Objective: To investigate the effects of pirfenidone on orbital fibroblasts (OFs) from patients with thyroid-associated ophthalmopathy (TAO) and its underlying mechanisms. Methods: OFs from patients with TAO were isolated and cultured in DMEM. Cells were divided into four groups and treated with 0, 250, 500 and 1 000 μg/ml pirfenidone for 24, 48 or 72 hours, respectively. Cell proliferation was detected by tetramethyl azo salt (MTT) assay, and cell viability was determined by trypan blue. Transforming growth factor (TGF) β1 mRNA level was determined by real-time fluorescence quantitative PCR (RT-qPCR). Type Ⅰ and type Ⅲ collagen secreted from cultured cells were measured by enzyme-linked immuno sorbent assay (ELISA). Results: (1) The primary cultured OFs had typical fibroblast spindle-like morphology. (2) MTT assay showed that pirfenidone treatment significantly inhibited the proliferation of OFs in a dose-dependent manner (P<0.05) with the proliferation rates of pirfenidone treated groups of -15.31%, -24.92%, -48.53% from 250, 500, 1 000 μg/ml after 72 h, respectively, in which the inhibition effect of 1 000 μg/ml pirfenidone was significantly different from the other two treated groups (P<0.05). There were no significant differences in the inhibitory effect of the same concentration group among different time points at 24 h, 48 h and 72 h (P>0.05). Trypan blue showed that the survival rate of OFs in different concentrations of pirfenidone from 0,250, 500, 1 000 μg/ml at 72 h were 78.37%, 79.21%, 78.24% and 76.28%, respectively. There were no significant differences between each drug treated and the control group (P>0.05). (3) RT-qPCR results showed that the mRNA expression levels of TGFβ1 at 250, 500, 1 000 μg/ml pirfenidone treated groups at 72 h were 0.760±0.010, 0.440±0.006, and 0.290±0.002, respectively. Compared with the control group (0.950±0.014), the differences were statistically significant (all P<0.05). Moreover, TGFβ1 mRNA expression level in 1 000 μg/ml pirfenidone treated group was significantly lower than those in the other two treated groups (all P<0.05). The secretion of type Ⅰ collagen (0.633±0.006, 0.527±0.003 and 0.402±0.008) and type Ⅲ collagen (0.511±0.003, 0.439±0.007 and 0.223±0.006) in 250, 500 and 1 000 μg/ml pirfenidone treated groups at 72 h were significantly lower than those in the control group (0.794±0.005, 0.527±0.007, all P<0.05). Type Ⅰ and type Ⅲ collagen secretion in 1 000 μg/ml pirfenidone treated group were significantly lower than those in the other two groups (P<0.05). Conclusions: Pirfenidone inhibits the cell proliferation, TGFβ1 expression and collagen secretion of OFs, which may contribute to the anti-fibrotic effect of pirfenidone.
目的： 观察吡非尼酮对甲状腺相关眼病（thyroid associated ophthalmopathy，TAO）患者眼眶成纤维细胞（orbital fibroblast，OF）功能的影响及相关机制。 方法： 体外培养TAO患者OF，不同浓度（0、250、500、1 000 μg/ml）吡非尼酮加入OF培养液中分别培养24、48、72 h，四甲基偶氮唑盐（MTT）比色法检测吡非尼酮对OF增殖的影响，台盼蓝染色法检测细胞存活状态。实时荧光定量PCR（RT-qPCR）检测转化生长因子β1（transforming growth factor β1，TGFβ1）的表达，酶联免疫吸附法（ELISA）检测吡非尼酮对Ⅰ、Ⅲ型胶原蛋白分泌的影响。 结果： （1）TAO眼外肌来源成纤维细胞原代培养成功并稳定传代；（2）各吡非尼酮药物浓度组均可抑制OF的增殖，250、500、1 000 μg/ml吡非尼酮处理72 h后各组增殖率分别为-15.31%、-24.92%、-48.53%，与对照组（0）比较差异均有统计学意义（P值均<0.05），1 000 μg/ml浓度组抑制作用与其他两组差异有统计学意义（P<0.05），同一浓度组作用不同时间的抑制作用差异无统计学意义（P>0.05）。250、500、1 000 μg/ml吡非尼酮处理72 h后成纤维细胞的存活率分别为79.21%、78.24%、76.28%，对照组细胞存活率为78.37%，差异无统计学意义（P>0.05）。（3）RT-qPCR结果显示，250、500、1 000 μg/ml吡非尼酮处理后各浓度组TGFβ1的mRNA相对表达量分别为0.760±0.010、0.440±0.006、0.290±0.002，与对照组（0.950±0.014）相比差异均有统计学意义（P值均<0.05），吡非尼酮1 000 μg/ml组TGFβ1 mRNA表达水平明显低于其他两试验组（P<0.05）。各浓度组不同时间段比较差异无统计学意义（P>0.05）。ELISA结果显示，250、500、1 000 μg/ml吡非尼酮处理72 h后各浓度组Ⅰ型胶原蛋白分泌量分别为0.633±0.006，0.527±0.003，0.402±0.008，Ⅲ型胶原蛋白的分泌量分别为0.511±0.003，0.439±0.007，0.223±0.006，均低于对照组（0.794±0.005，0.527±0.007，P值均<0.05），吡非尼酮1 000 μg/ml组Ⅰ、Ⅲ型胶原蛋白的分泌量明显低于250、500 μg/ml组（P<0.05）。同浓度组不同时间段比较，Ⅰ、Ⅲ型胶原蛋白的分泌量无明显变化，差异无统计学意义。 结论： （1）组织块贴壁法可成功培养TAO眼外肌来源成纤维细胞；（2）吡非尼酮抑制体外培养的TAO患者OF的增殖、TGFβ1的表达及胶原蛋白的分泌，吡非尼酮的抗纤维化作用可能与其相关。.
Keywords: Orbital fibroblast; Pirfenidone; Thyroid; Transforming growth factor.