Gastric cancer SGC7901 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA), inoculated in Dulbecco's modified Eagle's (high glucose) culture medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Clark Bioscience, Richmond, VA, USA) and cultured in a 5% CO₂ incubator at 37°C.

Prior to the experiment, the hyperbaric chamber was disinfected using UV techniques for 20 min, and cleaned using pure oxygen for 10 min. The cells were placed flat in the chamber under aseptic conditions for 90 min each time. The pressure of HBO was increased slowly to 0.2 MPa within 15 min and, after 60 min, the pressure was decreased to normal pressure within 15 min. Then, the cells were taken out of the chamber and cultured in the CO₂ incubator. During the experiment, the HBO chamber was kept ventilated with 95% oxygen at a flow rate of 2 l/min.

The cells were split into two groups. HBO group: Cells at log phase were subjected to HBO treatment once a day. Control group: SGC7901 cells at a log phase of their growth were cultured in the CO₂ incubator without HBO treatment. When cells of HBO group were receiving HBO treatment, this group of cells were removed from the incubator and maintained at room temperature.

Following trypsinization, cells at log phase were resuspended and seeded in a 96-well cell culture plate at a density of 5×10³ cells/well. Following treating the cells with HBO, 20 µl MTT (5 mg/ml, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) solution was added into each well after 48 h. The cells were cultured for a further 4 h in the incubator. Then, the supernatant was abandoned and 100 µl DMSO (Sigma-Aldrich; Merck KGaA) was added to each well. Following oscillation in a constant temperature culture vibration machine at 37°C for 10 min until the crystal was completely dissolved, the absorbance value (A value) at the wavelength of 490 nm was measured at a enzyme-linked immunosorbent assay reader (ELX800; BioTek Instruments, Inc., Winooski, VT, USA). The experiments were performed in triplicate and results are represented as the average value of three independent experiments.

Following trypsinization, cells at log phase were resuspended and seeded in a 6-well cell culture plate at a density of 3×10⁵ cells per well. At 48 h following HBO treatment, radioimmunoprecipitation assay buffer (25 mM HEPES, 1.5% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.5 M NaCl, 5 mM EDTA, 50 mM NaF, 0.1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride and 0.1 g/l leupeptin, pH 7.8) was used for cell lysis and total protein extraction. The quantitative determination of total protein concentration of each group was made by the bicinchoninic acid assay method (cat. no. P0009; Beyotime, Institute of Biotechnology, Haimen, China). The same amount of protein (50 µg) of each group was loaded onto 12.5% SDS-PAGE gels. Electrophoresis separated proteins were then transferred onto polyvinylidene difluoride membranes. Following blocking with 5% skimmed milk, membranes were incubated overnight with the primary antibody against LC3-phosphatidylethanolamine conjugate (dilution, 1:500; cat. no. sc-134226; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), binding immunoglobulin protein (BiP; dilution, 1:400; cat. no. sc-1051; Santa Cruz Biotechnology, Inc.) or CCAAT-enhancer-binding protein homologous protein (CHOP; dilution, 1:500; cat. no. sc-7351; Santa Cruz Biotechnology, Inc.) at 4°C. Membranes were then washed at room temperature and incubated for 2 h with IgG-horseradish peroxidase conjugate (dilution, 1:10,000; goat anti-mouse IgG; cat. no. AP124P), goat anti-rabbit IgG (dilution, 1:10,000; cat. no. AP132P) and rabbit anti-goat IgG (dilution, 1:10,000; cat. no. AP106P) all obtained from EMD Millipore (Billerica, MA, USA). Following another washing step (any unbound secondary antibody was removed by washing), membranes were treated with ECL visualization reagents (Thermo Fisher Scientific, Inc.) in the dark room. β-actin was used as the internal control in the experiment. Protein band intensities were determined by using the Quantity One software (version, 4.6.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiment was repeated three times and results were averaged. The ratio of the target protein band intensity to that of the internal control in each group was also calculated.

All statistical analysis was calculated using SPSS 16.0 statistical analysis software (SPSS, Inc., Chicago, IL, USA). The difference between control group and treatment group was evaluated using Student's t-test. P<0.05 was considered to be statistically significant.

An MTT assay was used for measuring cell proliferation following HBO treatment. The result indicated that the OD₄₉₀ value of SGC7901 cells following HBO treatment was significantly higher than that of control group with statistical difference (Fig. 1; P<0.05), which suggested that HBO treatment may promote the proliferation of gastric cancer SGC7901 cells.

As presented in Fig. 2, the expression level of autophagosome marker protein LC3-II following HBO treatment was significantly increased when compared with the control group (Fig. 2B; P<0.05). The result demonstrated that HBO treatment may significantly stimulate the autophagy of gastric cancer cells SGC7901.

Western blot analysis was used to evaluate the expression levels of oxidative stress-associated proteins BiP and CHOP following HBO treatment. The results are presented in Fig. 3. The expression level of BiP was significantly increased when compared with the control group (P<0.05). Conversely, the level of CHOP was significantly decreased (P<0.05). These data suggested that HBO treatment may promote SGC7901 cell survival and inhibit cell apoptosis caused by oxidative stress.