGF-β1 signaling cascade response in orbital fibroblasts. TSG-6 inhibited the production of inflammatory mediators and the release of ROS, and suppressed fibrosis of human orbital fibroblasts by downregulating the TGF-β1/Smad pathway. These data suggest a potential application of TSG-6 in the treatment of TED and provide a novel target for the treatment of TED.

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