Tissues acquisition and cell culture.
The surgical specimens of 4 subjects with GO (GO1-GO4) during decompression surgery were recruited. The disease was in the inactive stage in all the subjects who had achieved stable euthyroidism for at least 6 months prior to the surgery by using methimazole. All the subjects were precluded from radiotherapy and systemic corticosteroids for at least 1 month before the surgery. The primary cultures of orbital fibroblasts were collected from the surgical orbital tissues in an aseptic technique. Briefly, the orbital tissues were minced and digested with a sterile phosphate-buffered saline (PBS) containing collagenase (130 U/ml) and dispase (1 mg/ml) in an incubator filled with an atmosphere of 5% CO2 at 37 °C [6, 17]. After 24 hours, the digested orbital tissues were collected, and the resuspended in the cultured medium containing 10% fetal bovine serum (FBS), penicillin G (100 U/ml) and streptomycin sulfate (100 µg/ml). Cultured orbital fibroblasts were recruited between the 3rd and 5th passages whereby the cell cultures at the same passage number were selected for the same set of experiments. All study protocols were approved by the Institutional Review Board of Taipei Veterans General Hospital (TVGH) and were designed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all the subjects.
Chemicals and antibodies.
The recombinant protein for human TGF-β1 (#P01137), the mouse monoclonal antibodies against human TIMP-1 (#MAB970) and TIMP-3 (#MAB973) were acquired from R&D Systems, Inc (MN, USA), respectively. The rabbit polyclonal antibodies against the CTGF (#ab6992), fibronectin (#ab2413) and α-SMA (#ab5694) were acquired from Abcam Inc. (Cambridge, UK). The mouse monoclonal antibodies against the p38 (#ab31828), p38 phosphorylation (p-p38, #ab4822), JNK (#ab208035), JNK phosphorylation (p-p38, #ab76572), ERK (#ab184699) and ERK phosphorylation (p-p38, #ab201015) were all acquired from Abcam Inc. (Cambridge, UK). The secondary antibodies against rabbit (#A5795) and mouse (#A9044) as well as GAPDH (#G5262) and β-actin (#A5441) were acquired from Sigma-Aldrich (St. Louis, MO, USA) .
Western blot analysis.
The protein was extracted from cells lysate with lysis buffer containing a protease inhibitor cocktail (Bioman, Taipei, Taiwan) as our previous study . An aliquot of 60–100 µg proteins was separated on 10% SDS-PAGE and blotted onto the PVDF membrane (Amersham-Pharmacia Biotech Inc., Buckinghamshire, UK). After blocking with 5% skim milk, the PVDF membrane was incubated with the primary antibody, followed by a horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG antibody. The protein expression signals were detected by an enhanced chemiluminescence detection kit (Amersham-Pharmacia Biotech Inc., Buckinghamshire, UK) according to the manufacturer’s instruction, and were quantified by ImageScanner III with LabScan 6.0 software (GE Healthcare BioSciences Corp., Piscataway, NJ, USA).
MMP-2 and MMP-9 enzyme activity assay.
The MMP-2 and MMP-9 enzyme activities in the culture medium were measured from InnoZyme™ Gelatinase Activity Assay kit (Merck KGaA, Darmstadt, Germany) by a fluorogenic method according to the product instruction . Briefly, 100 µl cultured medium were diluted in activation buffer, and incubated for 3 hours with thiopeptide substrate specific for type IV collagenases (MMP-2 and MMP-9). The released fluorescence of the cleaved substrate of MMP-2 and MMP-9 (ex: 320 nm; em: 405 nm) was monitored. Triplicate tests were performed for each reaction in a 96-well, and the relative fluorescence unit ratios to control set were plotted.
The SPSS software and Microsoft Excel 2019 statistical package were used for statistical analysis. The results from the one-way ANOVA followed by an L.S.D. test and the Student's t test were obtained from three independent experiments, respectively. Data were presented as means ± S.D. and p value less than 0.05 was considered statistically significant.