Preparation of the medium containing hydrogen
Before initiation of the experiment, high-glucose DMEM was injected into an aluminium bag and stored in the refrigerator at 4℃. Using a high-pressure ventilation apparatus, hydrogen was infiltrated at 0.4MPa for 6 h until it dissolved completely to a saturation state in the medium with a concentration >0.6mmol/L. hydrogen was supplemented every three days to maintain the saturation concentration. The prepared hydrogen medium was stored in the refrigerator at 4℃.
Experimental grouping
PC12 cells in logarithmic growth phase were spread to a 24-well plate at a density of 1×104 cells/well. Confluent cells were divided into a normal control (NC) group, in which cells were normally cultured in 10% FBS+DMEM under 37℃and 5%CO2 conditions; a positive control (PC) group, in which cells were deprived with OGS for 12 h and restored with OGS for 1 h without intervention; and a hydrogen intervention (HI) group, in which cells were exposed to OGS for 12 h and restored in hydrogen-based medium for 1 h.
DAPI staining
The prepared cells in the three groups were washed with 1 x phosphate buffered saline (PBS) twice, fixed in 4% paraformaldehyde for 10 min, washed with 1×PBS twice, membrane-ruptured with 0.1% Triton X-100 for 10min, washed with PBS twice, added with 10ng/ml DAPI, cultured away from light at room temperature for 20 min, washed with PBS twice, observed in 6 randomly selected visual fields under a fluorescence microscope and photographed through the ultraviolet channel at 30ms of exposure time, whose magnification was x 400. Six pictures were selected from each group to calculate the cell apoptosis rate by counting the total number of cells and apoptotic cells.
Cell viability assay
PC12 cells in logarithmic growth phase were digested in 0.25% pancreatin. After centrifugation and counting, cells were spread to a 96-well plate at a density of 5×103 cells /well and cultured in a 37℃ 5%CO2 incubator for 24 h. After cells were treated as designated in each group, 10uL CCK8 was added to each well and cultured at 37℃ for 1 h. Optical density (OD) of each well was measured at 450nm to calculate the relative cell viability.
Detection of reactive oxygen species (ROS)
PC12 cells in logarithmic growth phase were digested in 0.25% pancreatin. After centrifugation and counting, cells were spread to a 6-well plate at a density of 2.5×105 cells /well and cultured in a 37℃ 5%CO2 incubator for 24 h. Cells in each group were harvested, added with H2DCF-DA probes and cultured away from light at 37℃ for 30 min. After removing the H2DCF-DA probe diluent, cells were washed with PBS, digested in 0.25% pancreatin and centrifuged at 500xg for 3min. Probes that were not mounted to the cells were washed off with serum-free medium thoroughly. Cells were re-suspended with serum-free medium, and fluorescence was collected and detected through the FL1-A channel of the flow cytometer.
Changes in endoplasmic reticulum (ER)-related signaling pathway proteins p-PERK, p-eIF2a, ATF4 and apoptosis-associated proteins
After activation of the ER apoptosis pathway protein PERK-eIF2a-ATF4, the protein expression levels of p-PERK, p-eIF2a and ATF4 were detected by Western-blot to compare differences in protein content between the three groups. Simultaneously, the relative content of caspase-12, cleaved-caspase-3 and CHOP/GADD153 in cells was compared between the three groups.
Statistical methods
All data are expressed as the mean ± standard deviation ( ±SD). Data treatment and statistical analyses were performed using SPSS 13.0 Software. Statistical treatment was performed using One-way ANOVA. Values of P<0.05/P<0.01 were considered statistically significant.