Mouse HT-22 hippocampal were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) (both Gibco; Thermo Fisher Scientific, Inc.) and 1% antibiotics, at 37°C in a humidified incubator with 5% CO₂. Brusatol (BR, Sigma-Aldrich; Merck KGaA) at 0.3 µg/ml was used as an Nrf2/HO-1 pathway inhibitor.

The OGD/R model is widely recognized as a model for studying cerebral ischemia at present. By injecting N₂, the concentration of CO₂ and O₂ in the incubator can be precisely controlled. The glucose in the culture medium of neurons can be deprived, thus creating the hypoxic and glucose-deficient environment for culturing neurons and simulating cerebral ischemia in vivo. The treatment of HT-22 cells by ODG/R was conducted as previously described (9). The mouse hippocampal HT22 cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) in a normoxic 5% CO₂ cell culture incubator at 37°C. OGD/R HT22 cells were used as an in vitro model of CIRI. Briefly, HT22 cells were cultured in glucose-free DMEM and then placed in hypoxic conditions (1% O₂, 94% N₂, 5% CO₂) at 37°C for 2 h. Thereafter, the medium was discarded, normal DMEM with glucose was added and the culture was continued for 24 h of reoxygenation under normoxic condition (95% air, 5% CO₂) to produce OGD/R. HT22 cells cultured in growth culture medium under normoxic condition served as a control.

The CCK-8 kit (Dojindo Molecular Technologies, Inc.) was used to detect cell proliferation. Briefly, HT-22 cells were seeded in 96-well plates (2×10⁴ cells/ml) and after the corresponding treatment, CCK-8 solution (10 µl) was added to each well. Subsequently, the plates were incubated for 2 h at 37°C and the absorbance of each well was detected at 450 nm using a microplate reader (BioTek Instruments; Agilent Technologies, Inc.).

The amount of malondialdehyde (MDA, cat. no. BMS222), superoxide dismutase (SOD, cat. no. BMS222) and glutathione (GSH, cat. no. PA5-18651) was evaluated using ELISA kits (BioSource International; Thermo Fisher Scientific, Inc.) according to the manufacturer's instruction. The cell supernatant was centrifuged for 5–10 min at 4°C, at 5,000 × g.

The cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology) and incubated for 30 min on ice. Total protein was quantified using a BCA protein assay kit. Following extraction of the total protein from the cells, 10% SDS-PAGE was used to separate the proteins (30 µg) and transferred to PVDF membranes, which were blocked with 10% skimmed milk for 1 h at room temperature. Following overnight incubation at 4°C with specific primary antibodies, the membranes were washed three times and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat no. AA24142, Cell Signaling Technology, Inc.) for 2 h at room temperature. The protein levels were visualized using the Super signal West Pico Chemiluminescent substrate (Pierce; Thermo Fisher Scientific, Inc.). Protein expression levels were semi-quantified using ImageJ software (version 146; National Institutes of Health) with GAPDH as the loading control. The following primary antibodies from Cell Signaling Technology, Inc. were used: Anti-Bax (1:1,000; cat. no. 14796S), anti-caspase3 (1:1,000; cat no. 700128), anti-Bcl-2 (1:1,000; cat. no. 14-1028-82), anti-Nrf2 (1:1,000; cat. no. 12721T), anti-HO-1 (1:1,000; cat. no. 86806S), anti-cleaved caspase3 (1:1,000; cat. no. PA5-38438), anti-NOX2 (1:1,000; cat. no. MA5-18052), anti-NOX4 (1:1,000; cat. no. PA5-53304) and anti-GAPDH (1:1,000; cat. no. MA5-15738).

After fixing 4% paraformaldehyde at room temperature for 20 min, the cells were washed twice with PBS. Then, 0.2% Triton X-100 was added to the cells and treated at room temperature for 5 min. The cells were collected and washed with PBS three times, and treated with 50 µl TUNEL assay solution (Roche Diagnostics GmbH) at 37°C in dark for 60 min and added with stop solution, followed by incubation with DAB solution and staining with hematoxylin and eosin for 5 min at room temperature. The cells were randomly selected for field observation under a light microscope (Carl Zeiss AG, magnification, ×200). The experiment was repeated three times.

All data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS v22.0 statistical software (IBM Corp.) and one-way ANOVA with Tukey's multiple comparison post hoc test was employed for comparison among groups. P<0.05 was considered to indicate a statistically significant difference.

CCK-8 was used to detect the effects of different concentrations of DHM (0, 10, 30, 100 and 300 µmol/l) on cell viability and no differences were found (18,19) (Fig. 1A). Subsequently, the different aforementioned concentrations of DHM were used in OGD/R-induced HT22 cells and the CCK-8 results revealed that the survival rate of OGD/R-induced HT22 cells was significantly decreased compared with that of the control group. Furthermore, the cell survival rate of the DHM+OGD/R group was increased in a dose-dependent manner, compared with the OGD/R group (Fig. 1B). It was found that 300 µmol/l DHM could significantly promote cell proliferation of OGD/R-induced HT22 cells, and DHM had no cytotoxic effect on cells at this concentration. Therefore, 300 µmol/l DHM was used for the subsequent experimentation.

The level of cellular oxidative stress was then investigated. The cells were grouped into either control, DHM, OGD/R, DHM+OGD/R groups, and compared with the control group. No differences were observed in the amount of MDA (Fig. 2A), SOD (Fig. 2B), GSH (Fig. 2C) and the expression levels of the oxidative stress proteins, NOX2 and NOX4 (Fig. 2D) in cells treated with DHM alone, while there was a significant increase in the OGD/R group. This indicated that OGD/R successfully induced oxidative stress in HT22 cells. Compared with that in the OGD/R group, the levels of MDA, and the expression levels of the oxidative stress proteins, NOX2 and NOX4 in the DHM+OGD/R group were significantly decreased and the expression of SOD and GSH were increased, indicating that DHM could inhibit the oxidative stress of OGD/R-induced HT22 cells.

TUNEL staining was used to detect the rate of apoptosis in cells. Compared with that in the control group, there was no change in the rate of apoptosis following DHM treatment in HT22 cells alone, while there was a significant increase in that of the OGD/R group (Fig. 3A), an increase in the expression levels of the pro-apoptotic proteins, Bax and cleaved caspase-3, and a decrease in the anti-apoptotic protein, Bcl-2 (Fig. 3B). This indicates that OGD/R successfully induced apoptosis of HT22 cells. Compared with that in the OGD/R group, cell apoptosis in the DHM+OGD/R group was decreased, accompanied by the decrease in the expression levels of Bax and cleaved caspase-3, and the increase in Bcl-2; indicating that DHM inhibited the apoptosis of OGD/R-induced HT22 cells.

The expression of Nrf2 and HO-1 in the OGD/R group was significantly decreased compared with the control group, while the expression of Nrf2 and Ho-1 in the DHM+OGD/R group was reversed compared with the OGD/R group. In addition, compared with the control group, the expression of Nrf2 and Ho-1 in the DHM group was significantly increased. It showed that Nrf2/HO-1 signaling pathway was inhibited after OGD/R induction, and that DHM could reverse the inhibitory effect of OGD/R on signaling pathway (Fig. 4A), indicating that the Nrf2/HO-1 signaling pathway was activated following DHM treatment. Subsequently, the Nrf2/HO-1 signaling pathway inhibitor, BR was added to the cells, and the protein expression levels of Nrf2 and HO-1 were detected using western blot analysis (Fig. 4B). Subsequently, the cells were divided into control, DHM, OGD/R, DHM+OGD/R, and BR+DHM+OGD/R groups. Compared with that in the DHM+OGD/R group, the levels of MDA (Fig. 5A), and the protein expression levels of NOX2 and NOX4 (Fig. 5D) in the BR+DHM+OGD/R group were significantly increased, the expression of SOD (Fig. 5B) and GSH (Fig. 5C) were decreased, indicating that BR could reverse the effect of DHM on OGD/R-induced oxidative stress in HT22 cells. In addition, compared with that in the DHM+OGD/R group, the rate of apoptosis in the BR+DHM+OGD/R was also increased (Fig. 6A), which was accompanied by an increase in the protein expression levels of Bax and cleaved caspase-3 (Fig. 6B and Table I), and the decrease in Bcl-2 (Fig. 6B); indicating that BR could also reverse the effect of DHM on OGD/R-induced apoptosis of HT22 cells. The results indicate that DHM inhibited oxidative stress and apoptosis of OGD/R-induced HT22 cells by activating the Nrf2/HO-1 signaling pathway.