Among the patients who visited our sleep disorders centre with a clinical suspicion of OSAHS, 46 male patients were enrolled in the study. All subjects underwent full-night polysomnography (Artisan, Rembrandt, USA), and 37 patients were diagnosed as having OSAHS, with an apnoea-hypopnoea index (AHI) > 5 (mild:AHI 5 ~ 15, n = 10; moderate: AHI 15 ~ 30, n = 11; severe: AHI > 30, n = 16). The AHI is the average number of apnoea and hypopnoea episodes per hour of sleep. No patient had previously been diagnosed with or treated for OSAHS. All subjects were free of any acute or chronic infection, cardiovascular diseases, and cancer and were taking no medications. This study was approved by the Ethics Committee of the First Affiliated Hospital of the Zhejiang University School of Medicine, Zhejiang University. Written informed consent was obtained from all subjects.
The presence and severity of OSAHS were determined by standard overnight polysomnography using electroencephalogram (EEG), electrooculogram (EOG), electrocardiogram (ECG), chin electromyogram, and measurements of oxygen saturation, airflow, and costal and abdominal movements when breathing. Apnoea was defined as a ≥90% decrease in airflow for at least 10 s relative to baseline. Hypopnoea was defined as a ≥50% decrease in airflow relative to baseline, with an associated ≥3% oxygen desaturation lasting at least 10 s . As measured by AHI, there were three levels of OSAHS: mild (5 ≤ AHI < 15), moderate (15 ≤ AHI < 30), and severe (AHI ≥ 30).
Measurement of IL-17A and IL-17 F levels
Blood samples were obtained from the antecubital vein on the morning after polysomnography and then centrifuged immediately at 3,000 rpm for 10 min. Plasma samples were stored at −80°C until use. The IL-17A and IL-17 F levels were determined quantitatively using a platinum enzyme-linked immunosorbent assay (eBioscience, San Diego, CA). The minimum detectable doses of IL-17A and IL-17 F were 0.23 pg/ml and 31.3 pg/ml, respectively, and the intra-assay coefficient of variation was <10%.
Because we found a correlation between the serum level of IL-17 and OSAHS, we conducted follow-up visits with the study subjects to conduct additional research. However, only 17 subjects received a follow-up appointment and participated further in our research.
Intracellular cytokines were studied by flow cytometry. FITC-labelled anti-human CD4 (eBioscience) and PE-labelled anti-human IL-17 (eBioscience) were used to detect the Th17 cells. PBMCs, obtained from heparinised peripheral whole blood (400 μl), were added to 1 ml of 1640 medium (PAA Laboratories, Pasching, Austria) and incubated for 6 h at 37°C with 5% CO2 in the presence of 50 ng/mL phorbol myristate acetate (PMA) (BioVision, Mountain View, CA), 500 ng/mL ionomycin (Fermentek, Jerusalem, Israel), and 1 μl of GolgiPlug (eBioscience). The cells were then stained with FITC-labelled anti-human CD4. After fixation and permeabilisation, the cells were stained with PE-labelled anti-human IL-17. Finally, the stained cells were analysed by flow cytometry using a FACS Calibur (BD Biosciences), and the results were analysed with CellQuest software (BD Biosciences).
Measurement of pulmonary arterial pressure (PAP)
Doppler echocardiography, using Vivid 7 (GE Vingmed Ultrasound, Horten, Norway) with a 2.5-MHz transducer (Accuson Cypress, Siemens, Germany), was performed by an experienced sonographer blinded to the study. The peak velocity of the tricuspid regurgitation jet was used to assess the PAP.
Statistical analysis was performed using SPSS statistical software (SPSS, version 16.0 for Windows; SPSS Inc., Chicago, IL). The data were analysed using the Kruskal-Wallis and Mann–Whitney tests. The Pearson test was used to assess the correlation between variables. Differences were considered significant at p < 0.05.