Insulin Growth Factor 1 Protects Neural Stem Cells Against Apoptosis Induced by Hypoxia Through Akt/Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase (Akt/MAPK/ERK) Pathway in Hypoxia-Ishchemic Encephalopathy.

BACKGROUND Insulin growth factor 1 (IGF-1) is reported to modulate cell growth and acts as potential therapy for traumatic brain injury. This study was designed to investigate the effect of IGF-1 on hypoxia-induced apoptosis in neural stem cells (NSCs). MATERIAL AND METHODS A hypoxia model was constructed using NSCs separated from the hippocampus of rat. NSCs were divided into four groups: cells under normoxic conditions that acted as controls (C group), cells under hypoxia (H group), cells under hypoxia with IGF-1 (HI group), and cells under hypoxia with IGF-1 as well as picropodophyllin (PPP), which acts as an inhibitor of the IGF-1 receptor (HIP group). The cell viability and apoptosis were respectively measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Finally, the phosphorylation levels of apoptosis-associated proteins and key kinases in the phosphatidylinositol-3-kinase (PI3K)/AKT and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways were detected by Western blot analysis. RESULTS In comparison with the H group, the cell viability was increased while the cell apoptosis was reduced by IGF-1 in the HI group. Besides, the expression levels of Bax, cytochrome c, and activated caspase-3 were all improved in the H group, and the remarkable differences were eliminated in the HI group compared with the C group. The expression level of Bcl-2 was the opposite. Additionally, down-regulations of phosphorylated AKT, MAPK, and ERK induced by hypoxia were all improved by IGF-1. All the influences of IGF-1 were weakened by addition of PPP. CONCLUSIONS IGF-1 increased cell viability while decreasing apoptosis in hypoxic NSCs through the PI3K/AKT and MAPK/ERK pathways.

Background

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acquired brain injury that mainly happens in newborn infants [1,2]. The severity, duration, and timing of the HIE antenatal injury are difficult to discern [3]. In live neonates, the incidence of HIE ranged from 0.1% to 0.8% in developed countries and even as high as 2.6% in underdeveloped countries [4]. In spite of widespread use of therapeutic hypothermia, 40% of neonates with HIE have neurological disability at 18–24 months of age [5].

Although the brain is 2% of the total body weight, it consumes 20% of the total body oxygen, which means that the brain has a relatively high rate of oxygen consumption [6]. In addition to the limited oxygen reserves in the brain, the central nervous system (CNS) is highly susceptible to anoxia and displays low tolerance to hypoxia [7]. Neural stem cells (NSCs) are the cells exhibiting stem cell properties that possess the ability to self-renew and to generate major cell types in the CNS [8,9]. Hypoxia disrupts the homeostasis of the microenvironment and plays a role in cell proliferation and differentiation. There are various overwhelming evidences confirming that a low oxygen level facilitates the survival of myoblasts [10], and hematopoietic [11], neural [12], and human embryonic stem cells [13]. However, long-term anoxia is considered to suppress cell growth of epithelial stem cells [14].

Insulin growth factor 1 (IGF-1) consists of 70 amino acid residues accompanied by three disulfide bridges [15]. In particular, IGF-1 is one of the neurotrophins that modulate cell growth [16]. IGF-1 has been proven to bind with the IGF-1 receptor (IGF-1R) and then activate the phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway, thereby facilitating cell proliferation and differentiation [17]. Recent studies illustrated that IGF-1 influenced early embryonic development via increasing the number of cells [18]. Moreover, IGF-1 is reported to be an essential growth factor for promoting proliferation and suppressing apoptosis of neural progenitor-like cells [19]. Rubovitch et al. also suggested that IGF-1 was a potential neuroprotective therapy for traumatic brain injury–induced damage [20]. Picropodophyllin (PPP) is a selective inhibitor of tyrosine phosphorylation of IGF-1R [21] and could effectively suppress the activation of IGF-1R [22]. To our knowledge, the modulating effect of IGF-1 on hypoxic NSCs has seldom been investigated. Hence, the aim of our research was to explore the effect of IGF-1 with or without addition of PPP on NSC apoptosis induced by hypoxia. The mechanism involved was also studied.

Material and Methods

Experimental animals

Pregnant Sprague-Dawley (SD) rats were purchased from the experimental animal center of Nantong University (Jiangsu, China). Every animal study was conducted in accordance with the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental protocols were approved by our local ethics committee. All efforts were made to minimize the number and suffering of rats involved in our study.

Cell culture

NSCs from the hippocampus were obtained as described previously [23]. Briefly, following anesthesia with chloral hydrate (2 mL/kg of body weight), the hippocampus was isolated from 14-day SD rat embryos and placed on ice. After digestion of the hippocampus with 0.125% trypsin (Gibco, Grand Island, New York, USA), the NSCs were dissociated into single-cell suspensions. The cell suspensions with a density of 1 × 104 cells/mL were cultured in 1: 1 Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, Carlsbad, California, USA) containing 2% B27 (Invitrogen, Carlsbad, California), basic fibroblast growth factor (bFGF) 10 ng/mL (Peprotech, Rocky Hill, New Jersey), epidermal growth factor (EGF) 10 ng/mL (Peprotech, Rocky Hill, NJ), and penicillin/streptomycin 100 U/mL (Beyotime, Shanghai, China). The cultures were maintained in a humidified incubator at 37°C, with 95% air and 5% CO2 (v/v). Five days later, neurospheres were centrifuged, resuspended, and plated on 96-well plates at 1–2 cells/well. Then, the digestion and passage were conducted again as before. In order to fetch neurospheres originating from a single primary cell, cells were passaged three times. Digested from the neurospheres, the NSCs used for subsequent experimentations were maintained under the same culturing condition with neurospheres.

Construction of hypoxia model

The hypoxia environment was made by placing NSCs in a humidified chamber (StemCell Technologies, Vancouver, Canada) with a gas mixture of N2, CO2, and O2 at 37°C. The percentage of the three gases was 94%, 5%, and 1%, respectively. In order to ascertain the most suitable duration of hypoxia for further experiments, the cells were transferred from the normoxic incubator to the hypoxic incubator for different durations (0, 2, 4, 6, 8, and 10 h). The control group was cultured in normoxic conditions all the time for the same durations.

IGF-1 and PPP treatments

NSCs were hypoxia cultured with IGF-1 at various concentrations (0, 10, 50, 100, or 200 ng/mL). After that, the cell viability of NSCs induced with different concentrations of IGF-1 was assayed. The group without IGF-1 induction acted as the control.

NSCs for further studies were divided into four groups, namely, the C, H, HI, and HIP groups. Cells in the C group were incubated without any treatment, cells in the H group were incubated with hypoxia treatment, cells in the HI group were incubated with hypoxia treatment accompanied by IGF-1, and cells in the HIP group were incubated with hypoxia treatment accompanied by IGF-1 and PPP 500 nmol/L.

Cell viability assay

The cell viability after different treatments was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay according to the standard method described before [24]. In brief, 20 μL of MTT (5 mg/mL) was added to each well of 96-well plates. After 4 h of incubation at 37°C, 200 μL of dimethyl sulfoxide (DMSO) (Sigma, St Louis, Missouri, USA) was added to terminate the reaction. At last, the plates were shaken vigorously for 10 min, and the levels of reduced MTT were measured by a plate reader (Biotek Synergy 2, Winooski, Vermont, USA) at a wavelength of 570 nm.

Cell apoptosis assay

The quantitative assessment of NSC apoptosis was conducted by flow cytometry using the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Beijing Biosea Biotechnology, Beijing, China). In brief, the cells were resuspended with a density of 1×105 cells/well in binding buffer after twice washing with cold phosphate-buffered saline (PBS). Then, both the adherent and floating cells were treated with 10 μL of Annexin V-FITC and 5 μL of PI according to the manufacturer’s instruction. Finally, the cells were measured with a flow cytometer (Beckman Coulter, USA), which distinguished apoptotic cells (Annexin-V positive and PI-negative) from necrotic cells (Annexin-V and PI-positive).

Western blot analysis

Proteins in NSCs were extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and protease inhibitors (Roche, Indianapolis, Indiana, USA) according to the manufacturer’s instruction. The protein contents were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, Wisconsin, USA). Equivalent amounts of proteins were loaded on 12% polyacrylamide gels and separated through sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE). Afterwards, the proteins on the gels were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA). The membranes carrying proteins were incubated for 1 h with blocking buffer that contained 0.1% Tween 20, 20 mM Tris-HC, and 137 mM NaCl (TBST) and 4% bovine serum albumin (BSA; Sigma-Aldrich, Sydney, NSW, Australia) at room temperature. After three rounds of rinsing with TBST, the membranes were incubated overnight at 4°C with primary antibodies against mammalian B cell lymphoma-2 (Bcl-2, 15071), Bcl-2-associated X protein (Bax, 14796), cytochrome c (11940), cleaved capase-3 (9661), pro capase-3 (9662), GAPDH (2118), phosphorylated mitogen-activated protein kinase (p-MAPK, 4370), MAPK (4695) (all from Cell Signaling Technology, Danvers, Massachusetts, USA), phosphorylated AKT (p-AKT, sc-101629), AKT (sc-8312), phosphorylated extracellular signal-regulated kinase (p-ERK, sc-16981-R), and ERK (sc-292838) (all from Santa Cruz Biotechnology, California, USA). Membranes were then incubated with second antibodies at room temperature for 2 h. After rinsing with TBST for three times, the membranes carrying blots and antibodies were transferred into the Bio-Rad ChemiDoc™ XRS system with addition of 200 μL of Immobilon Western Chemiluminescent HRP Substrate (Millipore). The signals were captured and the intensity of the bands was quantified using Image Lab™ Software (Bio-Rad, Shanghai, China). GAPDH was used as an internal control.

Statistical analysis

All results were collected from three independent experiments. Data from multiple experiments are presented as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad, San Diego, California, USA). The P values for multiple comparisons were calculated by one-way analysis of variance (ANOVA). A P value of <0.05 was considered statistically significant.

Results

To construct the hypoxia model of NSCs, the most suitable duration of hypoxia was evaluated by MTT assay. Figure 1 shows that when the hypoxia time lasted for 2 h, the cell viability was accelerated compared with control (F (5, 12)=94.78, P<0.01). With the extension of hypoxia time, the cell viability was decreased significantly in comparison with control (F (5, 12)=94.78, P<0.05 or P<0.001). Cell viability at 6 h was markedly decreased in comparison with that of cells at 4 h (F (5, 12)=94.78, P<0.01). However, the difference in cell viability between 6 h and 8 h, as well as that between 8 h and 10 h, was not significant. Thus, the hypoxia model was constructed with exposure to hypoxia for 6 h.

Figure 1

Effect of hypoxic duration on cell viability. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Data presented are the mean ±SD of three independent experiments. * P<0.05; ** P<0.01; *** P<0.001. The significance marked at the top of the columns refers to comparison with the C group.

IGF-1 enhanced cell viability of hypoxic NSCs

The most suitable concentration of IGF-1 induction for hypoxic NSCs was determined by MTT assay. When the concentration of IGF-1 reached 50, 100, and 200 ng/mL, the cell viabilities were all remarkably increased compared with control (F (4, 10)=51.35, P<0.001) (Figure 2A). Furthermore, it seemed that the cell viability was enhanced in a concentration-dependent manner when the concentration of IGF-1 was lower than 100 ng/mL. Besides, the induction was saturated with the addition of IGF-1 up to 100 ng/mL; thus, the concentration of IGF-1 used in the following experiments was 100 ng/mL.

Figure 2

Insulin growth factor 1 (IGF-1) improved cell viability in hypoxic neural stem cells (NSCs). Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. (A) Hypoxic NSCs were cultured under different concentrations of IGF-1. (B) NSCs were divided into four groups: the Control, Hypoxia, Hypoxia+IGF-1, and Hypoxia+IGF-1+PPP groups. Data presented are the mean ±SD of three independent experiments. ** P<0.01; *** P<0.001. The significance marked at the top of the columns refers to comparison with the C group.

MTT assays demonstrated that the cell viability of hypoxic NSCs was notably depressed compared with control (F (3, 8)=25.65, P<0.001) (Figure 2B). With the addition of IGF-1, the viability of hypoxic NSCs increased, resulting in no obvious difference in comparison with control. However, the addition of both PPP and IGF-1 markedly reduced the viability of NSCs in comparison with control (F (3, 8)=25.65, P<0.001). Hence, we reached the conclusion that IGF-1 promoted cell viability of hypoxic NSCs.

IGF-1 suppressed cell apoptosis of hypoxic NSCs

The apoptosis assays of the four groups of NSCs in Figure 3A and 3B suggested that the apoptotic rates of treated NSCs were all remarkably improved compared with control (F (3, 8)=101.9, P<0.001). Further comparison showed that IGF-1 suppressed hypoxia-induced apoptosis in NSCs (F (3, 8)=101.9, P<0.001), while PPP weakened the suppression that came from IGF-1 (F (3, 8)=101.9, P<0.001). We therefore concluded that IGF-1 suppressed cell apoptosis of hypoxic NSCs.

Figure 3

Insulin growth factor 1 (IGF-1) suppressed cell apoptosis induced by hypoxia in neural stem cells (NSCs). NSCs were divided into four groups: the Control, Hypoxia, Hypoxia+IGF-1, and Hypoxia+IGF-1+PPP groups. (A, B) NSCs were double stained by Annexin V-FITC and propidium iodide (PI), and then apoptotic cells were analyzed by flow cytometry. (C) The protein expression levels of apoptosis-associated factors were assayed by Western blot analysis. (D) The band intensity was assessed by Image Lab™ Software. The expression levels were all normalized by GAPDH. The expression level of activated caspase-3 was expressed as the relative intensity of cleaved/pro caspase-3. Data presented are the mean ±SD of three independent experiments. ** P<0.01; *** P<0.001. The significance marked at the top of the columns refers to comparison with the control group.

The protein expression levels of apoptosis-associated factors were further investigated by Western blot analysis. Figure 3C and 3D show that the expression levels of Bax, cytochrome c, and activated caspase-3 were all up-regulated in hypoxic NSCs compared with control (F (3, 8)=42.41, P<0.001; F (3, 8)=19.36, P<0.001; F (3, 8)=67.49, P<0.001). When the IGF-1 was added to the culture, the expression levels of these three proteins were all down-regulated, resulting in no significant difference between cells in the HI group and cells in the C group. When IGF-1 and PPP were both added to the culture, the expression levels of these three proteins were all up-regulated and were significantly different in comparison with control (F (3, 8)=42.41, P<0.001; F (3, 8)=19.36, P<0.01; F (3, 8)=67.49, P<0.001). In terms of Bcl-2, the expression level was just the opposite of that of the three proteins described above. All these results implied that IGF-1 suppressed the cell apoptosis of hypoxic NSCs.

IGF-1 activated PI3K/AKT and MAPK/ERK pathways

IGF-1 was added alone or in combination with its inhibitor PPP to hypoxic NSCs in order to explore the potential interactions between IGF-1 and apoptosis by Western blot analyses. Figure 4A and 4B show that the phosphorylation levels of AKT, MAPK, and ERK were all decreased in hypoxic NSCs compared with control (F (3, 8)=12.95, P<0.01; F (3, 8)=33.16, P<0.05; F (3, 8)=10.14, P<0.01). When the IGF-1 was added, the phosphorylation levels of AKT, MAPK, and ERK were all markedly improved, resulting in no significant difference or a marked increase in comparison with control (F (3, 8)=33.16, P<0.01). When the PPP was added, the up-regulation of p-AKT, p-MAPK, and p-ERK was eliminated, resulting in significant decreases in the phosphorylation levels of these three kinases in comparison with control (F (3, 8)=12.95, P<0.05; F (3, 8)=33.16, P<0.01; F (3, 8)=10.14, P<0.05). These results allowed us to reach a conclusion that IGF-1 inhibited hypoxia-induced apoptosis through activation of the PI3K/AKT and MAPK/ERK pathways.

Figure 4

Insulin growth factor 1 (IGF-1) activated the phosphatidylinositol-3-kinase (PI3K)/AKT and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways. Neural stem cells (NSCs) were divided into four groups: the Control, Hypoxia, Hypoxia+IGF-1, and Hypoxia+IGF-1+PPP groups. (A) Protein expression levels of kinases were assayed by Western blot analysis. (B) The band intensity was assessed by Image Lab™ Software. The phosphorylation rate was expressed as the relative intensity of phosphorylated kinases/kinases, and the final results were normalized by GAPDH. Data presented are the mean ±SD of three independent experiments. * P<0.05; ** P<0.01. The significance marked at the top of the columns refers to comparison with the control group.

Discussion

Acute mortality and chronic neurological disability are the main effects of HIE, which occurs in the fetus and newborn infant [25]. Although the use of hypothermia as a treatment strategy is widespread [26], the mortality and neurological disability from HIE have not obviously declined. Accelerating demands for effective HIE therapy are driving a sharp increase in the number of investigations. In this study, we constructed a hypoxia model to explore whether IGF-1 can protect NSCs from apoptosis. We found that IGF-1 significantly increased the cell viability in hypoxic NSCs, while the increase was eliminated when PPP was added. After that, we demonstrated that IGF-1 remarkably suppressed cell apoptosis in hypoxic NSCs, while the suppression declined when PPP was added. Furthermore, we explored the protein expression levels of apoptosis-associated factors and key kinases involved in the PI3K/AKT and MAPK/AKT pathways. The expression levels of Bax, cytochrome c, and activated caspase-3 were obviously down-regulated, while the expression level of Bcl-2 was prominently up-regulated by addition of IGF-1 in hypoxic NSCs. The effects of IGF-1 were reversed by addition of PPP. The studies on signaling pathway showed that IGF-1 activated the PI3K/AKT and MAPK/AKT pathways.

In general, hypoxia is considered to cause neural tissue damage in vivo and is involved in cell death, although a recent study indicated that hypoxia could facilitate proliferation of myoblast stem cells [10]. In our report, cell viability was also enhanced when the duration of hypoxia was 2 h, which was in line with the literature cited above. In addition, we found that cell viability was markedly decreased when NSCs were exposed to hypoxia for 4 h or more than 4 h. Moreover, the decreased cell viability of hypoxic NSCs could be reversed by IGF-1. The ability of IGF-1 to accelerate cell viability was also found in the previous literature. A report declared that IGF-1 promoted myoblast proliferation [27]. Furthermore, Yan et al. demonstrated that IGF-1 promoted cell proliferation of human periodontal ligament stem cells [28].

Previous research claimed that a long period of hypoxia might promote NSC apoptosis [29]. In this study, apoptosis assays implied that exposure to hypoxia for 6 h significantly promoted cell apoptosis. A majority of investigations revealed that the most common way to cell apoptosis was the mitochondrial pathway. Signals of cellular stress transferred to mitochondria resulted in mitochondrial outer membrane permeabilization (MOMP). The process of MOMP is orchestrated by the Bcl-2 family protein-protein interactions [30]. Both pro-apoptotic and anti-apoptotic proteins exist in the Bcl-2 protein family. Bax is one of the pro-apoptotic proteins that mediate MOMP, while Bcl-2 is one of the anti-apoptotic proteins that inhibit MOMP [31]. The process of MOMP triggers the release of cytochrome c. Subsequently, the caspase pathway is activated, which gives rise to activation of downstream orchestrations of apoptotic cellular dismantling and clearance [32]. Caspase-3 is the main effector caspase, which exists as an inactive proenzyme and undergoes proteolytic processing to produce an active enzyme [33]. In this report, the pro-apoptotic protein Bax, cytochrome c, and activated caspase-3 were all up-regulated in hypoxia NSCs, while the increase was eliminated by addition of IGF-1. Reasonably, the addition of PPP was observed to eliminate the function of IGF-1. The expression level of anti-apoptotic Bcl-2 was just opposite to that of the other three proteins. All the results suggested that IGF-1 could protect NSCs from hypoxia-induced apoptosis.

IGF-1 has been proven to bind tightly with IGF-1R. IGF-1R is activated with the binding and triggers multiple downstream signaling pathways. Among them, the PI3K/AKT and MAPK/ERK pathways are relevant to cell proliferation and survival [34]. IGF-1R affects the MAPK/ERK pathway via phosphorylating a sequence of protein kinases and then controls apoptosis. A report implied that inhibition of the MAPK/ERK pathway triggered apoptosis [35]. As for the PI3K/AKT pathway, activated IGF-1R activates PI3K and then the downstream phospholipids are produced. AKT is activated by phospholipids and then interacts with Bax and Bcl-2 [36]. These apoptosis-associated proteins thereby make a great difference regarding apoptosis. In agreement, we provided evidence that IGF-1 remarkably up-regulated the phosphorylation of AKT, MAPK, and ERK in hypoxic NSCs and that the up-regulation was eliminated by PPP. These results, as well as the reports described above, support the conclusion that IGF-1 protected NSCs from hypoxia-induced apoptosis through activating the MAPK/ERK and PI3K/AKT pathways.

Conclusions

We speculate for the first time that IGF-1 could protect NSCs from hypoxia-induced apoptosis through activation of the PI3K/AKT and MAPK/ERK pathways. More studies still must be done to examine in detail the interactions between IGF-1R and kinases. The findings might help in further exploring the clinical therapies for HIE.