reperfusion injury is mediated by the JNK pathway

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The protective effect of nordihydroguaiaretic acid on cerebral ischemia/reperfusion injury is mediated by the JNK pathway Yu Liu1 , Huan Wang1 , Yanmei Zhu, Li Chen, Youyang Qu, Yulan Zhu⁎ Department of Neurology, the Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin, Heilongjiang 150086, China

A R T I C LE I N FO

AB S T R A C T

Article history:

Nordihydroguaiaretic acid (NDGA) is a powerful antioxidant and/or lipoxygenase (LOX)

Accepted 13 January 2012

inhibitor which is isolated from Larrea tridentate. NDGA has been shown to have

Available online 24 January 2012

neuroprotective effects both in vitro and in vivo experiments. However, little is known regarding NDGA's protective mechanism in ischemia/reperfusion (I/R) injury. We

Keywords:

therefore investigated the potential protective effects of NDGA and explored the

Nordihydroguaiaretic acid

underlying mechanisms. Oxygen-glucose deprivation (OGD) was performed in cultured rat

12/15-lipoxygenase

cortical neurons for 60 min. The effect of NDGA on OGD induced cell death and apoptosis

Hydroxyeicosatetraenoic acid

was examined at 24 h after reperfusion. Western blot was used to analyze the expression

Cerebral ischemia/

of p-c-jun and p-JNK. Exogenous 5-, 12-, 15-hydroxyeicosatetraenoic acid (HETE) was added

reperfusion injury

respectively to the cells to investigate the contribution of the products of LOX to the c-Jun N-

JNK

terminal protein kinase (JNK) pathway. Rats were injected intraperitoneally with NDGA before being subjected to middle cerebral artery occlusion (MCAO). At 24 h after reperfusion, neurological deficit, brain infarct volume and the expression of p-c-jun and p-JNK were measured. The results showed that NDGA increased cell viability and inhibited apoptosis after OGD in neurons. NDGA suppressed the expression of p-c-jun and p-JNK in cortical neurons, whereas exogenous 12-, 15-HETE attenuated this effect. NDGA improved neurological deficit, reduced infarct volumes, and downregulated the overexpression of p-c-jun and p-JNK after MCAO and reperfusion. In conclusion, these results suggest that NDGA's protective effect against I/R injury is mediated by the suppression of JNK pathway. This effect is probably due to its 12/15LOX inhibitor property. © 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Nordihydroguaiaretic acid (NDGA) is a typical lignin isolated from the creosote bush, Larrea tridentata which is mainly

distributed in the arid regions of northern Mexico and Southwestern United States (Stege et al., 2011). NDGA has been identified to have multiple biological activities, such as antitumor, anti-inflammation and anti-virus (Lu et al., 2010). It

⁎ Corresponding author. Fax: + 86 451 86605656. E-mail address: [email protected] (Y. Zhu). Abbreviations: NDGA, nordihydroguaiaretic acid; LOX, lipoxygenase; I/R, ischemia/reperfusion; OGD, oxygen-glucose deprivation; HETE, hydroxyeicosatetraenoic acid; JNK, c-Jun N-terminal protein kinase; Aβ, amyloid β-peptide; AA, arachidonic acid; DMSO, dimethyl sulfoxide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; p-JNK, phospho-JNK p-c-jun, phospho-c-jun; TNF-α, tumor necrosis factor-α; MCAO, middle cerebral artery occlusion; MTT, 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide; TTC, 2, 3, 5-triphenyltetrazolium chloride 1 The first and second authors contribute equally to this paper. 0006-8993/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.01.031

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also exerts neuroprotective effects in the models of both cultured neurons and cerebral ischemic rats, probably due to its lipoxygenases (LOXs) inhibitor properties. Studies using cerebral neurons from rats or mice demonstrated that NDGA prevents neuronal injury caused by oxidative stress (Cardenas-Rodriguez et al., 2009; Lee et al., 2011). A study using hippocampal neurons from rats demonstrated that NDGA prevents against amyloid β-peptide (Aβ) induced neuronal apoptosis (Goodman et al., 1994). NDGA has also been proven to be a potent anti-ischemia/reperfusion (I/R) injury agent in animal models through various antioxidant pathways (Chu et al., 2010; Shishido et al., 2001). However, the detailed protective mechanisms and signaling pathways of NDGA are not clear. LOXs are non-heme iron dioxygenase enzymes that incorporate oxygen into specific sites of polyunsaturated fatty acids and are mainly classified as 5-,12- or 15-LOXs depending on the site of incorporation (Ivanov et al., 2010). The leukocyte-type 12-LOX and the 15-LOX-1 can form similar products from common substrates and are often referred to in some literatures as 12/15-LOX (Dobrian et al., 2011). LOXs play an important role in the pathogenesis of cerebral infarction, and recent studies have demonstrated that inhibition of LOXs showed protective effect against cerebral I/R injury (Dobrian et al., 2011; Pergola and Werz, 2010). Zhou et al. (2006) reported that cerebral ischemia leads to enhanced expression of 5-LOX, which might result in the expansion of ischemic brain damage. The 5-LOX inhibitors have been showed to protect brain against ischemic damage in an animal model of cerebral ischemia (Jatana et al., 2006; Tu et al., 2010). van Leyen et al. demonstrated increased concentrations of 12/15-LOX in the neurons surrounding an infarct in a murine model of transient focal ischemia, and showed that intraperitoneal injection of the 12/15-LOX inhibitor baicalein prior to the ischemic event leads to reductions in infarct size and brain edema (Jin et al., 2008; van Leyen et al., 2006). Thus inhibition of arachidonic acid (AA) metabolism could attenuate brain injury and provide therapeutic strategy for cerebral ischemia. The c-Jun N-terminal protein kinase (JNK) signaling pathway is implicated in neuronal apoptosis triggered by focal or global ischemia (Guan et al., 2006). Several studies have demonstrated that activation of JNK signaling may play a critical role in brain I/R injury (Gao et al., 2005; Guan et al., 2006; Ye et al., 2010). Therefore, JNK is an important therapeutic target for prevention of neuronal death induced by brain ischemia. Lebeau et al. reported that the 12/15-LOX metabolite, 12-hydroxyeicosatetraenoic acid (12-HETE) promotes c-Jun dependent neuronal apoptosis; blockade of 12-LOX expression protects cortical neurons from Aβinduced apoptosis through disruption of a c-Jun dependent apoptosis pathway (Lebeau et al., 2004). As a potent LOX inhibitor, NDGA can block the enzymatic activity of 12/15LOX and decrease the production of its metabolites (Lu et al., 2010). We hypothesized that NDGA's neuroprotective effect involves the inhibition of 12/15-LOX and subsequent suppression of JNK pathway after cerebral I/R injury. In this work, we provide evidence that this is indeed the case by using an oxygen-glucose deprivation (OGD) cell model and a focal cerebral I/R rat model.

2.

Results

2.1.

NDGA protects against OGD induced neuronal death

After 60 min of OGD and 24 h reoxygenation, cell viability decreased to 49.0± 2.63% of the control. NDGA treatment significantly reduced cell death induced by OGD and reperfusion (63.5± 3.16% of the control) (Fig. 1).

2.2. NDGA apoptosis

protects

primary

cortical

neurons

from

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was undertaken to determine the effect of NDGA on neuron apoptosis. OGD induced the increase of TUNEL-positive neurons and the addition of NDGA (20 μM) relieved the increase of TUNEL-positive cells induced by OGD when compared with DMSO (dimethyl sulfoxide) treated group (Fig. 2).

2.3. NDGA reduces JNK and c-jun activation in neurons and 12-HETE attenuates the effects We next investigated whether the addition of NDGA suppresses JNK pathway in cortical neurons. As shown in Fig. 3, treatment of rat cortical neurons with OGD and reoxygenation upregulated p-JNK and p-c-jun levels in DMSO + OGD group. Meanwhile, the administration of NDGA (20 μM) significantly suppressed the expression of p-JNK and p-c-jun compared with DMSO treated group. To examine whether the suppressive effects of NDGA on cJNK and p-c-jun are mediated with inhibition of 12/15-LOX we further explored the role of 12-HETE, the preferential

Fig. 1 – The effects of NDGA on neuronal survival in rat primary cortical neurons exposed to OGD and reoxygenation. Cortical neurons were pretreated with NDGA in glucose-free HBSS in a hypoxia chamber. Then the plates were restored to normoxic conditions. Control culture plates were exposed to oxygenated HBSS containing glucose in normoxic condition. All values are denoted as mean± SD from three independent batches of cells.*P < 0.05 versus the ODG+ DMSO group.

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Fig. 2 – TUNEL staining and effects of NDGA on OGD-induced apoptosis of rat cortical neurons. (A) The cells were double-stained with DAPI to identify nuclei, apoptotic neurons were labeled using TUNEL as indicated in the Experimental procedure. Then the cells were treated with TUNEL staining and imaged by fluorescent microscope. An overlay of both signals (Ovl) is also presented. Arrows indicate cells showing an overlay of TUNEL and DAPI signals. The content of TUNEL-positive cells was calculated as the ratio of TUNEL-positive cells to the total number of neurons. (B) Quantitative analysis of TUNEL-positive cell content in different groups. *P < 0.05 compared with DMSO treated group.

metabolite of 12/15-LOX in rat brain (Watanabe et al., 1993), on p-JNK and p-c-jun. The results showed that 12-HETE (1 μM) attenuated the inhibitory effects of NDGA on the expression of p-JNK and p-c-jun in cortical neurons subjected to OGD and reoxygenation (Fig. 3).

2.4. Effects of other HETE isoforms on p-JNK and p-c-jun in neurons We also administered 5-HETE or 15-HETE to observe whether these HETE isoforms could attenuate the inhibitory effect of

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Fig. 3 – Effects of NDGA on the JNK and c-jun activation in primary cortical neurons and 12-HETE attenuates the effects. NDGA significantly suppressed the expression of p-JNK and its downstream transcription factor p-c-Jun compared with the DMSO treated group. 12-HETE attenuated the inhibitory effects of NDGA on the expression of p-JNK and p-c-jun. (A) Representative photos of p-JNK and p-c-jun. (B) Quantitative analysis of the ratio of p-JNK to JNK and p-c-jun to c-jun. (*P < 0.05).

NDGA on JNK pathway. As illustrated in Fig. 4, the expression of p-JNK and p-c-jun in 15-HETE and NDGA treated group is notably higher than that in the single NDGA treated group.

Whereas, there were no significant changes of p-JNK and pc-jun in 5-HETE and NDGA treated group versus single NDGA treated group.

Fig. 4 – Effects of 5-HETE or 15-HETE on the activation of JNK and c-jun in primary cortical neurons. NDGA suppressed the expression of p-JNK and p-c-Jun compared with the DMSO treated group. 15-HETE attenuated the inhibitory effects of NDGA on the expression of p-JNK and p-c-jun. There were no significant changes of p-JNK and p-c-jun in 5-HETE and NDGA treated group. (A) Representative photos of p-JNK and p-c-jun. (B) Quantitative analysis of the ratio of p-JNK to JNK and p-c-jun to c-jun. *P < 0.05 compared with OGD + NDGA group.

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Table 1 – Neurological deficit scores. Score

Sham control I/R + DMSO I/R + NDGA

0

1

2

3

4

7 – –

– 1 5

– 4 2

– 2 –

– – –

Average score 0 2.14 ± 0.69 1.28 ± 0.48⁎

Compared with DMSO treated group (I/R + DMSO), neurological deficit scores were reduced in NDGA treated group (I/R + NDGA). (*P < 0.05; n = 7 per group)

2.5.

NDGA improves neurological deficits after I/R injury

Table 1 shows neurological scores after 90 min of MCAO and 24 h of reperfusion in the different experimental groups. Neurological deficit scores were significantly higher in I/R group compared with Sham control. NDGA treatment (35 mg/ kg per rat) significantly improved the neurological deficits in I/ R + NDGA group compared with I/R + DMSO group (Table 1).

2.6.

NDGA reduces cerebral infarct volume

There was no detectable infarction in the sham control group. The infarct volume percent of NDGA-treated group was 19.26 ± 3.68%, which was significantly smaller (*P < 0.05) than the DMSO-treated group (25.70 ± 3.63%, n = 6) (Fig. 5).

2.7. brain

NDGA inhibits the activation of JNK and c-jun in rat

To examine whether the protective effects of NDGA are mediated by the JNK pathway, Western blotting of rat brain homogenates was performed with antibodies specific for phosphorylated active forms of JNK and c-jun. I/R injury significantly induced the activation of phospho-JNK (p-JNK) and phospho-c-jun (p-c-jun) in the DMSO treated group and the expression of p-JNK and p-c-

Fig. 5 – Effects of NDGA on infarct volume at 24 h of reperfusion after 90 min of transient MCAO. Rats were pretreated with NDGA or DMSO 30 min before ischemia. Infarct volume in the brain section was significantly reduced by NDGA treatment (*P < 0.05 n = 6).

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jun was significantly decreased in the NDGA treated group (Fig. 6).

3.

Discussion

In this study, we have shown that NDGA protects against I/R injury both in vitro and in vivo. The study also provides evidence that the mechanism of this protective effect is associated with the inhibition of 12/15-Lipoxygenase and suppression of JNK pathway. Our study demonstrated that NDGA (20 μM) reduced neuron death and decreased the percentage of TUNEL-positive cells induced by OGD and reperfusion (Figs. 1, 2). Some reports indicate that NDGA induces apoptosis in a range of tumor cell lines, including breast cancer, pancreatic carcinoma, multiple myeloma and HL-60 cells(Park et al., 2004). On the contrary, others showed NDGA's capacity to block apoptosis either by tumor necrosis factor-α (TNF-α) or CD95 ligand(Meyer et al., 2008; Wagenknecht et al., 1998). Here we demonstrated for the first time that NDGA inhibits rat cortical neurons from apoptosis induced by OGD. The result provided a new evidence to support that NDGA showed different effects on apoptosis according to the different type of cell lines. We also demonstrated that NDGA significantly elevated neurological deficit scores (Table 1) and decreased infarction volume (Fig. 5) at 24 h of reperfusion after transient MCAO. These results demonstrated that NDGA has neuroprotective effects both in cell and animal models which agree with previous reports. Activation of JNK has been shown to play an important role in neuronal apoptosis after cerebral I/R injury. One study showed that JNK phosphorylates Bax and enhances its mitochondrial translocation, where it then augments proapoptotic caspase activation. Elevated phospho-JNK colocalizes with TUNEL positive apoptotic neurons in mouse focal cerebral ischemia and inhibition of JNK protects injured brain (Okuno et al., 2004). A number of neuroprotectants, including SP600125 (Guan et al., 2006), Edaravone (Wen et al., 2006), and Emodin (Liu et al., 2010) exert their protective effect by inhibiting the JNK pathway. Kuan et al. demonstrated that consistent with these pharmacologic data, ischemic brain injury is reduced in knockout mice lacking the neuronspecific JNK3 isoform (Kuan et al., 2003). The present study showed that NDGA's protective effect also relies on the inhibition of the JNK pathway (Figs. 3, 6), which agrees with the results of previous studies. Several reports have shown that 12/15-LOX increased rapidly in the tissue surrounding the infarct area following MCAO and reperfusion, suggesting that 12/15-LOX might be involved in I/R injury. Besides, 12/15-LOX has been described mainly in neurons and also in some glial cells throughout the cerebrum, basal ganglia, and hippocampus, and its metabolic product levels are increased in an experimental model of brain I/R injury (Pratico et al., 2004). Many studies have demonstrated that the 12/15-LOX products, 12-HETE and 15-HETE both stimulate JNK activity in different type of cells (Bleich et al., 1997; Wen et al., 1997).Lebeau et al. demonstrated that 12-HETE induces a concentration-dependent activation of JNK in cortical neurons. Inhibition of 12-LOX decreases the production of 12-HETE and suppresses JNK

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Fig. 6 – Effects of NDGA on JNK and c-jun activation in animal I/R model. Representative Western Blots showing significantly decreased expression of p-JNK and its downstream transcription factor p-c-Jun in the NDGA group compared with the DMSO treated group. (A) Representative photos of p-JNK and p-c-jun. (B) Quantitative analysis of the ratio of p-JNK to JNK and p-c-jun to c-jun. (*P < 0.05 n = 6).

dependent apoptosis (Lebeau et al., 2004). Our study showed that OGD and reperfusion induced activation of JNK and its downstream transcription factor c-jun, addition of NDGA suppressed the overexpression of p-JNK and p-c-jun (Fig. 3). As a potent LOX inhibitor, NDGA inhibited 12/15-LOX and reduced the production of endogenous 12-HETE and 15-HETE. Administration of the cortical neurons with exogenous12-HETE and 15HETE attenuates the effect of NDGA on JNK and c-jun activation (Figs. 3, 4), which suggested that NDGA's suppression effect on JNK pathway may be related to its 12/15-LOX inhibitor property. 5-HETE is another product of AA which is metabolized by 5-LOX (Radmark and Samuelsson, 2010). In the present study, data showed that unlike 12-HETE and 15-HETE, 5HETE shows no significant effect on the expression of p-JNK and p-c-jun (Fig. 4). NDGA is a non-selective LOX inhibitor that blocks the enzymatic activity of not only 12/15-LOX but also 5-LOX (Lu et al., 2010). A recent study demonstrated that NDGA protects I/R induced rat brain injury through inhibiting 5-LOX and inflammatory responses mediated by 5-LOX metabolites (Chu et al., 2010). Therefore, detailed mechanisms of protective effects of NDGA through 5-LOX inhibition pathway remain to be further defined. A range of experimental approaches has revealed that, as an effective antioxidant, NDGA may exert its neuroprotective effect through various mechanisms. It has been reported that the antioxidant activity of NDGA strongly diminishes neuron injury related cytokine secretion by dendritic cells (Aktan et al., 1993). Furthermore, NDGA has been identified as a compound capable of inducing glutamate uptake and upregulation of expression levels and activity of the glutamate transporter EAAT2 (GLT-1) in mice (Boston-Howes et al., 2008). NDGA has also been proven to be a potent agent that protects against oxidative stress in cerebellar neurons by activation of the nuclear factor erythroid-2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) axis (Guzman-Beltran et al., 2008). Undoubtedly, the antioxidant

ability of NDGA may also contribute to the protective effect on I/R injury we are observing. Taken as a whole, our results have shown that NDGA protects cerebral and neuronal I/R injury via the JNK pathway and the results of in vitro experiments have demonstrated that such an effect is likely to be mediated through the inhibition of 12/15-LOX. A complete understanding of the mechanisms of NDGA's protective effects will greatly facilitate the realization of its full clinical potential.

4.

Experimental procedure

4.1.

Materials and animals

NDGA and 5-,12-,15-HETEs were purchased from Cayman Chemical Company (Ann Arbor, Michigan, USA). Antiphospho JNK rabbit polyclonal antibody and anti-phospho cJun rabbit polyclonal antibody were obtained from Cell Signaling Technology (Beverly, MA, USA). Adult male Sprague–Dawley rats (body weight, 220–280 g) were used in the study. The experimental protocol and procedures were in accordance with the regulations of the ethics committee of Harbin Medical University.

4.2.

Primary neuronal culture

Primary cultures of cortical neurons were prepared from fetal day 17 Sprague–Dawley rats. The cortices were chopped into small pieces and exposed to a 0.125% trypsin solution for 30 min at 37 °C. Fetal bovine serum and trypsin inhibitor were used to stop digestion. Then tissues were mechanically dissociated by trituration with a fine-tipped pipette. Dissociated cells were seeded in 24-well plates previously coated with poly5 D-lysine at a density of 3 × 10 cells per well and then incubated

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in Neurobasal medium (Invitrogen, Carlsbad, CA) with 2% B-27 supplement, Glutamax (1:100) (Invitrogen, Calsbad, CA), penicillin, and streptomycin. The medium was changed 6 h after plating, and half of the medium was changed every 3 d. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 until their use.

4.3.

Oxygen-glucose deprivation (OGD) and reoxygenation

Cell cultures were rinsed twice with glucose-free Hank's balanced salt solution (HBSS, 5.4 mM KCl, 140 mM NaCl, 2 mM CaCl2, 10 mM HEPES, 30 μM glycine, pH 7.4). Cultured neurons were incubated in the pre-gassed HBSS buffer and then transferred into an anaerobic chamber which was previously flushed with mixed gas of 5% CO2 and 95% N2. Cells were maintained in the hypoxic chamber at 37 °C for 60 min. The OGD treatment was stopped by replacing HBSS with Neurobasal medium supplemented with B-27. The plate was placed back to normoxic conditions and incubated for 24 h for reoxygenation. Control culture cells were incubated with the HBSS buffer supplemented with 15 mM glucose in a humidified incubator with normoxia during the same period as the OGD cultures.

4.4.

Cell viability assay

NDGA (20 μM) was added to the culture medium 30 min before treatment with OGD. The concentration of NDGA was chosen on the basis of previous literature (Cardenas-Rodriguez et al., 2009; Lee et al., 2011).Neuronal cell injury was determined by 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide (MTT) assay. At 24 h of OGD and reoxygenation treatment in cultured cortical neurons, 10 μL of MTT(0.5 mg/ml) was added per well and incubated at 37 °C for 4 h. Absorbance was subsequently measured at 570 nm with a microplate spectrophotometer. Untreated cells were considered as control and the culture medium without cells in the presence of MTT solution was used as solution background. Cell viability was expressed as the percentage of the untreated control. For the MTT assay, there were eight samples in each group, and the experiment was repeated at least 3 times.

4.5.

TUNEL assay

The cells were fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4) at 24 h after reperfusion. Apoptotic cells were evaluated through TUNEL labeling using the in situ Death Detection Kit POD (Roche Applied Science, Mannheim, Germany). The TUNEL assay was performed according to the manufacturer's protocol. Stained slides were visualized by confocal laser scanning microscopy. Sixteen to eighteen fields in each coverslips were randomly selected and counted. TUNEL-positive cells were considered to be undergoing apoptosis. The percentage of apoptosis was determined above the total cell number with 4′, 6-diamidino-2-phenylindole (DAPI) staining.

4.6.

Middle cerebral artery occlusion (MCAO) model

The MCAO model was performed as described previously (Xu et al., 2008). Briefly, after a rat was anesthetized with an

79

intraperitoneal injection of chloral hydrate (400 mg/kg), it was placed in the supine position with the limbs taped to the operation table. After midline skin incision, the right external carotid artery was exposed, and its branches were ligated. A nylon thread coated with silicon was introduced into the internal carotid artery through the common carotid artery and advanced until faint resistance was felt. After 90 min of transient MCAO, blood flow was restored by withdrawal of the nylon thread to allow reperfusion. Sham-operated control rats received the same procedure except filament insertion. The rectal temperature was maintained at 37± 0.5 °C and all surgical procedures were performed under sterile conditions.

4.7.

Animal experimental groups and drug administration

In each experiment, rats were randomly divided into three groups (n = 6): (1) Sham-operated group (SH); (2) DMSOtreated I/R group; (3) NDGA-treated I/R group. NDGA-treated group was administered 35 mg/kg NDGA dissolved in DMSO intraperitoneally, 30 min before ischemia. The dose of the drug was decided by referring the previous report (Lambert et al., 2002), and our preliminary study. DMSO-treated group received equal volume of DMSO.

4.8.

Neurological function evaluation

A neurological test was administered by the same examiner blinded to the experimental groups at 24 h after reperfusion. The scoring system was based on the five-point scale system described by Cui et al. (2010). 0: normal spontaneous movements; 1: left front leg was flexed but no circling clockwise; 2: circling clockwise; 3 spin clockwise longitudinally; and 4: unconsciousness and no response to noxious stimulus.

4.9.

Evaluation of infarct volume

To examine the effect of NDGA on infarct size after MCAO and reperfusion, the rats were executed and forebrains were removed and divided into 6 coronal (2 mm) sections after 90 min of ischemia and 24 h of reperfusion with DMSO or NDGA (n = 6 for each group) treatment. The coronal sections were stained with saline containing 2% 2, 3, 5triphenyltetrazolium chloride (TTC) at 37 °C for 30 min, after which sections were fixed in 10% neutralized formalin. The infarction volume is presented as a volume percentage of the infarction compared with the contralateral hemisphere. The percent hemispheric infarct volume was calculated as described by Zhang et al. (2008).

4.10.

Western blot analysis

5-HETE, 12-HETE, 15-HETE was added respectively to the conditioned medium for 30 min at a final concentration of 1 μM. This concentration was chosen based on our preliminary experiments. Rat primary cortical neurons were harvested in a buffer containing Tris 50 mM (pH 7.4), NaCl 150 mM, 1% Triton X-100, EDTA 1 mM, and PMSF 2 mM. To assess the effects of NDGA on the expression of p-c-jun and p-JNK in vivo, NDGA treated rats were sacrificed after 90 min of ischemia and 24 h of reperfusion. The right hemisphere

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was quickly removed and pulverized into powder in liquid nitrogen. Brains were homogenized in the lysis buffer as described above and incubated for 30 min on ice. The cell or brain extracts were centrifuged at 13,000 rpm for 15 min at 4 °C, and the supernatants used for experiments. The protein concentrations in the supernatant were determined with the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA) with bovine serum albumin as standard. 50 μg of protein was subjected to 12% SDS-PAGE and then transferred to nitrocellulose membranes. Western blot analysis was carried out with antibodies against p-c-jun and p-JNK. Immunoreactive bands were identified, and a densitometric analysis was performed with an enhanced chemiluminescence detection system (Amersham, USA).

4.11.

Statistical analysis

Differences between groups were determined with the Student's t test for infarct volume; differences among groups were compared by one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test if there was a significant difference between groups. All the data are expressed as mean± SD.P<0.05 was considered statistically significant.

Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81171077), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20102307110009), and the Special Fund for Science and Technology Innovative Talents of Harbin City (No. 2010RFXXS022). We would like to thank Professor Daling Zhu from College of Pharmacy, Harbin Medical University for technical assistance.

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