Patients
We studied 102 eyes from 102 diabetics, who were complicated with dry eye and had more than an 8-year history of diabetes, seen at the Department of Ophthalmology, Affiliated Hospital of Yanbian University, from January 2018 through April 2018, and 117 eyes from 117 healthy control individuals. The DM group (54 women and 48 men; mean age: 52.03 ± 8.26 years; age range: 42–73 years) all met the diagnostic criteria for diabetes and diagnostic criteria for dry eye disease. The normal control group (60 women and 57 men; mean age: 55.12 ± 6.31 years; range: 40–69 years) had non-DM and non-DED. There was no significant difference in gender and age composition between the DM group and normal group (P = 0.078, P = 0.066). The institutional review board at Affiliated Hospital of Yanbian University approved this study, which followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from all patients after the nature and possible consequences of the study were explained.
The inclusion criteria and exclusion criteria for the normal group are based on the following. Inclusion criteria: no ocular irritation, no history of eye medication, Schirmer test (SIt) > 10 mm/5 min, tear film breakup time (BUT) >10 s, and no corneal fluorecein staining (CFS). Exclusion criteria: individuals who had an active ocular infection or inflammation, drug toxicity, contact lens wearing, ocular allergy, ocular surgery, and lid or lash abnormalities. Additionally, those with any ocular or systemic disease were excluded from this study.
The inclusion criteria and exclusion criteria for the DM group were based on the following : Inclusion criteria: 1. The diagnosis of diabetes was based on the criteria proposed by the 2014 American Diabetes Association (ADA) and included the following: (1) glycosylated hemoglobin (HbA1c) ≥ 6.5%, (2) fasting plasma glucose (FPG) ≥ 7.0mmol/L, (3) two hours postprandial blood glucose (2hPG) in oral glucose tolerance test (OGTT) ≥ 11.1mmol/L, and (4) blood glucose of patients with typical hyperglycemia or hyperglycemia crisis were randomly detected ≥ 11.1mmol/L. 2. A history of diabetes longer than 8 years. 3. The diagnosis of dry eye was based on the criteria included the following: (1) subjective symptoms: dryness sensation, foreign body sensation, burning sensation, red eye, many secretions, heavy sensation of the eyelids, visual fatigue, photophobia, lachrymation, itching sensation, eye pain, visual acuity fluctuation/ one or more of the first 6 symptoms needed to occur or persist on a regular basis, (2) BUT ≤ 5 s (+ +) or ≤ 10 s (+), (3) Sit ≤ 5 mm / 5 min (+ +) or ≤ 10 mm/ 5 min (+), and (4) CFS score ≥ 1. According to the above mentioned criteria, the patients subjective symptoms and positive findings of DED on ocular examination (1 strong positive finding or 2 positive findings) were diagnosed as having DED. Exclusion criteria: The DM group included patients who had no ocular diseases and systemic diseases affecting tear secretion other than dry eye disease and diabetes.
BUT
Sodium fluorescein filter paper was used to touch the lower eyelid conjunctival sac of the tested subjects. The time, in seconds, between the last complete blink and the appearance of the first corneal black spot was measured and recorded.
CFS
After the tear film rupture test was performed, corneal epithelium staining was observed, and the staining area was graded on anumerical scale of 0–3, with 0 representing no staining; 1, scattered in dot staining or less than 5 staining points; 3, high density spot or flake staining or massive or filamentous staining; 2, between 1 and 3. The score ranges from 0 to 12.
SIt
A standard Schirmer test strip was placed in the lateral canthus with the eyes closed for an additional 5 min. The test paper was taken out, and the length of wetting of the strip was measured using a millimeter scale. The intensity of its staining was compared with that of a color standard.
Dry eye symptom
All subjects finished completing the dry eye symptom questionnaire (Ocular Surface Disease Index, OSDI), which was created by the International Dry Eye Workshop, under guidance of the same researcher. There were 12 problems, with a score of 0~100, 0~12 representing no symptoms, 13~32 moderate symptoms, and 33~100 severe symptoms.
Conjunctival impression cytology
A piece of 6 mm nitrocellulose filter (Mil-lipore) was applied tightly to the upper palpebral conjunctiva of the subject for approximately 5 s. Then, it was removed from the conjunctiva and attached to 5 × 5 mm size slides. The expression levels of NF-κB and TGF-β1 in the conjunctiva were observed by periodic acid-Schiff staining (PAS) and immunohistochemical staining.
Tear collection
Basal tear samples were collected atraumatically from the inferior tear meniscus of subjects using glass capillary tubes (Corning, New York, NY) or micropipettes (Eppendorf, Hamburg, Germany), and they were stored at –70℃ until further examination. Care was taken to not irritate the conjunctiva, cornea, or lid margin.
Preparation of single cell suspension
Collecting the conjunctiva of the two groups within 3 hours using conjunctival polyethersulfone filter membrane (cutting the membrane in half and placing it on the unexposed conjunctiva separately on the superior temporal side and superior nasal side). Then, the filter membranes of two groups were transferred into RPMI1640 cell culture fluid (which included 10% fetal bovine serum, 100 UI/ml penicillin, 100 UI/ml streptomycin, and 2 mM glutamate). The collected cells were immediately stored at 4°C to prevent degradation of the samples. To collect more cells from the membrane, each test tube was stirred for 10-15 min; Then, the cell suspension was removed, transferred to a clean test tube and centrifuged (1600 r/min, 8 min, 4°C).
Western-blot
The expression levels of TGF-β1, NF-κB and interleukin-17 (IL-17) were detected by western-blot. The single cell suspensions of two groups that were prepared were washed 2 times by 4°C precooled phosphate buffer solution, and 400μl cell lysate RIPA was added. Then, the suspensions were cracked on ice for 30 min and centrifuged for 30min (12×103r.min-1). Then, the supernatant of the protein was collected and packed for storage. The sample protein was then subjected to polyacrylamide gel electrophoresis. This experiment was repeated three times.
Flow cytometric
The supernatant of the cell suspension was removed, and the heavy suspension was placed in 0.3 ml medium. Then, the cells were counted by flow cytometry. The same threshold was used for analysis on a FACS Calibur to avoid cell debris. The same voltage was used for the forward scattering (FSC) and lateral scattering (LSC). Each specimen was analyzed for 60 s.
Enzyme-linked immunosorbent assay (ELISA)
The IL-lβ, interleukin-6 (IL-6) and TNF-α levels in tears of subjects were measured using commercial ELISA kits (Quantikine; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Statistical analysis
SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used to analyze the data. The Mann–Whitney U test and Student's t were used to compare results between groups and Spearman correlation coefficients were calculated for the correlation between levels of inflammatory cytokines and parameters of tear film and ocular surface. P < 0.05 was considered statistically significant.