Coenzyme Q10 protects neural stem cells against hypoxia by enhancing survival signals

brain research 1478 (2012) 64–73

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Research Report

Coenzyme Q10 protects neural stem cells against hypoxia by enhancing survival signals$ Jinse Park1,a,b, Hyun-Hee Park1,a, Hojin Choia, Young Seo Kima, Hyun-Jeung Yuc, Kyu-Yong Leea, Young Joo Leea, Seung Hyun Kima, Seong-Ho Koha,d,n a

Department of Neurology, Hanyang University College of Medicine, Seoul, Korea Department of Neurology, Haeundae Paik Hospital, Inje University, Busan, Korea c Department of Neurology, Bundang Jesaeng General Hospital, Seongnam, Korea d Department of Translational Medicine, Hanyang University Graduate School of Biomedical Science & Engineering, Seoul, Korea b

ar t ic l e in f o

abs tra ct

Article history:

Recanalization and secondary prevention are the main therapeutic strategies for acute

Accepted 15 August 2012

ischemic stroke. Neuroprotective therapies have also been investigated despite unsuccess-

Available online 21 August 2012

ful clinical results. Coenzyme Q10 (CoQ10), which is an essential cofactor for electron

Keywords:

transport in mitochondria, is known to have an antioxidant effect. We investigated the

Coenzyme Q10

protective effects of CoQ10 against hypoxia in neural stem cells (NSCs). We measured cell

Hypoxia

viability and levels of intracellular signaling proteins after treatment with several

Neural stem cells

concentrations of CoQ10 under hypoxia-reperfusion. CoQ10 protected NSCs against

Neuroprotection

hypoxia-reperfusion in a concentration-dependent manner by reducing growth inhibition and inhibiting free radical formation. It increased the expression of a number of survivalrelated proteins such as phosphorylated Akt (pAkt), phosphorylated glycogen synthase kinase 3-b (pGSK3-b), and B-cell lymphoma 2 (Bcl-2) in NSCs injured by hypoxiareperfusion and reduced the expression of death-related proteins such as cleaved caspase-3. We conclude that CoQ10 has effects against hypoxia-reperfusion induced damage to NSCs by enhancing survival signals and decreasing death signals. & 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Hypoxia-induced neuronal cell death is the main cause of ischemic stroke. Although early revascularization is the most

important treatment for acute ischemic stroke, it is only possible in less than 5% of stroke patients. With the growing understanding of the mechanism of neuronal cell death in ischemic stroke, interest has intensified in the

$ Jinse Park and Hyun-Hee Park participated in the acquisition, organization, and review of data, and drafting of the manuscript. Hojin Choi, Kyu-Yong Lee and Young Joo Lee participated in the acquisition, organization, review and interpretation of the data. Seong-Ho Koh participated in the review and interpretation of data. This article received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. The authors have no potential conflicts of interest to report concerning this article, and have nothing to disclose. n Correspondence to: Department of Neurology, Hanyang University College of Medicine, 249-1, Hanyang University Guri Hospital Gyomun-dong, Guri-si, Gyeonggi-do, 471-701, Korea. Fax: þ82 31 560 2267. E-mail address: [email protected] (S.-H. Koh). 1 These two authors have contributed equally to this work.

0006-8993/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2012.08.025

brain research 1478 (2012) 64–73

role of neuroprotection. For several decades, numerous studies have tried to identify the neuroprotective mechanism of ischemic neuronal damage after acute ischemic stroke. Despite unsatisfactory results in clinical studies, many therapeutic agents such as antioxidant, anti-apoptoic, anti-inflammatory and anti-excitotoxic agents have been explored. Oxidative stress induced by hypoxia is well known to be one of the major mechanisms leading to neuronal destruction (Lin et al., 2004). Oxidative stress is induced by the production of reactive oxygen species (ROS) including free radicals and peroxides and is closely related to apoptosis and necrosis in ischemic injury. Mitochondria are well known to be a major source of ROS production (Fleury et al., 2002). Coenzyme Q10 (CoQ10) is essential for mitochondrial oxidative phosphorylation and adenosine triphosphate (ATP) production. Coenzyme Q10 is also a potent free radical scavenger in lipid and mitochondrial membranes. Much evidence has accumulated that it is an important antioxidant and has protective effects in many neurodegenerative diseases (Beal, 1999; Shults et al., 2002; Spindler et al., 2009). Theoretically, administration of CoQ10 in acute stroke patients is a logical neuroprotective treatment due to its reduction of ROS production. However, few studies have investigated the effects of CoQ10 in an acute stroke model. This study focused on the effects of CoQ10 in neural stem cells (NSCs) after ischemic stroke. NSCs are well known to play an important role in endogenous neurogenesis following cerebral ischemia. However, hypoxia induces cell death of NSCs as well as neurons. Our purpose is to investigate neuroprotection of CoQ10 in NSCs under a hypoxic condition.

2.

Results

2.1.

Expression of HIF-a under hypoxic condition

To confirm that NSCs were affected in intracellular level after hypoxic injury, we measured levels of hypoxia-inducible factor-alpha (HIF-1a) by Western blotting. HIF-1a, which is known to be key regulator of hypoxia response, is suggested to have an important role following ischemic stroke. It is generally accepted that HIF-1a is accumulated under hypoxia. In our experiment, HIF-1a expression rapidly increased in response to hypoxia after 4 h and remained elevated at 8 h and peaked at 24 h (Fig. 1).

2.2. Effect of hypoxia and CoQ10 on the viability of neural stem cells To confirm the effect of the hypoxic condition used in the present study on NSCs viability, we incubated NSCs in the anaerobic chamber with different exposure time (0, 2, 4, 8, and 24 h). Cell viability was measured with trypan blue staining (TBS) and LDH assay. In the hypoxic condition, cell viability was significantly reduced in a time-dependant manner (Fig. 2A and B). Because cell viability was decreased

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Fig. 1 – Expression of HIF-1a in response to hypoxia. npo0.05 by Tukey’s test after one-way ANOVA (compared with the control group (no hypoxia)), (n¼ 5).

drastically after exposure of hypoxia for 24 h, we chose 8 h as an optimal exposure time. To evaluate the effect of CoQ10 itself on NSCs, we treated NSCs for 8 h with several concentration of CoQ10 (0.01, 0.1, 1, 10, and 100 mM) without hypoxia. We found that CoQ10 is itself cytotoxic at high concentrations of 10 and 100 mM. Cell viability was decreased with more than 10 mM CoQ10 treatments in TBS and LDH assay (Fig. 2C and D). To evaluate the effect of CoQ10 on NSCs under hypoxic condition, NSCs were exposed to hypoxia for 8 h and were treated simultaneously with several concentration of CoQ10 (0.01, 0.1, 1, and 10 mM). Cell viability was measured by TBS and LDH assay. Compared to NSCs under hypoxic condition without treatment, viability of NSCs treated with CoQ10 under hypoxic condition was gradually increased in a concentration-dependent manner up to 1 mM. However, the viability was not increased with the treatment of more than 10 mM CoQ10 (Fig. 2E and F). To investigate the role of the PI3K/Akt pathway in the neuroprotective effects of CoQ10, the NSCs were separated into four groups: control (group 1), hypoxia for 8 h (group 2), hypoxiaþ1 mM CoQ10 for 8 h (group 3), and 10 mM LY294002 for 9 hþhypoxia and 1 mM CoQ10 for 8 h (group 4). In this study, pretreatment with the PI3K inhibitor in group 4 resulted in an approximately 16% decrease in cell viability as compared with group 3 (Fig. 2G).

2.3.

Anti-apoptotic effects of CoQ10 in hypoxic condition

DAPI and TUNEL stainings were performed to examine the effect of CoQ10 on apoptosis. As shown in Fig. 3, the

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Fig. 2 – Effects of hypoxia and coenzyme Q10 on NSC viability. po0.05 and po0.01 (when compared with the control group), #po0.05 (compared with NSCs under hypoxic condition without CoQ10), and $ po0.05 (compared with NSCs under hypoxic condition and 1 lM CoQ10), (n ¼5).

percentage of apoptotic cells under a hypoxic condition was markedly increased under an 8-h hypoxic condition (po0.01) but was significantly decreased with the treatment of CoQ10

(0.1, 1, and 10 mM) (po0.05) (Fig. 3). Treatment of 1 mM CoQ10 resulted in the greatest decrease in the percentage of apoptotic cells.

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Fig. 3 – Anti-apoptotic effect of CoQ10 on NSCs during exposure to hypoxic conditions for 8 h. NSCs were stained with DAPI and TUNEL staining. The data are presented as % of TUNEL-positive cells7SD (B) or apoptotic cells7SD (C). Each treatment group was compared with the other groups using Tukey’s test after one-way ANOVA (n ¼ 5). po0.05 (compared with control group); #po0.05 (compared with NSCs under hypoxia alone), ##po0.01 (compared with NSCs under hypoxia alone).

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2.4. Effects of CoQ10 on proliferation of neural stem cells under hypoxia Because CoQ10 could be involved in regulation of cellular process, the effects of CoQ10 on proliferation of NSCs were examined. The BrdU assay showed that NSCs treated with CoQ10 promoted cell proliferation in a concentrationdependent manner when compared with the control group (Fig. 4).

2.5. Effect of CoQ10 on free radical production by hypoxia in NSCs Theoretically, CoQ10 might provide antioxidant action against hypoxia by reducing free radical production. To evaluate free radical production, NSCs were treated with hypoxia for 8 h and assessed using DCF fluorescence (Fig. 5). Treatment with hypoxia for 8 h significantly increased free radical production in NSCs compared with the control group (po0.01). Combined treatment with CoQ10 (0.1, 1 and 10 mM) drastically decreased free radical production without regard to concentration. We found the amount of free radical in NSCs treated with CoQ10 is lower than in the control group not treated with hypoxia.

2.6. Effects of CoQ10 on levels of intracellular signaling proteins To confirm the effects of CoQ10 on several intracellular signaling proteins, we measured the expression levels of Akt, GSK3-b, Bcl-2, and caspase-3. The results of Western blotting showed that the immunoreactivities (IRs) of phosphorylated Akt (Ser473), phosphorylated GSK-3b (Ser9), and Bcl-2 in NSCs were significantly

Fig. 4 – Effect of CoQ10 on proliferation of NSCs injured by hypoxia. The data are means (absorbance 460 nm)7SD from five independent experiments. The treatment groups are compared with the control group using Tukey’s test after one-way ANOVA (n¼ 5). BrdU assay shows that treatment with coenzyme Q10 (CoQ10) restores the proliferation of NSCs inhibited by hypoxia. po0.05 and po0.01 (vs. control group), #po0.05 (vs. the group treated with only hypoxia).

increased with the treatment of CoQ10 in a concentrationdependent manner up to 1 mM when compared with NSCs treated only with hypoxia (Fig. 6A–C). In contrast, treatment of CoQ10 significantly decreased the expression of cleaved caspase-3 in a concentration-dependent manner up to 1 mM (Fig. 6D).

3.

Discussion

Neural stem cells (NSCs) are multipotent cells that can differentiate to various cell types in nervous system. It is well known that NSCs are presented in subventricular zone near lateral ventricle (SVZ) and subgranular zone of hippocampus (SGZ) (Nakayama et al., 2010) (Kallur et al., 2006; Willaime-Morawek and van der Kooy, 2008). In the last decades, neural stem cells have been attracting interest in potential utility of therapeutic strategy for acute ischemic stroke. NSCs are activated by ischemic hypoxia, migrate toward injured lesions, and then may be involved in the recovery of the damaged area (Jin et al., 2003). However, NSCs can be also damaged by hypoxia and our results also reveal that NSCs are vulnerable to hypoxia (Figs. 1 and 2(A and B). It has been reported that hypoxia caused inhibition of survival signals and activation of death signals in NSCs (Zhao et al., 2007). One such pathway includes bcl-2 family protein members, up-regulation of HIF-a and activation of caspase-3 (Walls et al., 2009). Describing in detail, initiation and execution of apoptosis following hypoxia are complex and diverse groups of proteins are involved in the apoptotic pathway (Broughton et al., 2009). For one example, hypoxia inhibits phosphoinositide 3-kinase (PI3K) and activates glycogen synthase kinase (GSK)-3b (Chung et al., 2008) as also shown in our study (Fig. 4). Cellular damage associated with hypoxia includes impaired metabolism, energy failure, free radical production, disturbed calcium homeostasis, and activation of proteases. Among these pathomechanisms of ischemic injury, dysfunction of mitochondria might contribute to ROS production. Subsequently, ROS generation is known to inhibit the Akt pathway, leading to apoptosis (Pan et al., 2010). We assumed that targeting mitochondrial dysfunction, which is involved in these mechanisms, could have potential utility as therapeutic agents in ischemic stroke. Much research in recent years has focused on mitochondrial dysfunction in ischemic neuronal injury. Potential agents such as CoQ10, creatine, and Schiller peptide (SS-31) were proven to improve mitochondrial dysfunction. Among them, studies on CoQ10 have been performed most frequently and CoQ10 is in clinical trials for many neurodegenerative diseases such as Parkinson disease (PD) and Huntington disease (HD) (Yang et al., 2009). CoQ10 participates in electron transfer within the inner mitochondrial membrane, leading to adenosine triphosphate (ATP) production (Matthews et al., 1998). In the inner mitochondrial membrane, CoQ10 accepts electrons from complexes I and II and transfers them to complex III. CoQ10 also increases expression of proteins with an antioxidant effect by inhibiting ROS production (Shults and Haas, 2005). These action mechanisms might contribute to the neuroprotective effects of

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Fig. 5 – Effect of CoQ10 on free radical production by hypoxia in NSCs. The data are means (% of control)7SD from five independent experiments. The treatment groups were compared using the Tukey test after a one-way ANOVA (n ¼10). Neural stem cells (NSCs) were treated with hypoxia for 30 min to measure free radicals generated during exposure to hypoxia using the fluorescent probe DCFH-DA (see Section 4). Production of free radicals was significantly increased in the NSCs treated with hypoxia. Combined treatment with CoQ10 reduced free radical production in a concentration-dependent manner. po0.05 (when the level of free radicals was compared with that of the control group); #po0.05 (when the level of free radicals was compared with that of NSCs treated with only hypoxia).

CoQ10. CoQ10 was reported to reduce striatal injury by MPTP toxicity in mice (Beal, 1999) and was proven to reduce reactive oxygen species in models of oxidative stress (Somayajulu et al., 2005). In regard to neurological diseases, most studies on the neuroprotective effects of CoQ10 have been confined to neurodegenerative disease and only a few studies have reported the neuroprotective effects of CoQ10 against hypoxia in neuronal cells: namely, it has been reported that the neuroprotective effects of CoQ10 were observed in animal models of cerebral ischemia (Ostrowski, 1999) and administration of CoQ10 prevented ischemic damage in rat model of ischemic stroke (Abd-El-Fattah et al., 2010; Grieb et al., 1997). Nevertheless, there has been no report about the effects of CoQ10 on NSCs under hypoxia. We hypothesized that CoQ10 might have antiapoptotic effect and prevent hypoxic damage of NSCs. The PI3K/Akt pathway might be contributed with this mechanism. In this study, we observed that viability was decreased and expression of HIF-a was increased in NSCs after exposure of hypoxia (Figs. 1 and 2(A and B). Treatment of CoQ10 increased cell viability of NSCs under hypoxic condition in a concentrationdependent manner up to 1 mM (Fig. 2E and F, and Fig. 3). Overall, 1 mM CoQ10 was the best optimal dosage for neuroprotection of NSCs under hypoxic condition in the present study. Pretreatment of LY294002, a PI3K inhibitor, partially blocked the neuroprotective effects of CoQ10 (Fig. 2G), which indicates that the activation of the PI3K/Akt plays important roles in the neuroprotective effects of CoQ10 against hypoxia. BrdU assay to analyze proliferative activity of NSCs showed that CoQ10 restored apoptosis after hypoxia (Fig. 4). In the

studies to confirm the neuroprotective mechanisms of CoQ10 against hypoxia injury in NSCs, we also found that treatment of CoQ10 effectively attenuated free radical production by hypoxia injury in NSCs (Fig. 5), significantly enhanced survival signaling proteins such as phosphorylated Akt, phosphorylated GSK-3b, and Bcl-2, and markedly inhibited death signaling proteins such as cleaved caspase-3 (Fig. 6). These findings supported that CoQ10 have a neuroprotective role in NSCs after ischemic injury by diminishing ROS production and inhibiting apoptosis. There were some limitations in this study: (1) because this study was performed under in vitro conditions, these results might be different from those under in vivo conditions or in clinical trials where more various factors were involved; (2) the dosage of CoQ10 used in our study might be slightly high so it might not be physiologically relevant; (3) we could not confirm the precise point of action of CoQ10 in NSCs under a hypoxic condition; and (4) we could not use the NSCs from the same origin in all studies. In conclusion, we have shown that CoQ10 have neuroprotective effects on NSCs against hypoxia and that the mechanism of neuroprotection was associated with the inhibition of apoptosis by attenuating free radical production. Neuroprotection of NSCs as well as neuron and glial cells is also important in acute ischemic stroke considering its potential role. These neuroprotective effects of CoQ10 on NSCs might provide the experimental basis for the clinical use for the treatment of ischemic cerebrovascular disease. Furthermore, our result might be theoretical ground for the use of CoQ10 in transplantation of NSCs, which is great concern.

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Fig. 6 – Immunoreactivities (IRs) of pAkt, pGSK-3b, Bcl-2, and Cleaved caspase-3 in neural stem cells (NSCs). Immunoreactivities (IRs) were assessed by western blotting. Data were expressed as ratio of the simultaneously assayed control group’s value and were compared using Tukey’s test after a one-way ANOVA (n¼ 5). Representative ECL radiographs of the immunoblots demonstrate that combined treatment with 1 lM CoQ10 increased the IRs of pAkt (A), pGSK-3b (B), and Bcl-2 (C) and decreased the IRs of cleaved caspase-3 (D) when compared with NSCs under hypoxia alone. po0.05 (when compared with control group), #po0.05 (when compared with NSCs under hypoxia without treatment). ##po0.01 (when compared with NSCs under hypoxia without treatment).

4.

Experimental procedure

4.1.

Materials

To create hypoxia, we used an anaerobic chamber (Anaerobic System Model 1025, Thermo Forma, Marietta, OH, USA).

Protein protease inhibitor cocktail, trypan blue solution, insulin, and DNase I were obtained from Sigma-Aldrich (St. Louis, USA). CoQ10 (Ubidecarenone) was a generous gift from Daewoong Bio Inc. (Korea). CoQ10 was dissolved in methyl chloride to 500 mM, diluted with DMSO to 100 mM, and then diluted in culture medium to the desired concentrations.

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4.2. Culture of neural stem cells, and production of a hypoxic environment All procedures using animals were performed in accordance with Hanyang University guidelines for the care and use of laboratory animals. Every effort was made to minimize the number of animals used and suffered. All the animals were used only once. Neural stem cells (NSCs) were isolated from rodent embryonic brains, cultured, and expanded. The protocol of NSC culture was previously described and widely used in previous studies (Chojnacki and Weiss, 2008; Currle et al., 2007; Studer et al., 1998). Briefly, rat embryos were decapitated at embryonic day 12 (E12) and the brains were rapidly removed and placed in a petri-dish half-filled with ice-cold Hank’s balanced salt solution (HBSS; 137 mM NaCl, 5.4 mM KCl, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 5.6 mM glucose, and 2.5 mM HEPES) (Gibco BRL, NY, USA). Single cells were dissociated from the whole cerebral cortexes, lateral ganglionic eminences, and ventral midbrains of fetal rats. The cells were plated at 2  104 cells/cm2 on culture dishes precoated with poly-L-ornithine/fibronectin in Ca2þ/Mg2þ-free PBS (GIBCO, Grand Island, NY, USA) and cultured in N2 medium (DMEM/F12, 4.4 IM insulin, 100 mg/l transferrin, 30 nM selenite, 0.6 IM putrescine, 20 nM progesterone, 0.2 mM progesterone, 0.2 mM ascorbic acid, 2 nM L-glutamine, 8.6 mM D(þ) glucose, 20 nM NaHCO3 (Sigma, St. Louis, MO)) supplemented with basic fibroblast growth factor (BFGF;10 ng/ml, R&D Systems, Minneapolis, MN). Cultures were maintained at 37 1C under a humidified 5% CO2 atmosphere for 4–6 days. Hypoxia was achieved in the anaerobic chamber. A gas mixture containing CO2 (5 mol%), O2 (0.2 mol%), and N2 (94.8 mol%) was flushed through the chamber for 2, 4, 8, and 24 h. This procedure maintained a non-fluctuating hypoxic environment below 1 mol% O2 (Li et al., 2003). To see whether the NSCs were affected by hypoxia delivered by this procedure, we measured hypoxic-inducible factor-alpha (HIF-a), which is well-known to be induced by hypoxia, by western blotting. To evaluate the effect of CoQ10 itself on NSCs, they were treated with several concentrations of CoQ10 alone for 8 h. NSCs were treated with several concentrations of CoQ10 in hypoxic chamber for 8 h, as described above. Finally, to examine the role of the PI3K/Akt pathway in the neuroprotective effects of CoQ10, we also treated NSCs with 10 mM LY294002, a PI3K inhibitor, 1 h prior to the treatment with CoQ10 and hypoxia. The NSCs were divided into the following four groups: control (group 1), hypoxia for 8 h (group 2), hypoxiaþ1 mM CoQ10 for 8 h (group 3), and 10 mM LY294002 for 9 hþhypoxia and 1 mM CoQ10 for 8 h (group 4).

4.3. Trypan blue staining and the lactate dehydrogenase (LDH) release assay for measuring cell viability For trypan blue staining, 10 ml samples of cells were incubated with 10 ml of trypan blue solution for 2 min. Unstained live cells were counted with a hemocytometer. A colorimetric assay kit (Roche Boehringer–Mannheim, IN, USA) was used to quantify LDH released from cultured NSCs according to the

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manufacturer’s instructions. Cell viability was assessed using an ELISA plate reader at 490 nm with a reference wavelength of 690 nm. All results were normalized to the OD of an identical well without cells.

4.4.

DAPI and TUNEL staining to evaluate apoptosis

NSCs were seeded on collagen-coated 13 mm diameter glass cover slips and treated (1) with CoQ10 without exposure to hypoxia, (2) with exposure to hypoxia for 8 h, or (3) with exposure to hypoxia in the presence of 1 mM CoQ10 for 8 h. The, cells were then rinsed twice with PBS, air-dried, and fixed with 4% paraformaldehyde in PBS for 1 h at room temperature. Apoptotic cell death was identified by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) (Roche Boehringer–Mannheim, IN, USA). To monitor intact, condensed, and fragmented nuclei, TUNEL-stained cells were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI, Sigma, Saint Louis, MO, USA; 100 mg/ml in PBS) for 20 min, washed several times with PBS, and mounted on glass slides with Moviol 4-88 solution. The cells were observed under an Olympus fluorescence microscope with the appropriate excitation wavelengths for TUNEL and DAPI staining (Jiang et al., 2000).

4.5.

BrdU cell proliferation assay

After exposure to hypoxia for 8 h with different concentrations of CoQ10, NSCs were collected immediately. Then, NSCs were incubated in BrdU-labeling medium (10 mM BrdU) for 1 h, cell proliferation was measured using a BrdU Labeling and Detection Kit (Roche Boehringer–Mannheim, IN, USA), according to the manufacturer’s instructions. The results were calculated as the proportion of stained cells among 1000 cells counted under a light microscope at 400  magnification.

4.6.

Determination of free radical production

To measure free radical production, NSCs were exposed to hypoxia for 8 h and then incubated with the fluorescent probe 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) (Molecular Probes Inc., Eugene, OR, USA) for 30 min. Then, NSCs treated with different concentrations of CoQ10 under hypoxia and the control group were incubated with 0.1 DCFHDA and 1 mM CoQ10. After incubation at 37 1C for 30 min, we washed the cells with PBS three times. DCFH-DA freely crosses cell membranes and is hydrolyzed by cellular esterases to 20 ,70 -dichlorodihydrofluorescein (DCFH2), which is oxidized to the fluorescent 20 70 -dichlorofluorescein (DCF) in the presence of peroxides; this means that DCF fluorescence mainly indicates the level of intracellular hydrogen peroxide not superoxide. The accumulation of DCF in cells is measured as an increase in fluorescence at 525 nm, when the sample is excited at 488 nm by a Microplate Fluorescence Reader (FL600) (D.I. Biotech Ltd., Seoul, Korea) (Lee et al., 2011).

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4.7.

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Western blot analyses

Levels of phosphorylated Akt (pAkt) (Ser473), phosphorylated glycogen synthase kinase-3b (GSK-3b) (Ser9), B cell lymphoma-2 (Bcl-2), and cleaved caspase-3 (Asp175) were analyzed by Western blotting. Western blotting of several intracellular signals was performed immediately after treatment for 8 h. Briefly, 5  106 cells were washed twice in cold PBS, incubated for 10 min on ice in lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 100 mg/ ml phenyl methyl sulfonyl fluoride (PMSF), 50 ml/ml aprotinin, 1% Igepal 630, 100 mM NaF, 0.5% sodium deoxy choate, 0.5 mM EDTA, 0.1 mM EGTA); unbroken cells and nuclei were pelleted by centrifugation for 10 min at 2000  g and the lysates were cleared by centrifugation at 10,000  g. The antibodies used were: anti-pAkt (1:500, Cell Signaling, Beverly, MA, USA), anti-pGSK-3b (Ser9) (1:1000, Santa Cruz Biotech, Santa Cruz, CA, USA), anti-Bcl-2(1:1000, cell signaling,), and anti-cleaved caspase-3 (Asp 175) (1:1000, cell signaling, Beverly, MA, USA). The membranes were washed with Trisbuffered saline containing 0.05% Tween-20 (TBST), and then processed using HRP-conjugated anti-rabbit antibody or antimouse antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA) followed by ECL detection (Amersham Pharmacia Biotech,) (Lee et al., 2009). The blots were quantified with an image analyzer (Bio-Rad, Quantity One-4,2,0).

4.8.

Statistical analysis

All data are presented as means7standard deviations of five or more independent experiments. The viabilities of different treatment groups were compared with Tukey’s test after oneway ANOVA. Levels of apoptosis and free radicals in different treatment groups, and the western blotting results, were compared using Tukey’s test after two-way ANOVA. P-values less than 0.05 were considered statistically significant.

Acknowledgments This work was supported by the NanoBio R&D Program of the Korea Science and Engineering Foundation, funded by the Ministry of Education, Science and Technology (2007-04717) and a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A101712).

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