Recombinant human EPO (rHuEPO) was purchased from CJ Cheiljedang Corporation (Seoul, Korea). Poly-D-lysine was from Sigma (Saint Louis, MO, USA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Duchefa Biochemie (Haarlem, the Netherlands). Hanks' balanced salt solution (HBSS), Neurobasal medium, B27 supplement, glutamax I, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid were obtained from GibcoBRL (Grand Island, NY, USA). Dulbecco's modified eagle's medium (DMEM, high glucose-4,500 mg/L, low glucose-1,000 mg/L), fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin, and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Hyclone Laboratories (Logan, UT, USA). Albumin bovine was obtained from Affymetrix (Santa Clara, CA, USA). Antibodies against phospho-p44/42MAPK (p-ERK1/2), p44/42MAPK (ERK1/2), phospho-p38 MAPK (p-p38 MAPK), p38 MAPK, phospho-SAPK/JNK (p-SAPK/JNK), SAPK/JNK were all purchased from Cell Signaling Technologies (Beverly, MA, USA). Secondary goat anti-rabbit IgG-horseradish peroxidase (HRP) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Clarity Western Enhanced Chemiluminescence (ECL) Substrate was purchased from Bio-Rad Laboratories (Hercules, CA, USA).

This study was performed under the approved animal use guidelines of the Catholic University of Daegu, DCIAFCR-150910-6-Y. Primary neuronal cells from cerebral cortices were prepared as previously described²²⁾. Embryonic day 16 mice were harvested by cesarean section from anesthetized pregnant ICR mice. Cerebral cortices were isolated and dissociated by trypsin digestion at 37℃ for 1 minute. After washing with HBSS, the cells were triturated with a fire-polished Pasteur pipette. The cell suspension was centrifuged at 1,000 rpm at 25℃ for 5 minutes. The pellet was resuspended in Neurobasal medium supplemented with 2% B 27 and 0.5 mM glutamax, 100-U/mL penicillin, and 100-lg/mL streptomycin. The cell suspension was filtered through a 70-µm strainer. The cells were seeded at a density of 2×10⁶ cells/mm2 in each dish precoated with 50-µg/mL poly-D-lysine. Cultures were maintained in a Neurobasal medium at 37℃ in 95% humidified air and 5% CO₂. Three days after plating, the medium was changed. Fifty percent of the medium was changed twice per week.

Primary astrocytes were prepared from postnatal day 1 (P1) or 2 (P2) ICR mouse pups as previously described²³⁾. Cerebral cortices were isolated and dissociated by trypsin digestion at 37℃ for 5 minutes. After washing with HBSS, the cells were titrated with a fire-polished Pasteur pipette. The cell suspension was centrifuged at 1,000 rpm at 25℃ for 5 minutes. The pellet was resuspended astrocyte culture medium (DMEM, high glucose+10% FBS+1% penicillin/streptomycin). The cell suspension was filtered through a 70-µm strainer. Cells were seeded in T75 flasks precoated with 50 µg/mL poly-D-lysine. Cultures were maintained in culture medium at 37℃ in 95% humidified air and 5% CO₂. After 7 to 8 days, the microglial cells were removed by shaking the T75 flask (at 180 rpm) for 30 minutes in a shaker water bath to obtain purified astrocytes. The supernatant was removed, and fresh culture medium was added to the T75 flask. Then the flask was shaken for 6 hours at 240 rpm to detach the oligodendrocytes from the astrocytes layer. The T75 flask was washed once with PBS and treated with 0.25% trypsin-EDTA. After the astrocytes detached from the T75 flask, they were plated in dishes and incubated at 37℃ in 95% humidified air and 5% CO₂. The medium was changed twice per week.

In the first set of experiments, cell viabilities were measured by the MTT assay to determine the optimum concentration. Cells were plated in 96-well plates and treat with different concentrations of EPO for 48 hours before a hypoxic insult. The medium was removed and replaced with the 0.5-mg/mL MTT (final concentration). After incubation for 6 hours and 15 hours, at 37℃ in the dark, the MTT solution was removed, and the formazan dye was extracted with 100-µL dimethyl sulfoxide. Absorbance was measured with a microtiter plate enzyme-linked immunosorbent assay reader at 540 nm.

In the second set of experiments, cells were assigned into the normoxic (N, n=4) group, hypoxia (H, n=4) group, and hypoxia-pretreated with EPO (H+EPO, n=4) group. In the N group, the medium was replaced with DMEM with high glucose and then incubated under normoxic conditions (95% humidified air and 5% CO₂) at 37℃ for 6 hours or 15 hours. This group was used as a negative control during the process. In the H group, the medium was replaced with DMEM containing low glucose. Afterward, the cells were placed in a hypoxia chamber (Billups-Rothenberg Inc., San Diego, CA, USA) and flushed with 1% O₂ (premixed 1% O₂, 5% CO₂, 94% N₂) at the rate of 30 L/min for 4 minutes. In the H+EPO, the medium was replaced with DMEM containing low glucose and then pretreated with 0.1 U/mL (neuronal cells) or 10 U/mL (astrocytes) for 48 hours. The chamber was sealed and incubated at 37℃ for 6 hours or 15 hours.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis was performed to measure the degree of cellular apoptosis using an in situ ApoBrdU DNA fragmentation assay kit (BioVision, Mountain View, CA, USA), following the manufacturer's instructions. Cell pellets were harvested, washed twice with PBS, and fixed in 1% paraformaldehyde for 15 minutes on ice. After washing with PBS, the cells were incubated with 70% ethanol for a minimum of 30 minutes on ice. The ethanol was carefully aspirated, and the cells were washed with Wash Buffer. Then, the cells were incubated with DNA Labeling Solution for 60 minutes at 37℃. After rinsing with Rinse Buffer, the cells were incubated with the Antibody Solution for 30 minutes at room temperature (RT) in the dark. Thirty minutes after the incubation was complete, Propidium Iodide/RNase A solution was added to the cells, and the cells were incubated for 30 minutes at RT in the dark. Cellular fluorescence was then measured using the Gallios Flow Cytometer (Beckman Coulter, Brea, CA, USA). The 2 dyes in use were propidium iodide (for total cellular DNA) and fluorescein (for apoptotic cells).

The activity of caspase-3 was measured using a colorimetric assay kit (Biovision, San Francisco Bay Area, CA, USA) according to the manufacturer's instructions. Cultured cells were concentrated at 300×g for 5 minutes. The pellet was resuspended in a cell lysis buffer and incubated on ice for 10 minutes. The cells were centrifuged for 1 minute in a microcentrifuge (10,000×g). 2X Reaction Buffer was added to the supernatant, and the supernatant proteins were incubated for 1–2 hours at 37℃ with 200 µM DEVD-p-nitroanilide (pNA). The color change was measured using a microtiter plate reader at 405 nm.

Cell extracts were prepared with radioimmunoprecipitation assay lysis buffer (ROCKLAND Immunochemicals, Pottstown, PA, USA) and protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Samples were diluted in 5×sodium dodecyl sulfate (SDS) gel-loading buffer (60 mM Tris-HCl pH 6.8, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoehanol and 0.1% bromophenol blue) and boiled at 95℃ for 10 minutes. Protein lysates (30 µg) were separated by 12% SDS-polyacrylamide gel electrophoresis. After transferring to polyvinylidene difluoride membrane (Millipore, Bed ford, MA, USA) at a constant voltage of 10 V for 27 minutes, membranes were incubated with 5% albumin bovine in Tris-buffered saline plus 0.1% Tween-20 (TBST) at RT for 1 hour. The blots were incubated overnight with primary antibodies against p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, p-SAPK/JNK, SAPK/JNK. Next, the membranes were washed in TBST and incubated with secondary goat anti-rabbit IgG-HRP at 1:2,000 dilution at RT for 1 hour. Detection of a signal was performed with the ECL plus kit. The intensities of the western blot bands were measured by densitometry using Multi Gauge Software (Fuji Photofilm, Tokyo, Japan).

Data were analyzed using the IBM SPSS Statistics ver. 22.0 (IBM Co., Armonk, NY, USA). Examined data were assessed using t tests and analysis of variance. In each test, the data were expressed as the mean±standard deviation with Excel 2007. Statistical significance was defined as when the P value was less than 0.05.

The MTT assay was performed and measure the relative cell viabilities after treatment with rHuEPO at different concentration to determine the most effective concentration of the drug for cell viability (Fig. 1). The results showed that the best cell viabilities were achieved at EPO 0.1 U/mL and 10 U/mL in the neuronal cells and the astrocytes respectively.

The TUNEL assay was performed by flow cytometry in neuronal cells (Fig. 2) and the astrocytes (Fig. 3) to determine whether EPO reduces apoptotic rates against a hypoxic injury. Hypoxia induces apoptosis in both neuronal cells and astrocytes. This results showed that the astrocytes were more resistant to hypoxic injury compared with the neuronal cells. While the apoptotic rates were 9.91% and 7.12% in the neuronal cells and astrocytes, respectively after 6-hour exposure to hypoxia, as time progresses till 15 hours, the neuronal cells undergo more profound apoptosis (61.07%) compared with the astrocytes (10.54%). After 6-hour exposure to hypoxia, the apoptotic rate was not reduced and rather increased with EPO pretreatment (9.91% in the H and 16.44% in the H+EPO) in the neuronal cells. However, after 15-hour exposure to hypoxia, the apoptotic rate was reduced by about two-third with EPO pretreatment (61.07% in the H and 42.93% in the H+EPO, P<0.05).

In the astrocytes, apoptotic rates were similar with EPO pretreatment after 6 hours after hypoxia exposure (7.12% in the H and 6.01% in the H+EPO). After 15-hour exposure to hypoxia, the apoptotic rate was decreased with EPO pretreatment (10.54% in the H and 6.43% in the H+EPO, P<0.05).

The effects of EPO on neuronal apoptosis induced by hypoxia were evaluated by measuring cleaved caspase-3 activity (Fig. 4). In both neuronal cells and astrocytes, the caspase-3 activity was significantly increased after 15 hours exposure to hypoxia (P<0.05). Pretreatment with EPO attenuates significantly this increase of caspase-3 activity induced by hypoxia in both cell groups (P<0.05).

Western blot was performed at baseline, 6 hours, and 15 hours to examine the expressions of MAPKs and phosphorylated MAPKs to explore the relationship between the effects of EPO on survival and the MAPK pathways in the neuronal cells (Fig. 5) and the astrocytes (Fig. 6).

In the neuronal cells, the phosphorylation of ERK1/2 and SAPK/JNK was gradually increased with a peak at 15 hours exposure to hypoxia. The phosphorylation of p38 MAPK has increased persistently both 6- and 15-hour exposure to hypoxia. Pretreatment with EPO did not affect the phosphorylation of p38 MAPK and SAPK/JNK whereas significantly attenuates a decreased of the phosphorylation of ERK1/2 after 15-hour exposure to hypoxia (P<0.05).

In the astrocytes, the phosphorylation of ERK1/2 and SAPK/JNK was increased after both 6 hours and 15 hours exposure to hypoxia. The phosphorylation of p38 MAPK was increased after 6-hour exposure to hypoxia and then decreased after 15-hour exposure to hypoxia. Pretreatment with EPO did not affect the phosphorylation of JAPK/JNK whereas significantly attenuates a reduction in the phosphorylation of ERK1/2 and p38 MAPK after 15-hour exposure to hypoxia (P<0.05).