Daphnetin protects hippocampal neurons from oxygen-glucose deprivation–induced injury

2.1 Cell culture

Ten Sprague-Dawley rats (Charles River, Beijing Vital River Laboratory Animal Technology Co., Ltd., China) were used for the preparation of primary hippocampal neurons as described previously.13 Briefly, cerebral hippocampi were separated from brains and dissected in HBS. After mechanical dissociation and trypsinization, the cells were plated in dishes that were pretreated with 0.1% poly-d-lysine (Sigma-Aldrich, St. Louis, MO). After incubating for 2 hours, cells were cultured in neurobasal medium supplemented with 2% B-27, 1% penicillin-streptomycin, and 0.25% glumax for 7 days. Hippocampal neurons were then purified by density gradient centrifugation, and identified by neuron-specific enolase immunofluorescence (data not shown). All experiments were carried out according to the National Institute of Health Guide for the Care and Use of Laboratory Animals.

2.2 Oxygen-glucose deprivation/reperfusion model and treatment

After 7 days, the primary hippocampal neurons were subjected to oxygen-glucose deprivation (OGD), through incubating in serum and glucose-free medium with 5% CO₂ and 95% N₂ at 37°C for 3 hours. Then the cells were incubated with normal medium containing serum and glucose in normoxic conditions for 24 hours. For the daphnetin treatment group, cells were pretreated with 10, 20, 40 μM daphnetin (≥97%, Sigma) for 12 hours before OGD/reperfusion (OGD/R) stimulation. The experiment was repeated three times.

2.3 Cell transfection

Small interfering RNA targeting Nrf2 (si-Nrf2; Invitrogen, Thermo Fisher Scientific, Waltham, MA) that can silence the complementary target messenger RNA of Nrf2 was transfected into the hippocampal neurons using Lipofectamine 2000 (Invitrogen). After 24 hours, the transfection efficiency was tested using Western blot analysis. The experiment was repeated three times.

2.4 Cell viability assay

Approximately 5 × 10³ cells/well hippocampal neurons were seeded in 96-well plates and incubated for 12 hours with or without the presence of daphnetin. Then, the cells were treated with OGD/R stimulation, and then cell viability was evaluated with the cell counting kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. Finally, the absorbance at 450 nm was measured with a microplate reader (Bio-Tek Instruments, Winooski, VT). The experiment was repeated three times.

2.5 Lactate dehydrogenase assay

The hippocampal neurons injury was assessed by detecting the lactate dehydrogenase (LDH) activity with a commercial LDH Cytotoxicity Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China). Hippocampal neurons (5 × 10³ cells/well) were plated in 96-well plates and subjected to OGD/R stimulation with or without the presence of daphnetin. After different treatments, 20 μL of cell supernatant was collected for the detection of LDH activity according to the manufacturer's instructions. The absorbance was measured spectrophotometrically at 450 nm using a microplate reader (Bio-Tek). The values are shown as percentages of the total LDH. The experiment was repeated three times.

2.6 Measurement of intracellular reactive oxygen species

Intracellular reactive oxygen species (ROS) was determined using a fluorescence probe 2′,7′-dichlorofluorescin-diacetate (DCFH-DA; Sigma). As described by the manufacture's protocol, in the presence of ROS, DCFH-DA can be oxidized to the 2′,7′-dichlorofluorescin, which is highly fluorescent. After treatment, hippocampal neurons were incubated with DCFH-DA in the dark for 30 minutes. Then, the fluorescence intensity of the cells was analyzed using a fluorospectrophotometer with the excitation/emission of 485/525 nm. The experiment was repeated three times.

2.7 Measurement of malondialdehyde, superoxide dismutase, and glutathione peroxidase

After treatments, the cell lysates of the hippocampal neurons were prepared using cell lysis buffer (Beyotime Biotechnology, Shanghai, China). The superoxide dismutase (SOD) activity was measured using a SOD assay kit with WST-8 method (Beyotime). The O₂^- produced by xanthine oxidase can react with WST-8 to produce formazan dye. The reaction can be blocked by SOD because it can react with the O₂^-; therefore, the production of formazan dye is correlated with the SOD activity. After incubation, the absorbance was measured at 450 nm. The data are expressed as the fold activity relative to the control. The measurement of malondialdehyde (MDA) level in cell lysates was measured using a lipid peroxidation MDA assay kit (Beyotime). MDA can react with thiobarbituric acid (TBA) in special conditions to produce MDA-TBA. The levels of MDA-TBA can be detected at 535 nm using a colorimetric method. The unit of MDA concentration is nmol/mg protein.

The glutathione peroxidase (GPx) activity in cell lysates was assessed by a reaction with the GPx detection working solution from the kit (0.25 mM NADPH, 2.1 mM reduced GSH, 0.5 unit/mL glutathione reductase, and 300 μM tert-butyl hydroperoxide), in which the reduction in NADPH absorbance was indicative of the GPx activity, detected at 340 nm after an initial delay of 15 seconds and monitored every 10 seconds for 1 minute at 25°C. The data are expressed as the fold activity relative to the control. The experiment was repeated three times.

2.8 Cell apoptosis assay

The cell apoptosis of hippocampal neurons was analyzed using an enzyme-linked immunosorbent assay (ELISA)–based cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) on the principle of the quantification of apoptosis-specific DNA fragmentation. DNA fragmentation was measured by using a microplate reader to measure the absorbance at 405 nm. The experiment was repeated three times.

2.9 Western blot assay

The cytosolic and nuclear proteins were prepared using a celLytic nuclear extraction kit (Sigma). The protein concentration was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific). After separation by 10% SDS-PAGE, the proteins were transferred to polyvinylidene difluoride membranes (Thermo Fisher Scientific). The membranes were blocked with 5% skim milk in tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) buffer for 1 hour at room temperature, and then incubated overnight at 4°C with the following antibodies: anti-bax (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-bcl-2 (1:1,000 dilution, Santa Cruz), anti-cleaved-caspase-3 (1:1000 dilution; Cell Signaling Technology, Boston, MA), anti-Nrf2 (1:500 dilution; Abcam, Cambridge, MA), anti-HO-1 (1:500 dilution; Abcam), anti-lamin B1 (1:1500 dilution; Abcam), and anti-GAPDH (1:1000 dilution; Santa Cruz). The membranes were incubated with horseradish peroxidase–conjugated secondary antibodies (1:3000 dilution; Cell Signaling Technology) at room temperature for 1 hour. An enhanced chemiluminescence detection system (Thermo Fisher Scientific) was used to visualize the protein bands. The intensity of the bands was analyzed using quantity one software (Bio-Rad, Hercules, CA). The experiment was repeated three times.

2.10 Statistical analysis

The quantitative data are shown as the mean ± SEM. A one-way analysis of variance followed by a Dunnett's post hoc test were applied for analyzing the multiple comparisons. The statistical analysis was performed using the SPSS 18.0 (SPSS, Chicago, IL), and the statistical significance was set at P < 0.05.

3.1 Daphnetin ameliorates OGD/R–induced neuron injury

We first evaluated the effect of daphnetin on the cell viability of hippocampal neurons. CCK-8 assay showed that daphnetin did not affect cell viability until the concentration up to 40 μM (Figure 1A). Thus, the concentrations of 10, 20, 40 μM were selected for the following study. Next, we investigated the role of daphnetin in the OGD/R–induced neuron injury. Figure 1B shows that daphnetin inhibited OGD/R–induced inhibition of hippocampal neurons viability in a dose-dependent manner. Figure 1C revealed that daphnetin dose-dependently reduced OGD/R–induced LDH release in hippocampal neurons.

3.2 Daphnetin represses OGD/R–induced oxidative stress in hippocampal neurons

The effect of daphnetin on OGD/R–induced oxidative stress was investigated by detecting the levels of ROS and MDA, as well as the activities of SOD and GPx. ROS and MDA production was significantly increased in the OGD/R–induced cells relative to the control cells. However, the induction caused by OGD/R was markedly inhibited by daphnetin in a dose-dependent manner (Figures 2A and 2B). The activities of SOD and GPx were lower than that in the control cells. Pretreatment with daphnetin increased the activities of SOD and GPx when compared with the OGD/R–induced cells (Figures 2C and 2D).

3.3 Daphnetin suppresses OGD/R–induced cell apoptosis in hippocampal neurons

We next tested whether daphnetin affects the cell apoptosis of hippocampal neurons after OGD/R stimulation with an ELISA-based cell death detection kit. Figure 3A shows that the percentage of apoptotic cells was higher in the OGD/R–induced cells as compared to the normal cells. After daphnetin pretreatment, the percentage of apoptotic cells was decreased relative to the OGD/R-induced cells. Moreover, we detected the expressions of bax, bcl-2, and cleaved caspase-3 with Western blot analysis. Figure 3B shows that OGD/R induction caused significant increases in the expressions of bax and cleaved caspase-3, while it led to a reduction in the expression of bcl-2. Daphnetin pretreatment mitigated the changes in the expressions of bax, bcl-2, and cleaved caspase-3, which were caused by OGD/R induction.

3.4 Daphnetin induces the activation of Nrf2/HO-1 pathway in hippocampal neurons induced by OGD/R

To explore the effect of daphnetin on the activation of the Nrf2/HO-1 pathway, the expressions of Nrf2 in nuclear fraction, and the HO-1 in the whole cell lysates were measured by Western blot analysis. As indicated in Figure 4A, the expression of Nrf2 nucleus was increased in the OGD/R–induced hippocampal neurons, indicating that OGD/R slightly induced the nuclear translocation of Nrf2. The HO-1 expression in the whole cell lysates was increased in the OGD/R–induced hippocampal neurons compared with the control cells. However, daphnetin elevated the nuclear translocation of Nrf2 and HO-1 expression relative to the OGD/R–induced hippocampal neurons.

To further evaluate the role of the Nrf2/HO-1 pathway, the Nrf2 was silenced by si-Nrf2. Western blot analysis showed that the expressions of Nrf2 and HO-1 were dramatically decreased after transfection with si-Nrf2 (Figure 4B). Si-Nrf2 attenuated the inhibitory effects of daphnetin on cell viability, ROS production, and cell apoptosis (Figures 4C-E). The results indicated that the Nrf2/HO-1 pathway was implicated in the neuroprotective effects of daphnetin.