Cell culture and transfection
Human neurons were obtained from the BeNa Culture Collection (BNCC, Beijing China) and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) containing 10% fetal bovine serum (FBS) and 1% Penicillin and Streptomycin. Cells were grown at 37°C in 95% air and 5% CO2. RNA interfering assays were carried out by transfecting neuronal cells with siRNA designed against Nrf2 (si-Nrf2). Transfection was carried out using a Lipofectamine2000 (Thermo Fisher Scientific, Waltham, USA). The primer sequences were as follows, si-Nrf2-forward-5’-GAGACUACCAUGGUUCCAA (dTdT)-3’, si-Nrf2-reverse-5’-UUGGAACCAUGGUAGUCUC (dTdT)-3’; si-NC-forward-5’-CCUACGCCACCAAUUUCGU-3’, si-NC-reverse-5’-ACGAAAUUGGUGGCGUAGG-3’.
Construction of a model for OGD/R
We constructed an OGD/R model in accordance with previous studies . Neurons (5×105 cells/ml) were then incubated at 37°C in 95% air and 5% CO2 for 24 h. Thereafter, neurons were maintained in glucose-free Dulbecco's Modified Eagle's Medium (DMEM) under hypoxic conditions (3% O2, 5% CO2 and 92% N2) for 8 h. Thereafter, the medium was replaced with fresh medium containing glucose (20%). Neurons were then cultured for 24 h under normal conditions. An additional group of neurons were cultured in normal media and under normal conditions to form a control group.
The Cell Counting Kit-8 (CCK-8) assay
Cell survival was evaluated at 24 h post-OGD/R induction by performing a CCK-8 assay. In brief, 1×104 cells were seeded into 96-well plates and cultured over night at 37°C. The following morning, 10 µl of CCK-8 solution (Beyotime Biotechnology) was added into each well and cultured at 37°C for another 2 h. Finally, cell survival was calculated by detecting the absorbance at 450 nm with a microplate reader (BMG Labtech GmbH).
Co-IP assays were conducted using a method that was described previously . In order to perform Co-IP for α2-AR and/or Nrf2 proteins, we used anti-α2-AR and anti-Nrf2 agarose beads to pull down α2-AR and Nrf2, respectively. Mouse or rabbit purified IgGs were used as negative controls. The supernatants were transferred to new microcentrifuge tubes for western blot analysis.
For western blotting, neurons were first homogenized in Radio-Immunoprecipitation Assay (RIPA) buffer. Then, equal amounts of total protein were separated by 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. Membranes were then blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and probed with primary antibodies against Nrf2 (ab62352), HO-1 (ab13243), NQO-1 (ab28947), SOD (ab80946), Bax (ab32503), Bcl-2 (ab185002), Cleaved Caspase 3 (ab2302), α2-AR (ab198394) and GAPDH (ab181602). Thereafter, proteins were probed with species-specific HRP-conjugated secondary antibodies. All antibodies were obtained from Abcam company (USA). Immunoreactive bands were visualized using enhanced chemiluminescence reagents (ECL) and the optical density of each band was quantified by mage LabTM Software (Bio-Rad, Hercules, CA, USA)
The detection of MDA, SOD and GSH-PX
Specific Activity Assay Kits (Cayman Chemical, USA) were used to determine the levels of MDA, SOD and GSH-Px in neurons. When the assays were complete, the absorbance at 450 nm was read via a spectrophotometric assay and a microplate reader (Syntron, USA).
TdT-mediated dUTP Nick-End Labeling (TUNEL) staining
The extent of apoptosis in neurons was using an In Situ Cell Death Detection Kit (TUNEL fluorescence FITC kit, Roche). Neurons were grown in 24-well culture plates. The neurons were then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 5 min on ice. Next, 50 μl of the TUNEL reaction was added to the samples and cultured for 60 min at 37°C. The nuclei were then stained with DAPI. TUNEL staining was then visualized by fluorescence microscopy (Leica, Germany).
All data analysis involved SPSS version 20.0 (Chicago, IL, USA) and all experiments were carried out in triplicate. Data are shown as mean ± standard deviation (SD). For comparisons between two independent groups, we used the Student's t-test. When comparing differences among three or more groups, we used one-way analysis of variance (ANOVA). Statistical significance was set to P < 0.05.