Subjects
A total of 16 patients who were diagnosed with TAO based on Bartley’s criteria[19] ,
were enrolled in the present study. We used the Bartalena Standard classification[20] for moderate-to-severe selection of TAO. Standard ophthalmic examinations such as visual acuity, slit-lamp examination, fundus examination, tear film break-up time, fluorescein staining and Schimer tests were performed for both TAO and control patients.
Exclusion criteria included patients suffering from ocular surface disorders, such as dry-eye syndrome, allergic ocular surface diseases or pterygium prior to development of TAO; a history of medication which may affect ocular surface parameters[21], such as rheumatoid arthritis drugs, topical anti-glaucoma drugs; wearing of contact lenses; patients with a history of steroid treatment or radiation therapy; and patients with other diseases that are related with TAO such as diabetes. Ophthalmopathy was evaluated based on the clinical activity score (CAS) followingcomplete examination of the eyes. Patients with CAS ≥ 3/7 were defined as having active phase TAO, whereas, CAS ≤ 2/7 were defined as having inactive phase TAO[22]. Based on these criteria, 16 patients were classified as active phase TAO, while,16 patients with inactive TAO were from the same patients with active TAO after corticosteroid treatment. In addition, 18 age-matched and sex-matched healthy volunteers were enrolled as the control group. There was no signifificant difference between the ocular parameters and disease course in our experiments.
The study was approved by the Ethics Committee of Inhuman Hospital affiliated
with Shanghai Jiaotong University School of Medicine, Shanghai, China. All experiments were performed in strict accordance to the Declaration of Helsinki. Informed consent was obtained from each participating patient prior to taking part in the study.
Tear collection and protein extraction
Unstimulated tear samples were collected from 32 eyes of the 16 TAO patients and 36
eyes of 18 healthy volunteers using disposable 10μl microcapillary pipettes (Microcap 10μl; Drummond Scientific, Broomall, PA, USA) via the inferior meniscus. Tear samples were placed into 0.5 ml Eppendorf tubes and stored at -80℃ for subsequent investigation.
Eotaxin-1 concentration was measured using Human Eotaxin-1 ELISA Kit (Abcam, UK) according to the manufacturer’s instructions. The samples were divided into three groups: 16 patients with active TAO, 16 patients with inactive TAO-derived from the active TAO patients after corticosteroid treatment, and 18healthy volunteers. For the experiment, tear samples were diluted 10 times with assay diluents. A total volume of 100μl from each sample was used for the assay.
Detection of protein expression in tears via western blot assay
Western blot was performed according to standard procedures. Briefly, total protein
was quantified using BCA protein assay kit (Tiangen Biotech Co., Ltd, China) and equal amounts of protein was loaded into each well for separation via SDS-PAGE. Proteins were then transferred onto a PVDF membrane (Millipore, USA), blocked for
1 h at room temperature, then incubated with relevant antibodies at 4℃overnight. The
membranes were then incubated with the HRP-conjugated secondary antibody for 1 h
at room temperature and immunoreactive bands were reacted with ECL substrate and
visualized under chemiluminescence imaging analyzer (GE, USA). GAPDH was used
as the internal control to normalize protein loading. Eotaxin-1 primary antibodies was
purchased from Santa Cruz Biotechnology and used at 1:2000 dilution. Western blot images were analyzed using Quantity One software (Bio-Rad, USA).
Statistical analysis
Eotaxin-1concentration was expressed as mean ± standard derivation. Statistical analysis of the data was performed per multivariate ANOVA and Bonferroni’s adjusted test for multiple comparisons, automatically applied by computer program (System). The multiplication factor was calculated based on the number of comparisons applied. Statistical software (IBM SPSS Statistics 25.0; SPSS inc ChicaTAO, IL) was used for analysis, and P<0.05 was considered as statistically significant.