Hypoxic-ischemic encephalopathy is a significant cause of neonatal morbidity and mortality. Erythropoietin (EPO) is emerging as a therapeutic candidate for neuroprotection. Therefore, this study was designed to determine the neuroprotective role of recombinant human EPO (rHuEPO) and the possible mechanisms by which mitogen-activated protein kinase (MAPK) signaling pathway including extracellular signal-regulated kinase (ERK1/2), JNK, and p38 MAPK is modulated in cultured cortical neuronal cells and astrocytes.
Primary neuronal cells and astrocytes were prepared from cortices of ICR mouse embryos and divided into the normoxic, hypoxia (H), and hypoxia-pretreated with EPO (H+EPO) groups. The phosphorylation of MAPK pathway was quantified using western blot, and the apoptosis was assessed by caspase-3 measurement and terminal deoxynucleotidyl transferase dUTP nick end labeling assay.
All MAPK pathway signals were activated by hypoxia in the neuronal cells and astrocytes (
Pretreatment with rHuEPO exerts neuroprotective effects against hypoxic injury reducing apoptosis by caspase-dependent mechanisms. Pathologic, persistent ERK activation after hypoxic injury may be attenuateed by pretreatment with EPO supporting that EPO may regulate apoptosis by affecting ERK pathways.
Hypoxic-ischemic encephalopathy (HIE) is one of the most important causes of neonatal brain injury. Despite the introduction of hypothermia treatment, HIE still contributes to significant morbidity and mortality in newborn infants
Among, erythropoietin (EPO) has attracted more attention as a neuroprotective agent because it is already used safely to treat anemia even in newborn infants
Therefore, this study is designed to determine whether EPO has neuroprotective effects against hypoxic injury through cell apoptosis assays. Also, it examined how EPO regulates apoptosis in the MAPK signaling pathway including extracellular signal-regulated kinase (ERK1/2), p38 MAPK and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in the cultured cortical neuronal cells and astrocytes.
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 described
Primary astrocytes were prepared from postnatal day 1 (P1) or 2 (P2) ICR mouse pups as previously described
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
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 (
The TUNEL assay was performed by flow cytometry in neuronal cells (
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,
The effects of EPO on neuronal apoptosis induced by hypoxia were evaluated by measuring cleaved caspase-3 activity (
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 (
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 (
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 (
In this study, the effects of EPO on hypoxia-induced neuronal apoptosis and the underlying mechanisms involving MAPK pathways are examined using cultured neuronal cells and astrocytes.
Hypoxia induced apoptosis in both the neuronal cells and astrocytes with caspase-3 activation. Since the caspase-dependent cell damage after neonatal hypoxic injury does not peak until 12–24 hours
Pretreatment with rHuEPO decreased apoptotic rates in both neuronal cells and astrocytes after 15-hour exposure to hypoxia accompanying caspase-3 inactivation indicating that rHuEPO exerts neuroprotective effect via caspase-3 dependent mechanisms. Also, this study showed that rHuEPO attenuated ERK1/2 activation induced by 15-hour hypoxia suggesting the neuroprotective effect of EPO seems to be transduced via the ERK-mediated mechanism.
Since the activation of SAPK/JNK was not affected in both neuronal cells and astrocytes by rHuEPO pretreatment in this study, this result supports the SAPK/JNK pathways themselves may not be involved in the EPO-mediated neuroprotective mechanism. In agreement with this study, Kwon et al.
In the case of p38 MAPK, rHuEPO decreased activation of p38 MAPK in only the astrocyte after exposure of 15-hour hypoxia. These results suggest that the response of each MAPK are cell-specific. The activation of p38 MAPK tended to decrease as time progressed regardless of rHuEPO pretreatment while the role of EPO in p38 MAPK pathway in astrocytes does not seem to be important pathways for EPO-neuroprotection.
ERK is one of the MAPK pathways, and activated by growth factors, mitogen stimuli, glutamate receptor activation and intracellular calcium increases
The role of ERK activation may be via multiple upstream signaling mechanisms depending on factors such as cell type, culture conditions, and the injurious mechanisms
Short, transient activation by growth factors under a physiologic condition, ERK activation are associated with neuroprotection while prolonged, persistent activation could induce cell death
Since this study did not use the ERK inhibitor, it was not determined whether the ERK inhibition initiated by EPO was direct causative mechanisms for neuroprotection or resultant indirect phenomenon. Kwon et al.
In conclusion, rHuEPO exerts neuroprotective effects against hypoxic injury reducing apoptosis by caspase-dependent mechanisms. Pathologic, persistent ERK activation after hypoxic injury may attenuate by pretreatment EPO supporting that EPO may regulate apoptosis by affecting ERK pathways. This study may contribute to determining the possible mechanisms of the role of each MAPK pathways in the cytoprotective effect of rHuEPO.
This work was supported by the grant of Research Institute of Medical Science, Catholic University of Daegu (2016)