Patients & samples
Forty adult patients with severe asthma and twenty control subjects were recruited from the Affiliated Changzhou No.2 People’s Hospital of Nanjing Medical University. Patients with severe asthma were diagnosed based on the ERS/ATS guidelines. The inclusion criteria were as follows: (1) FEV1≥40% predicted, and FEV1≥55% predicted after bronchodilator; (2) less than 6 times of acute exacerbation of asthma in the last 6 months; (3) no hospitalization for asthma in the last 6 months; (4) no tracheal intubation due to asthma in the last 1 year; (5) oral administration of prednisolone less than 20mg per day; (6) non-smoking patients. Participants with cardiac insufficiency, abnormal liver function, pulmonary embolism, coinfection, tuberculosis and blood system diseases were not included. At the period of drawing blood, all enrolled patients were in a stable clinical condition without acute attack of asthma. Severe asthmatic patients were randomly divided into 2 groups: DXM group (n=20) and GSPE+DXM group (n=20). Twenty healthy volunteers had no history of asthma, no other allergic and immune system diseases, and were non-smokers. The detailed information of all subjects was displayed in Table 1. Heparinized peripheral venous blood from each subject was collected in a sterile vacuum tube between 7:00 and 9:00 AM.
Cell separation and culture
PBMCs were enriched by Ficoll-Hypaque (TBD, Tianjin, China) gradient centrifugation within 4 hours. 10ml whole blood was transferred into 50ml centrifuge tube, added 10ml PBS solution to dilute. Two 15ml centrifuge tubes were added 5ml Ficoll solution, then the diluted blood was gently added to the upper ficoll of the two centrifuge tubes (each centrifuge tube 10ml each diluted blood), centrifuged at 2000rpm for 20min. PBMC cells layer was white. The cells could be sucked into another clean 15ml centrifuge tube with a pipette, added PBS to 15ml, centrifuged at 1500rpm for 15min. The supernatant was removed after centrifugation, then the medium was added for the same operation. The cell viability determined by trypan blue exclusion assay was greater than 95%. PBMCs were cultured in RPMI 1640 (Gibco, Invitrogen, UK) containing 10% foetal bovine serum and 1% penicillin, streptomycin. The diluted PBMCs were distributed in 1ml aliquots into 24-well plates (1.0×105/well) and incubated at 37°C with 5%CO2. GSPE (50μg/ml) were added to the plates of GSPE+DXM group. After 16 hours, dexamethasone (DXM, 1μM) was added to the culture medium. Following 1 hour incubation to allow adherence, the cell culture supernatants and cell pellets were collected and stored at -80°C for further detection. The PBMCs of control group were cultured alone without any reagents.
Enzyme linked immunosorbent assay (ELISA)
The levels of interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 were detected by ELISA kit (Biolegend, USA) according to the manufacturer’s instructions. The independent experiment was repeated 3 times.
Quantitative real-time PCR (qRT-PCR)
Total RNA from human PBMCs was extracted using Qiagen RNeasy Kit (Qiagen, Valencia, CA, USA). The cDNA was synthesized by reverse transcription using random primers and MultiScribe Reverse Transcriptase (Applied Biosystems, Foster City, CA, USA). qRT-PCR was carried out on ABI 7000 Taqman system (Applied Biosystems, Foster City, CA, USA). Nrf2, GCLM, iNOS and HDAC2 primers and probes were purchased from Applied Biosystems (Foster City, CA, USA). PCR reaction conditions were: pre-denaturation at 95°C for 3min, denaturation at 95°C for 15s, annealing at 60°C for 30s, 40 cycles in total, and dissolution curve analysis at 60°C for 10s and 95°C for 10s. Three multiple Wells were set for each group and β-actin was used as housekeeping gene. The sequences of primers of each target gene were shown in Table 2.
Protein extraction and Western blot
Total protein extracts from PBMCs were harvested using Qproteome Mammalian Protein Prep Kit (Qiagen, German). Protein concentrations were measured using the BCA protein assay (Beyotime, Shanghai, China). We loaded 15ug samples per lane on 10% SDS-PAGE gel for separation, and then transferred them to a PVDF membrane and incubated in blocking solution (5% skimmed milk in Tris-buffered saline) for 2h at room temperature. After washing three times with TBS and Tween (0.1% Tween-20, 100mM Tris-HCl, and 150mM NaCl, pH7.5), the membrane was incubated with the first antibodies of Nrf2, GCLM, iNOS and HDAC2 (1:1000 dilution) at 4°C overnight with gentle shaking. After complete elution, the membrane was incubated with HRP-conjugated secondary antibodies (1:5000 dilution, 2.5% BSA, PBS, 0.1% Tween-20) for 1h at room temperature. The proteins were quantified using ECL Plus Substrate (Amersham Biosciences) with β-actin as the gatekeeper protein.
Determination of GSH and Nrf2-antioxidant response element (ARE) binding ability
GSH was measured by using Glutathione Fluorometric Detection Kit (Biovision, Mountain View, CA, USA), and Nrf2-ARE binding ability was measured by using TransAM Nrf2 Transcription Factor ELISA Kit (Active Motif, Rixensart, Belgium) according to the manufacturer’s instructions.
Differences among each group were accessed by one-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparison tests. Data were expressed as the mean ± standard error of the mean (SEM). A P value <0.05 was considered statistically significant. All calculations were carried out by using SPSS 17.0 software.