Ko JaeSang, Chae Min Kyoung, Lee Joon H, Lee Eun Jig, Yoon Jin Sook
Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea.
Myung-gok Eye Research Institute, Konyang University College of Medicine, Seoul, Korea.
Invest Ophthalmol Vis Sci. 2017 May 1;58(5):2544-2553. doi: 10.1167/iovs.16-20684.
To investigate the effect of sphingosine-1-phosphate (S1P) on fibrosis in orbital fibroblasts in Graves' orbitopathy (GO).
Orbital fibroblasts were cultured from orbital adipose/connective tissues of patients with GO and healthy control subjects. Effects of treatment with TGF-β and cigarette smoke extract (CSE) on S1P receptor (S1PR) messenger RNA (mRNA) and S1P expression were evaluated by real-time polymerase chain reaction and Western blotting. To evaluate the role of S1P in fibrosis, cells were pretreated with W146 (S1PR1 antagonist); JTE013 (S1PR2 antagonist); FTY720 (S1PR1 modulator); or 5C (sphingosine kinase-1 blocker) for 1 hour before stimulation with TGF-β, CSE, or IL-1β. Expression of fibrosis-related proteins (collagen Iα, fibronectin, and α-smooth muscle actin [SMA]) and tissue remodeling-related proteins (matrix metalloproteinases [MMPs] and tissue inhibitor of metalloproteinase [TIMP]-1) was then evaluated by Western blotting.
Expression levels of S1PR mRNA and S1P in GO orbital fibroblasts increased upon TGF-β and CSE treatment. Treatment with S1PR blockers and 5C inhibited TGF-β and CSE-induced expression of collagen Iα, fibronectin, and α-SMA, as well as IL-1β-induced expression of MMP-1, MMP-2, MMP-9, and TIMP-1. Exogenous S1P treatment without profibrotic stimulants upregulated collagen Iα, fibronectin, α-SMA, MMP-1, MMP-2, MMP-9, and TIMP-1 expression in a dose-dependent manner.
Blocking of S1PR activity and inhibition of S1P synthesis led to decreased expression of fibrosis and tissue remodeling-related proteins in primary cultures of orbital fibroblasts derived from patients with GO. Thus, modulation of S1P activity might have therapeutic potential in the suppression of fibrosis in GO.
研究1-磷酸鞘氨醇（S1P）对格雷夫斯眼病（GO）眼眶成纤维细胞纤维化的影响。
从GO患者和健康对照者的眼眶脂肪/结缔组织中培养眼眶成纤维细胞。通过实时聚合酶链反应和蛋白质印迹法评估转化生长因子-β（TGF-β）和香烟烟雾提取物（CSE）处理对S1P受体（S1PR）信使核糖核酸（mRNA）和S1P表达的影响。为了评估S1P在纤维化中的作用，在用TGF-β、CSE或白细胞介素-1β（IL-1β）刺激前1小时，用W146（S1PR1拮抗剂）、JTE013（S1PR2拮抗剂）、FTY720（S1PR1调节剂）或5C（鞘氨醇激酶-1阻滞剂）预处理细胞。然后通过蛋白质印迹法评估纤维化相关蛋白（I型胶原蛋白α、纤连蛋白和α-平滑肌肌动蛋白[SMA]）和组织重塑相关蛋白（基质金属蛋白酶[MMPs]和金属蛋白酶组织抑制剂[TIMP]-1）的表达。
TGF-β和CSE处理后，GO眼眶成纤维细胞中S1PR mRNA和S1P的表达水平升高。用S1PR阻滞剂和5C处理可抑制TGF-β和CSE诱导的I型胶原蛋白α、纤连蛋白和α-SMA的表达，以及IL-1β诱导的MMP-1、MMP-2、MMP-9和TIMP-1的表达。在没有促纤维化刺激物的情况下，外源性S1P处理以剂量依赖性方式上调I型胶原蛋白α、纤连蛋白、α-SMA、MMP-1、MMP-2、MMP-9和TIMP-1的表达。
阻断S1PR活性和抑制S1P合成导致GO患者来源的眼眶成纤维细胞原代培养物中纤维化和组织重塑相关蛋白的表达降低。因此，调节S1P活性可能对抑制GO中的纤维化具有治疗潜力。
