Neuroprotective effect of the stearic acid against oxidative stress via phosphatidylinositol 3-kinase pathway

Chemico-Biological Interactions 160 (2006) 80–87

Neuroprotective effect of the stearic acid against oxidative stress via phosphatidylinositol 3-kinase pathway Ze-Jian Wang a , Guang-Mei Li a , Bao-Ming Nie b , Yang Lu b , Ming Yin a,∗ a b

School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, PR China

Received 26 October 2005; received in revised form 16 December 2005; accepted 16 December 2005 Available online 31 January 2006

Abstract Stearic acid is a long-chain saturated fatty acid consisting of 18 carbon atoms without double bonds. In the present study, we reported the neuroprotective effects and mechanism of stearic acid on cortical or hippocampal slices insulted by oxygen–glucose deprivation, NMDA or hydrogen peroxide (H2 O2 ) in vitro. Different types of models of brain slice injury in vitro were developed by 10 min of oxygen/glucose deprivation, 0.5 mM NMDA or 2 mM H2 O2 , respectively. After 30 min of preincubation with stearic acid (3–30 ␮M), cortical or hippocampal slices were subjected to oxygen–glucose deprivation, NMDA or H2 O2 . Then the tissue activities were evaluated by using the 2,3,5-triphenyltetrazolium chloride (TTC) method. Population spikes were recorded in randomly selected hippocampal slices. Stearic acid (3–30 ␮M) dose-dependently protected brain slices from oxygen–glucose deprivation, NMDA and H2 O2 insults. Its neuroprotective effect against H2 O2 insults can be completely blocked by wortmannin (inhibitor of PI3K) and partially blocked by H7 (inhibitor of PKC) or genistein (inhibitor of TPK). Treatment of cortical or hippocampal slices with 30 ␮M stearic acid resulted in a significant increase in PI3K activity at 5, 10, 30 and 60 min. These observations reveal that stearic acid can protect cortical or hippocampal slices against injury induced by oxygen–glucose deprivation, NMDA or H2 O2 , and its neuroprotective effects are via phosphatidylinositol 3-kinase dependent mechanism. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Stearic acid; Brain ischemia; Anoxia; NMDA; Hydrogen peroxide

1. Introduction Stroke is a leading cause of death and disability in older people. Although several types of neuroprotectant drugs have been developed in clinical trials, few are in fact clinically effective. Energy failure, excitatory amino acid release, free radical and caspase-dependant cell death are known to contribute to stroke injury in the cortex, striatum and hippocampus. Therefore, new

∗

Corresponding author. Tel.: +86 21 62932228; fax: +86 21 62945529. E-mail address: [email protected] (M. Yin).

potential target should be identified for stroke therapy [1]. Epidemiological, clinical and biochemical studies have shown that different types of dietary fatty acids can modify the risks of many chronic diseases, such as cardiovascular diseases, stroke and inflammatory diseases. But the molecular and cellular mechanisms by which dietary fatty acids exert such effects are still not well understood [2,3]. The brain is rich in diverse fatty acids (saturated, monounsaturated and polyunsaturated) with chain lengths ranging from 16 to 24 carbons. Previously, exploration of long-chain fatty acids has mainly placed emphasis on investigating their fate after metabolized by cells. For example, intracellular fatty acids are processed

0009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2005.12.008

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into phospholipids, possibly affecting certain membrane functions, or are used to supply energy after decompositions by mitochondria. The body’s preferred energy source is glucose, which comes from carbohydrates, but it can also use fatty acids from fats and amino acids from proteins. However, the brain relies almost exclusively on glucose for oxidative ATP production [4]. Stearic acid is a long-chain fatty acid consisting of 18 carbon atoms without double bonds. It is biochemically classified as a saturated fatty acid for the purposes of food labeling and dietary recommendations. The behavior of stearic acid is especially unique with respect to its effects on serum cholesterol levels. Studies in humans and experimental animals have suggested that ingestion of stearic acid has a neutral or cholesterol-lowering effect, which is in contrast to the effects of lauric, myristic and palmitic acids [5]. In addition, a beneficial effect of stearic acid on clotting factors can result in a less thrombogenic state [6]. However, the molecular and cellular mechanisms by which it exerts these effects are still unclear. More and more evidences suggest that fatty acids can directly and indirectly modulate signaling pathways at multiple levels, which play important roles in regulating and executing cells death, survival or proliferation [3,7]. Elucidating the mechanism of this modulation can help us better understand how different types of dietary fats modify potential risks for stroke. Therefore, we hypothesize that the stearic acid may work as a modulator of signaling molecules to protect neuron from insults. Its neuroprotective mechanism has been primarily explored as well. 2,3,5-triphenyltetrazolium chloride (TTC) has been widely used in the assessment of brain ischemia. This method of quantitative measurement of extracted red formazan, by the solvent extraction and colorimetry on the brain slices incubated with TTC solution, has been introduced recently. This can be used as a simple, objective and sensitive technique in the assessment of brain ischemia in vitro. In the present study, we evaluated the effects of stearic acid on brain slice injury induced by OGD, NMDA or H2 O2 , using the TTC method.

2. Material and methods 2.1. Experimental animals Male Sprague-Dawley (SD) rats (150 ± 20 g, grade II, certification no. SCXK 2004-0005) were purchased from Shanghai SLAC Laboratory Co. Ltd.

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2.2. Reagents and drugs 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), wortmannin, [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenyl-thio) butadiene] (U0126), genistein, MK-801, RpcAMPs, 2,3,5-triphenyltetrazolium chloride (TTC) and N-methyl-d-aspartic acid (NMDA), were purchased from Sigma; Stearic acid (Chemical Reagent Co. Ltd., China) and all other reagents used were of analytical grade. Polyclonal anti-PI3K p85 subunit antibody was purchased from Santa Cruz Biotechnology; Stearic acid dissolved in ethanol was diluted in artificial cerebrospinal fluid (ACSF) before use. The final ethanol concentration in ASCF was less than 0.5%. 2.3. Brain slices preparation and drug exposure The slices were made as described by Bickler with several modifications [8,9]. SD rats were decapitated, whole brains were quickly removed and immersed in iced ACSF, which had the following composition (in mM): NaCl 119, KCl 2.5, CaCl2 2, MgSO4 1, NaH2 PO4 1.25, NaHCO3 26.2, glucose 10 (final pH 7.4). Brains were cut coronally into 400 ␮m thick sections with a vibrating tissue slicer (ZQP-86, Xiangshan, Zhejiang, China). Cortical and hippocampal slices were quickly isolated from the appropriate sections. Before being transferred to an experimental chamber, all slices were incubated in ACSF bubbled with 95% O2 and 5% CO2 at 32–34 ◦ C for 90 min recovery. After the process, several hippocampal slices were taken randomly into experimental chamber to monitor their viability by using cell recording. The stimulating electrode was planted in Schaffer collateral-commissural pathway of hippocampal slices and the recording electrode, in the CA1 region. Slices were incubated with different agents in oxygenated ACSF respectively for 3 h, and then the activities of the slices were evaluated by using the TTC staining method. 2.4. In vitro injury models of brain slices Brain slices were transferred to experimental chambers and randomly assigned to one of the following groups: (I) control group, in which slices were immersed in oxygenated ACSF at 34 ◦ C; (II) OGD group, in which slices were made anoxic by switching the ACSF into the glucose-free ACSF equilibrated with 95% N2 /5% CO2 . After 10 min insult, slices were reoxygenated in ACSF for 2 h; (III) OGD + stearic acid group, in which slices were incubated with different concentrations of stearic acid (3–30 ␮M) 30 min prior

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to and during OGD application; (IV) NMDA group, in which slices were subjected to 0.5 mM NMDA with magnesium-free artificial cerebrospinal fluid for 15 min (which had the following composition [in mM]: NaCl 143, KCl 5.4, CaCl2 1.8, NaH2 PO4 1.0, HEPES 2.4, glucose 5.6, pH 7.4). The decreased glucose concentration in artificial cerebrospinal fluid made brain slices more venerable to NMDA insult. Slices were reoxygenated in ACSF for 2 h; (V) NMDA + stearic acid group, in which slices were incubated with different concentrations of stearic acid (3–30 ␮M) 30 min prior to and during NMDA application; (VI) H2 O2 group, in which slices were subjected to 2 mM H2 O2 for 30 min; (VII) H2 O2 + stearic acid group, in which slices were incubated with different concentrations of stearic acid (3–30 ␮M) 30 min prior to and during H2 O2 application. 2.5. TTC staining [10–12] Slices were immersed in 2% TTC solution in a covered water bath shaker at 37 ◦ C for 1 h, the wet weight was measured after rinsed twice by saline. An extracted solution (50:50, mixture of ethanol/dimethylsulfoxide) was added with proportion of 20 ml per 1 g of slices. After 24 h extraction in a dark box, the extracted liquid was added to 96-well plates (200 ␮l per well), the absorbance (A) at 490 nm of each well was measured by the ELISA reader (Elx800uv , Bio-TEK). Percentage of tissue injury was calculated from the following equation: % tissue injury = 100% × [1 − (Ainjury / Acontrol )]. 2.6. Influence of inhibitors of different pathways on the neuroprotective effects of stearic acid [13]

had the following composition: Tris–HCl 50 mM (pH 7.0), Na3 VO4 1 mM, EGTA 1 mM, NaF 50 mM, PMSF 1 mM, 1% Nonidet P40, 20 ␮g/ml of leupeptin, 20 ␮g/ml of peptin and 20 ␮g/ml of aprotinin. The protein concentration of lysates was determined by the BCA method. The tissue lysates were centrifuged in a refrigerated microcentrifuge at 10,000 rpm for 10 min to remove debris; Supernatants were incubated with a 1:250 dilution of rabbit anti-p85 antibody for 1 h at 4 ◦ C, followed by protein A-Sepharose for an additional hour at 4 ◦ C. Immunoprecipitates were washed once with lysis buffer and thrice with PBS and resuspended in assay buffer (25 mM HEPES, 5 mM MgCl2 and 1 mM EGTA, pH 7.0). The assay mixture was composed of 10 ␮g lipids, 1:1:1 phosphatidylinositol–phosphatidylinositol 4,5-bisphosphate–phosphoserine and 150 ␮M ATP with 25 ␮Ci [␥-32 P] ATP. After incubation for 20 min at 37 ◦ C, the reaction was stopped. To measure PI3K activity, a TLC-based assay was employed using phosphatidylinositol 4-5-biphosphate (PIP2 ) as a substrate. The PI (3, 4, 5) P3 was quantitated by liquid scintillation counting. 2.8. Statistical analysis Four to six brain slices from three rats were used for each experimental group, and data collected from three to six independent experiments were expressed as means ± S.D. SPSS statistical software 10.0 for Windows was used and statistical analysis was evaluated by using one-way ANOVA and the SNK test. Statistical significance was assumed if P < 0.05. 3. Results 3.1. Brain slices preparation and drug exposure

Wortmannin (100 nM; an inhibitor of PI3K), H7 (50 (M; an inhibitor of protein kinase C), RpcAMPs (10 (M; an inhibitor of protein kinase A), genistein (50 (M; an inhibitor of PTK) or U0126 (20 (M; an inhibitor of ERK1/2) was incubated respectively with brain slices 1 h prior to and during OGD, NMDA or H2 O2 application. Brain slices were treated with different inhibitors in the presence or absence of stearic acid, to understand the molecular mechanisms by which stearic acid (30 ␮M) sustains neuronal survival. 2.7. Biochemical analysis of PI3K activity [14,15] Brain slices were quickly frozen in liquid nitrogen. The frozen tissues were sonicated in lysis buffer, which

The hippocampus is very sensitive to hypoxic and chemical hypoxia. Hypoxia and tissue injury are both associated with a decreased amplitude or disappearance of population spikes [8,9] (Fig. 1). Population spikes (PS) in CA1 regions of randomly selected hippocampal slices indicated the recovery of brain slices after incubation in ACSF for 90 min. These recovered slices were used for the subsequent experiments. None of the brain slices exhibited significant tissue damage (P > 0.05) after being incubated with different concentrations of stearic acid (3–30 ␮M), MK-801 (10 ␮M), RpcAMPs (10 ␮M), genistein (50 ␮M), H7 (50 ␮M), wortmannin (100 nM) or U0126 (20 ␮M) in ACSF for 3 h (Table 1).

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Fig. 1. Population spikes were recorded in hippocampal slices. Population spikes were recorded before and after 10 min OGD insult. Slices were prepared from animals weighing 150 ± 20 g (0.2 mA, 0.05 Hz, 0.1 ms in duration). (A) PS before OGD insult; (B) disappearance of PS after OGD insult.

3.2. Effects of stearic acid on brain slices insulted with OGD, NMDA or H2 O2

stearic acid treated groups insulted by H2 O2 (P < 0.05, Table 2).

After they were subjected to 10 min of OGD and 2 h of post-incubation, the activities of the brain slices were reduced by approximately 50%. There was a significant difference between the OGD and controls group (P < 0.01). Stearic acid dose-dependently protected against the decreases in tissue activity induced by OGD insult. Approximately 60% of tissue activity remained after brain slices were subjected to 15 min of NMDA and 2 h of post-incubation. Stearic acid dosedependently protected against the decreases in tissue activity induced by the NMDA insult. After they were subjected to 30 min exposure to H2 O2 , the activities of the brain slices were reduced by approximately 50%. A dose-dependently protection was also found in the

3.3. Influence of certain inhibitors on the neuroprotective effects of stearic acid

Table 1 Effects of agents on tissue activity of brain slices after 3 h incubation in ACSF Group

Dose

TTC (OD490 ) Cortical slices

Control Stearic acid

MK801 U0126 RpcAMPs Wortmannin H7 Genistein

– 3 ␮M 10 ␮M 30 ␮M 10 ␮M 20 ␮M 10 ␮M 100 nM 50 ␮M 100 ␮M

0.95 0.94 0.95 0.97 0.92 0.92 0.92 0.95 0.94 0.92

± ± ± ± ± ± ± ± ± ±

0.05 0.04 0.05 0.03 0.05 0.06 0.04 0.04 0.07 0.08

Hippocampal slices 0.55 0.53 0.54 0.55 0.54 0.54 0.51 0.56 0.54 0.54

± ± ± ± ± ± ± ± ± ±

0.05 0.05 0.02 0.03 0.02 0.02 0.03 0.05 0.03 0.02

Brain slices were incubated with different reagents in ASCF for 3 h, the viability of brain slices was evaluated by TTC staining method. n = 6. Data are mean ± S.D. b, P < 0.05 vs. control.

Certain selective cell-permeable kinase inhibitors provide useful tools to understand the molecular mechanisms by which stearic acid sustains neuronal survival. Those inhibitors did not significantly aggravate

Table 2 Effects of stearic acid on injury brain slices induced by three different insults Group

TTC (OD490 ) Cortical slices

Control OGD OGD + 3 ␮M stearic acid OGD + 10 ␮m stearic acid OGD + 30 ␮M stearic acid OGD + 10 ␮M MK-801 NMDA NMDA +3 ␮M stearic acid NMDA + 10 ␮M stearic acid NMDA + 30 ␮M stearic acid NMDA + 10 ␮M MK-801 H2 O2 H2 O2 + 3 ␮M stearic acid H2 O2 + 10 ␮M stearic acid H2 O2 + 30 ␮M stearic acid H2 O2 + 10 ␮M MK-801

0.98 0.52 0.64 0.78 0.92 0.80 0.60 0.78 0.84 0.92 0.86 0.49 0.60 0.73 0.85 0.62

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.07 0.05 c 0.06 c,e 0.09 c,f 0.12 a,f 0.06 b,f 0.05 c 0.08 c,e 0.05 b,f 0.04 a,f 0.04 b,f 0.05 c 0.05 c,e 0.06 c,f 0.08 b,f 0.08 c,e

Hippocampal slices 0.58 0.28 0.33 0.40 0.49 0.41 0.33 0.41 0.48 0.52 0.50 0.27 0.34 0.41 0.47 0.33

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.03 c 0.03 c 0.04 c,f 0.05 b,f 0.06 c,f 0.03 c 0.03 b,e 0.06 c,f 0.04 c,f 0.04 c,f 0.05 c 0.05 c 0.06 c,e 0.05 b,f 0.05 c,e

Brain slices were incubated with stearic acid 30 min prior to and during OGD, NMDA or H2 O2 application. The viability of brain slices was evaluated by using TTC staining method. n = 6. Data are mean ± S.D. b, P < 0.05 vs. control group; c, P < 0.01 vs. control group; e, P < 0.05 vs. injury group; f, P < 0.01 vs. injury group.

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Table 3 Influence of inhibitors on injury brain slices induced by three different insults

mainly mediated by the activation of PI3K signaling pathway.

Group

3.4. Biochemical analysis of PI3K activation

TTC (OD490 ) Cortical slices

Control OGD OGD + wortmannin OGD + H7 OGD + genistein OGD + RpcAMPs OGD + U0126 NMDA NMDA + wortmannin NMDA + H7 NMDA + genistein NMDA + RpcAMPs NMDA + U0126 H2 O2 H2 O2 + wortmannin H2 O2 + H7 H2 O2 + genistein H2 O2 + RpcAMPs H2 O2 + U0126

0.92 0.46 0.42 0.43 0.44 0.48 0.47 0.55 0.52 0.54 0.54 0.56 0.55 0.45 0.42 0.44 0.45 0.47 0.44

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.04 c 0.05 c,d 0.06 c,d 0.03 c,d 0.05 c,d 0.04 c,d 0.04 c 0.05 c,d 0.03 c,d 0.05 c,d 0.05 c,d 0.04 c,d 0.05 c 0.05 c,d 0.06 c,d 0.03 c,d 0.05 c,d 0.04 c,d

Hippocampal slices 0.57 0.27 0.24 0.25 0.24 0.29 0.29 0.35 0.32 0.32 0.35 0.36 0.35 0.25 0.24 0.24 0.24 0.24 0.24

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.05 c,d 0.05 c,d 0.04 c,d 0.03 c,d 0.05 c,d 0.04 c,d 0.04 c 0.04 c,d 0.06 c,d 0.06 c,d 0.05 c,d 0.04 c,d 0.04 c 0.05 c,d 0.04 c,d 0.03 c,d 0.05 c,d 0.04 c,d

Brain slices were cultured with inhibitors 1 h prior to and during OGD, NMDA or H2 O2 application. The viability of brain slices was evaluated by using TTC method. n = 6. Data are mean ± S.D. c, P < 0.05 vs. control; d, P > 0.05 vs. injury group.

the severity of the tissue injury in the absence of stearic acid during insults at a given concentration (P > 0.05) (Table 3). Cotreatment of brain slices with 50 ␮M H7 or 50 ␮M genistein in the presence of 30 ␮M stearic acid during OGD and H2 O2 insults resulted in approximately a 14% decrease in tissue activity for H7 group and approximately an 18% decrease for genistein group (P < 0.05). Meanwhile, cotreatment of brain slices with H7 (50 ␮M) or genistein (50 ␮M) in the presence of stearic acid (30 ␮M) during NMDA insult, had resulted in a decrease in tissue viability by approximately 8–10%. When slices were treated with RpcAMPs (10 ␮M) or U0126 (20 ␮M) during pre-incubation, the protection against OGD, NMDA and H2 O2 insults afforded by stearic acid was not abolished (Table 4). Wortmannin, a potent selective irreversible inhibitor of phosphatidylinositol 3-kinase (PI3K), can serve as a powerful tool to investigate the activation of PI3K signaling pathways. As shown in Table 4, wortmannin significantly reversed the protective effects of stearic acid against OGD and NMDA insults. Additionally, neuroprotective effects of stearic acid were completely blocked by wortmannin during H2 O2 insult. Those results indicate that its neuroprotective effect may be

Treatment of homogenate of brain slices with stearic acid for 5–60 min did not significantly change the activity of PI3K directly (data not shown). However, treatment of brain slices with 30 ␮M stearic acid resulted in a statistically significant increase in PI3K activity at 5, 10, 30 and 60 min (Fig. 2). 4. Discussion Experiments using brain slices have the advantages of both in vivo and in vitro. They not only maintain anatomic relations and natural synaptic connection in vitro, but also eliminate such in vivo variables as blood flow, temperature, ionic environment and closely match in vivo conditions. Therefore, an increasing numbers of brain slice models have been used to study brain function and neuron protection. In this study, we used three different damage models to reflect the pathological characteristics of different phases of ischemia/refusion injury (metabolism disorder, toxic amino acid and oxidative stress) [9]. The present study is the first to demonstrate that stearic acid can dose-dependently protect rat brain slices against OGD, NMDA and H2 O2 toxicity. In CNS pathologies including stroke, trauma or Alzheimer’s disease, nerve cell death occurs as a result of cell injury. Consequently, survival-signaling cascades may provide targets for neuroprotective therapies against these conditions. Two well-studied mitogenic pathways are the phosphoinositide 3-kinase (PI3K)-Akt pathway and the Ras-Raf-MEK-ERK pathway, which are central to cell survival signals respectively [16–18]. Additionally, activation of protein kinase A, protein kinase C (DAG/PKC) and tyrosine protein kinase (TPK) were also involved in the promotion of neuronal survival [19–21]. As an early consequence of OGD associated with brain ischemia, neuronal aerobic metabolism and ATP production are severely influenced. The decrease in energy production and malfunction of Na+ /K+ -ATPase leads to loss of active ion transport, imbalance of transmembrane electrochemical ionic gradients, which promotes presynaptic glutamate release and impaired uptake [22]. Because the brain does not rely on stearic acid oxidative metabolism for the production of ATP, the neuroprotective effects provided by stearic acid against OGD insult may result from the blockage of excitatory amino acid (EAA) receptors, presynaptic depression of glutamate release, promotion of glutamate uptake or

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Table 4 Influence of inhibitors on the protective effect of stearic acid against injury brain slices induced by three different insults Group

TTC (OD490 ) Cortical slices

Control OGD OGD + stearic acid OGD + stearic acid + wortmannin OGD + stearic acid + H7 OGD + stearic acid + genistein OGD + stearic acid + RpcAMPs OGD + stearic acid + U0126 NMDA NMDA + stearic acid NMDA + stearic acid + wortmannin NMDA + stearic acid + H7 NMDA + stearic acid + genistein NMDA + stearic acid + RpcAMPs NMDA + stearic acid + U0126 H2 O2 H2 O2 + stearic acid H2 O2 + stearic acid + wortmannin H2 O2 + stearic acid + H7 H2 O2 + stearic acid + genistein H2 O2 + stearic acid + RpcAMPs H2 O2 + stearic acid + U0126

0.92 0.48 0.85 0.60 0.71 0.68 0.85 0.86 0.57 0.86 0.74 0.79 0.76 0.86 0.85 0.45 0.80 0.47 0.67 0.63 0.80 0.78

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.06 c 0.05 a 0.05 c,e 0.06 c,f 0.04 c,f 0.05 a,f 0.04 a,f 0.04 c 0.04 a 0.05 c,f 0.04 b,e 0.05 b,e 0.05 a,f 0.04 a,f 0.05 c 0.05 b 0.05 c,d 0.06 c,f 0.04 c,e 0.05 b,f 0.04 b,f

Hippocampal slices 0.57 0.27 0.49 0.36 0.40 0.39 0.47 0.49 0.35 0.51 0.43 0.46 0.45 0.46 0.45 0.25 0.49 0.28 0.38 0.36 0.44 0.47

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.04 c 0.05 a 0.04 c,e 0.04 c,f 0.03 c,f 0.05 a,f 0.05 a,f 0.04 c 0.04 a 0.04 c,f 0.04 b,e 0.06 b,e 0.05 a,f 0.04 a,f 0.04 c 0.05 a 0.05 c,d 0.04 c,f 0.04 c,e 0.06 a,f 0.05 a,f

Brain slices were cultured with stearic acid (30 ␮M) in the absence of or in the presence of H7 (50 ␮M), RpcAMPs (10 ␮M), genistein (100 ␮M), U0126 (20 ␮M) or wortmannin (100 nM) during OGD, NMDA or H2 O2 insult. The viability of tissue was evaluated by using TTC method. n = 6. Data are mean ± S.D. a, P > 0.05 vs. control group; b, P < 0.05 vs. control group; c, P < 0.01 vs. control group; d, P > 0.05 vs. injury group; e, P < 0.05 vs. injury group; f, P < 0.05 vs. injury group.

Fig. 2. Effect of stearic aid on PI3K activity in brain slices. Brain slices were treated with stearic acid (30 ␮M) for 5, 10, 30 and 60 min, respectively. Then, brain slices were sonicated in lysis buffer after quickly frozen in liquid nitrogen. PI3K activation was measured by immunoprecipitated using an anti-p85 antibody. The PIP3 was quantitated by liquid scintillation counting. (A) cortical slices; (B) hippocampal slices. n = 3. Data are mean ± S.D. * P < 0.05 vs. control.

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defense against oxidative stress [1]. The excitotoxicity of NMDA in brain slices is mediated through ion channel and oxidative stress. Our results suggest that neuroprotective effects of stearic acid are related to the blockage of excitatory amino acid (EAA) receptors and (or) defense of oxidative stress. Persistent activation of NMDA receptors can significantly decrease the level of intracellular glutathione (GSH). Reactive oxygen species (ROS) will accumulate strikingly in very short time as the decreasing of GSH [23]. Oxidative stress-induced neuronal cell death has been implicated in acute cerebral hypoxia/ischemia and chronic neurodegenerative diseases. Hydrogen peroxide (H2 O2 ), a by-product of oxidative stress, has been implicated in triggering apoptosis and (or) necrosis in various cell types including cultured neurons. Here we tested the hypothesis that stearic acid may provide protection against H2 O2 insult. We found that the brain slices were significantly protected by stearic acid, which suggests that neuroprotective effects of stearic acid are mediated, at least in part, by anti-oxidative mechanism. MK-801, a highly potent, selective and non-competitive NMDA receptor antagonist, can significantly protect brain slices against OGD or NMDA insult (P < 0.01). However, it was less effective than stearic acid in neuroprotection against H2 O2 insults (P < 0.05). These results suggest that persistent activation of NMDA receptor more or less contributes to neuronal damage induced by three different insults. We found that wortmannin completely blocked the protective effect of stearic acid against H2 O2 insult, while H7 or genistein can only partially block its neuroprotective effects. It has been known that TPK or PKC can respectively affect the activation of PI3K or the phosphorylation of subunit of Akt [20,21]. These observations reveal that neuroprotective effect of the stearic acid against oxidative stress is via phosphatidylinositol 3-kinase pathway. We found that the incubation of stearic acid with homogenate of brain slices did not activate PI3K, while the treatment of brain slices with stearic acid (30 ␮M) resulted in a significant increase in PI3K activity at 5, 10, 30 and 60 min. These results imply that stearic acid promotes PI3K activity through indirect pathways. Because Akt is a major downstream PI3K target, reported to play a key role in phosphorylation of caspase-9, we were interested in the possible relationship between stearic acid and caspases [17,20]. In animal models of transient global cerebral ischemia, caspase-9 release from mitochondria and accumulation in nuclei was observed in hippocampal and other vulnerable neurons exhibiting early post-ischemic changes preceding apoptosis [24]. Caspase-9 is a caspase upstream of caspase-3 and its acti-

vation is stimulated by Apaf-1/cyt-c and inhibited by Akt signals. The Akt protein kinase is activated by a variety of growth factors via a PI3-kinase-dependent pathway. Oleamide (100 ␮M), structurally related to stearic acid without
9-cis double bond, significantly decrease the K+ deprivation-induced apoptosis and its actions were well parallel with the attenuation of caspase-3 activity [25]. Studies in more detail on the possible relationship between stearic acid and caspases in brain slices are in progress in our laboratory. Vasoactive intestinal peptide (VIP, 28aa) and pituitary adenylate cyclase activating peptide (PACPA, 28 or 38 aa) are two important neuropeptides in the maintenance of neuronal survival. However, neuropeptides are difficult to get across the blood–brain barrier when administered peripherally, which limits the clinical uses. A useful strategy for this problem is to find small molecules mimicking the parent peptide activity while offering applicable benefit in better bioavailability and stability. It has been reported that a short peptide fragment, carrying a stearyl moiety, can not only provide a significant bioavailability and stability, but also capture the activities of the seven-fold larger parent peptide [26]. Thus, understanding of stearic acid may provide a novel avenue for drug design for abundant neurodegenerations. In conclusion, stearic acid can protect brain slices (cortical or hippocampal) against OGD, NMDA or H2 O2 insult and its neuroprotective effect against oxidative stress is via phosphatidylinositol 3-kinase dependent mechanism. Acknowledgments This project was supported by the Shanghai Natural Scientific Research Fund (grants number: 03ZR14056). References [1] P. Lipton, Ischemic cell death in brain neurons, Physiol. Rev. 79 (1999) 1431–1568. [2] N.G. Bazan, Lipid signaling in neural plasticity, brain repair, and neuroprotection, Mol. Neurobiol. 32 (2005) 89–103. [3] D.H. Wang, H.R. Sang, Receptor-mediated signaling pathways: potential targets of modulation by dietary fatty acids, Am. J. Clin. Nutr. 70 (1999) 545–556. [4] M.J. McArthur, B.P. Atshaves, A. Frolov, W.D. Foxworth, A.B. Kier, F. Schroeder, Cellular uptake and intracellular trafficking of long chain fatty acids, J. Lipid Res. 40 (1999) 1371–1383. [5] D.J. Baer, J.T. Judd, P.M. Kris-Etherton, G. Zhao, E.A. Emken, Stearic acid absorption and its metabolizable energy value are minimally lower than those of other fatty acids in healthy men fed mixed diets, J. Nutr. 133 (2003) 4129–4134. [6] T. Tholstrup, P. Marckmann, J. Jespersen, B. Sandstrom, Fat high in stearic acid favorably affects blood lipids and factor VII coagu-

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