Antifibrotic effect of pirfenidone on orbital fibroblasts in patients with thyroid-associated ophthalmopathy and its mechanisms / 中华内科杂志

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 11Ⅲ 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 μ-g/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 11Ⅲ 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.

The effect of colchicine on the proliferation of human orbital fibroblast in thyroid associated ophthalmopathy in vitro / 中华实验眼科杂志

Background The orbital fibroblasts (OFs) in thyroid associated ophthalmopathy (TAO) play important roles in the proliferative and inflammatory response.Seeking the drug which inhibit OFs growth is of a vital significance for the prevention and treatment of TAO.Research documented that colchicine has an anti-fibrosis effect.But its influence on OFs of TAO patient is few known.Objective This study was to investigate the effect of colchicine on growth and apoptosis of OFs in vitro.Methods The retroobital connective tissue was obtained form 3 TAO patients and cultured using explant method.OFs were passaged and identified by immunochemistry,and 3-8 genetaions of cells were used in the study.Colchicine at the concentrations of 1 × 10-8,1 × 10-7,1 × 10-6,1 × 10-5,1 ×10-4 mol/L was added into the RPMI 1640 with 10％ fetal bovine serum(FBS) to incubated the cells for 24,48 and 72 hours respectively,and only RPMI 1640 was used to culture the cells as the control group.Cell counting kit-8 (CCK-8)was used to detect the absorbance value (A450) of OFs for the evaluatuion of OFs and the inhibitory rate of colchicine to OFs.The colchicine of 1 ×10-6,1 ×10-5,1 × 10-4 mol/L was added into the culture medium for 48 hours,and then the apoptotic rate of the cells and the cell percentage in various cellular cycle was assayed by flow cytometry(FCM).The expression of transforming growth factor-β (TGF-β)in the cells was detected by immunochemistry to assess the influence of colchicine on the serection of the cells.Results Cultured cells showed the spindle-like in shape and the cell number was significantly increased with the incubation time.After incubated with 1 × 10-4,1 × 10 5,1 × 10-6,1 ×10-7,1 × 10-8 mol/L colchicines,the A450 values were gradually reduced with the increase of the concentrations of colchicine(F ion =62.004,P＜0.05),and significant differences were found between different contrations of colchicine groups(all P＜0.05).Aslo,gradually declined A450 values of the cells were seen with the lapse of culture time among the groups(Ftime =459.582,P＜0.05).The inbitory rate of colchicine to the cells was elevated with the increase of concentrations.The apoptotic rates of the cells were (1.73 ± 0.15) ％,(21.04 ± 4.56) ％,(31.84 ±6.21)％and(35.32±5.56)％ in the control group and 1 × 10-6,1 × 10-5,1 × 10 4 mol/L colchicine groups respectively,with statistically significant difference among the 4 groups (F =83.905,P＜0.05).With the increase of concentrations of colchicines,the cell percentage in G2 +M phase lessened gradually,showing significant difference among the control group and the 1 × 10-6,1 × 10-5,1 × 10-4 mol/L colchicine groups (F =20.443,P＜0.05).The expression of the TGF-β in the cells was (97.60± 2.09) ％ in the control group,and that in the 1 × 10-4 mol/L colchicine group was (44.43 ± 3.96) ％,presenting a significant difference between them (t =65.330,P ＜ 0.05).Conclusions Colchicine can induce apoptosis of OFs and inhibit the prolilferation of OFs in a time-and dose-dependent manner probably by decreasing the TGF-β secretion

The Antifibrotic Effects of alpha-Tocotrienols in Primary Cultured Orbital Fibroblasts from Thyroid-Associated Ophthalmopathy Patients

PURPOSE: To evaluate the antiproliferative and antifibrotic effects of alpha-tocotrienols in primary cultured orbital fibroblasts from thyroid-associated ophthalmopathy (TAO) patients. METHODS: Orbital fibroblasts were cultured (5 eyes from TAO patients, 5 eyes from normal patients) and classified into a control group, alpha-tocotrienol group and alpha-tocopherol group. The cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The proliferation of orbital fibroblasts was measured using the Click-iT(TM) assay. The collagen production of the control and alpha-tocotrienol groups was measured using a hydroxyproline assay. RESULTS: The alpha-tocotrienol and alpha-tocopherol groups showed no cytotoxicity up to 150 microm in orbital fibroblasts from TAO and normal patients. The proliferation of orbital fibroblasts from TAO and normal patients was significantly inhibited with alpha-tocotrienol at 80 microm and 120 microm. The collagen production of orbital fibroblasts from TAO patients was significantly inhibited with alpha-tocotrienol at 120 microm. CONCLUSIONS: The results from the present study indicate that non-toxic concentrations of alpha-tocotrienol have significant antiproliferative and antifibrotic effects on orbital fibroblasts from TAO patients.

Effect of Gangliosides Mixture on Differentiation of Orbital Fibroblasts into Adipocytes

PURPOSE: To investigate the role of gangliosides in the differentiation of orbital fibroblasts into adipocytes, a component in the pathogenesis of Graves' ophthalmopathy. METHODS: Orbital tissues were obtained during orbital surgery for subjects without Graves' ophthalmopathy or other inflammatory orbital disease, and orbital fibroblasts were primarily cultured from each obtained tissue. Morphological examination of orbital fibroblasts was performed after treatment with commercially available gangliosides mixture (Gmix) comprised of several subtypes. To determine the effect of Gmix on the differentiation of orbital fibroblasts into adipocytes and the differentiation-related genes, Oil Red-O staining and RT-PCR were performed. RESULTS: The treatment with Gmix induced the morphological changes, which at least in part were explained with the differentiation of orbital fibroblasts into adipocytes in accordance with the increase of mRNA level of genes known to be related to adipogenesis, whereas dermal fibroblasts and preadipocytes were irresponsive to the same treatment. CONCLUSIONS: The results from the present study suggest gangliosides may have a role in pathologic mechanisms of Graves' ophthalmopathy by the induction of differentiation of orbital fibroblasts into adipocytes.