Antifibrotic effect of pirfenidone on orbital fibroblasts in patients with thyroid-associated ophthalmopathy and its mechanisms

Abstract

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.