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Introduction
Epidemiological, clinical, and biochemical studies have
shown that different types of dietary fatty acids can modify
the risks of stroke[1,2], but the molecular and cellular
mechanisms by which dietary fatty acids exert such effects are still
not well understood. Free fatty acids have been focused on
for their potential neuroprotection more than ever in
pharmacological research. Besides being crucial biomolecules in
metabolic processes, they serve as the substrates for cell
membrane biogenesis (glyco and phospholipids), and the
precursor molecules of intracellular signaling such as
prosta-glandins, leukotrienes, thromboxanes, and the platelet
activating factor. Polyunsaturated fatty acids have been
implicated in the prevention of various human diseases,
including obesity, diabetes, coronary artery disease, stroke, and
inflammatory and neurological
diseases[3]. Only a few articles have reported the effects of saturated fatty acids on
human health. Saturated fats are usually regarded as
unhealthy, but nutritionists believe that the type of
saturated fat is also important.
Stearic acid is a long-chain fatty acid consisting of 18
carbon atoms without double bonds. It is present in fairly
constant proportions in beef, pork, lamb, and veal
(approximately 9%_12% of total fatty acid content), with lower
proportions found in poultry (approximately 6%_7% of total
fatty acid content)[4]. It is biochemically classified as a
saturated fatty acid, both for the purpose 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. Stearic acid, one of the most
common fatty acids in brain phospholipids, originates in the
circulation and is sequestered from blood by the brain along
with precursor fatty acids[6]. Recently, it has been found
that several fatty acids can function as natural ligands of
peroxisome proliferator-activated receptors (PPAR) to
regulate lipid homeostasis. PPAR are members of the nuclear
receptor superfamily of ligand-dependent transcription
factors. The PPAR subfamily comprises 3 isotypes:
PPARα, PPARβ, and PPARγ. Although a precise biological role for
PPARγ remains unclear, PPARα and PPARγ have modulatory roles in various cellular responses, including immune
and inflammatory responses. Various fatty acids can
regulate gene expression at micromolar concentrations through
direct interactions with PPARα or PPARγ. Recently, it has
been reported that ligands of PPARα or PPARγ can
significantly protect the brain from ischemic or oxidative
insult[7,8]. Therefore, we hypothesize that stearic acids may function
as natural ligands of PPAR to protect neurons from ischemic
insult. We have reported that the neuroprotective effects of
stearic acid against toxicity of oxygen/glucose deprivation
or glutamate on rat cortical or hippocampal
slices[9]. In the present study, we evaluated the effects of stearic acid on
cortical neurons insulted by glutamate
NaN3 or hydrogen peroxide
(H2O2).
Materials and methods
Experimental animals Pregnant Sprague-Dawley (SD)
rats (300±20 g, grade II, Certification
No SCXK 2004-0005), were purchased from the Shanghai Laboratory Animal Co,
Ltd (Shanghai, China).
Reagents and drugs Poly-D-lysine, bisphenol A diglycidy
ether (BADGE), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe
nyltetrazolium bromide (MTT), dizocilpine maleate (MK801),
2-thibarbituric acid (TBA), and
{3-[1(p-chlorobenzyl)-5-(isopropyl)-3-t
-butylthiondol-2-yl]-2, 2-dimethylpropanoic acid, Na} (MK886) were purchased from Sigma (St Louis,
MO, USA). Fetal calf serum was purchased from FuMeng
Gene Bio-Engi Res and Dev Co, Ltd Inc (Shanghai, China);
Dulbecco's modified Eagle's medium (DMEM)-F12, neurobasal medium and B27 were from Gibco (Logan, UT,
USA); The LDH kit, superoxide dismutases (SOD) kit,
glutathione peroxidase [GSH-Px], and the catalase (CAT) kit were
purchased from Jiancheng Institute of Biotechnology
(Nan-jing, China). The bicinchoninic acid (BCA) protein assay kit
was from Pierce (Rockford, IL, USA). Neuron-specific
enolase (NSE) antibody and streptavidin biotin complex kits were
purchased from Boster Biological Technology (Wuhan,
China). Polyclone antibody to PPARγ and SP1 were
purchased from ALEXIS Biochemicals (San Diego, CA, USA)
Cytosine arabinoside was purchased from Shanghai
Hua-Lian Pharmaceutical Cooperation (Shanghai, China). Stearic
acid (Chemical Reagent Co, Ltd, Shanghai, China) and all
other reagents were of analytical grade. Stearic acid
dissolved in ethanol was diluted in culture medium before use.
The final ethanol concentration in the culture medium was
less than 0.5% (Table 1).
Cell culture Primary rat cultured cortical neurons were
prepared from embryonic d 18 Sprague-Dawley rats. The
brains were removed aseptically from the skulls, the meninges
excised carefully, and the cerebral cortex was dissected in
iced dissection solution, which had the following
composition (in mmol/L): NaCl 113, KCl 3,
CaCl2 1, MgCl2 6,
NaHCO3 25,
NaH2PO4 1, and glucose 11 (final pH 7.2_7.4). The
bilateral cerebral cortex were collected and mechanically
frag-mented, then digested with 0.25% trypsin (Sino-American
Biotechnology Co, Shanghai, China), and 0.2 mg/mL DNase
I (Sino-American Biotechnology Co, Shanghai, China), and
incubated for 10 min at 37 ºC. Following trypsinization, the
cells were washed twice in DMEM-F12 culture medium
containing 15% heat-inactivated fetal calf serum. Dissociated
cells were plated in 35 mm dishes or 96-well plates precoated
with 0.1% poly-l-lysine at densities of
1×106/mL, 2 mL/dish, or 100 µL/well. The culture was maintained in a humidified
incubator with a 5% CO2 atmosphere at 37 ºC. The medium
was changed into medium consisting of neurobasal
modified with B27 supplement after the cells were attached to the
culture plate. After 1 d in the culture, cytosine arabinoside (5
µmol/L) was added to the medium to inhibit glial proliferation.
Experiments were performed on d 6 of culturing. The
purification rates were evaluated according to the cell count and
NSE immunohistochemistry.
Cell viability Cell viability was evaluated by using a
colorimetric assay with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe
nyltetrazolium bromide (MTT), an indicator of mitochondrial
respiratory chain activity. Briefly, the cells were pretreated
with stearic acid over a wide dose range at various times
prior to and during subjected to glutamate,
NaN3 or hydrogen peroxide insult. The viability of cells was determined
using MTT assay after 24 h cotreatment. The 0.5 mg/mL
MTT solution was added to each well and the cells were
incubated for a further 4 h at 37 ºC. Then, the media were
removed and formazan crystals were solubilized with DMSO.
The optical density of each well was measured at 490 nm
(OD490) by using an ELISA reader (Bio-Tek ELX800uv,
Bio-Tek Instrument Inc, Winooski, VT, USA). The cells without
stearic acid treatment served as the controls. The release of
LDH from the intracellular compartment into the
surrounding medium was expressed as an indicator of cell death. LDH
activity in the extracellular medium was monitored by using
a commercial kit. Data were expressed as percent of LDH
release of the positive control (0.1% Triton X-100 treated
group).
Glutamate uptake assay in cortical
neurons[10] Cortical neurons were plated on 24-well plates. After stimulation
with or without stearic acid for 24 h, the cells were washed
and equilibrated in Krebs buffer solution [(mmol/L NaCl
120.6, KCl 5.9, NaH2PO4 1.2,
MgCl2 1.2, NaHCO3 15.4,
CaCl22.5, C6H12O6 11.5, pH=7.4)] for 20 min at 37 ºC. Glutamate
uptake was initiated by the addition of 5 mmol/L (0.2
Ci/mmol) L-[3H] glutamate. After 10 min incubation, the
reaction was stopped by washing the cells 3 times with ice-cold
PBS. The cells were then lysed in 1 mol/L NaOH at 37 ºC for
30 min, and the radioactivity in the cell lysates was
quantified by scintillation counting. The protein content was
determined in aliquots of each lysates by the BCA method.
Lipid peroxidation[11] The lipid peroxidation of the cells
was determined by measuring thiobarbituric acid reactive
substances (TBARS). The cells were lysed with 4 mL fulvic
acid (0.167 mol/L) and 0.5 mL 10% phosphotungstic acid,
and then centrifuged at 4000×g for 10 min. The precipitation
was resuspended with 1.5 mL distilled water and 0.5 mL TBA
reagent (1:1, v/v) mixture of 0.67% TBA and acetic acid. The
reaction mixture was heated at 95 ºC for 1 h. After cooling, 2
mL of n-butanol was added, and the mixture was shaken
vigorously for 30 s. After centrifugation at
3000×g for 10 min, the n-butanol layer was used for fluorometric
measurement with lex 515 nm and
lem 553 nm using a fluorescence spectrophotometer. The value of fluorescence was
calculated by comparing with standards prepared from
1,1,3,3-tetraethoxypropane.
Analysis of activity of antioxidant enzymes in cortical
neurons For the assay of antioxidant enzymes, the cultures
were washed with iced PBS buffer and then homogenized.
The homogenate was centrifuged at 10
000×g for 15 min at 4 ºC. The supernatant was used as test sample for the
enzyme assay. The protein concentration of the supernatant
was determined by the BCA method. The total SOD and
Cu/Zn SOD were measured by the guidance of the kits,
respectively. The control (C) consisted of all the reagents
except the supernatant (2%, w/v), while the blank (B)
consisted of buffer and the supernatant without any reagents.
The absorbance of T, C, and B was measured at 550 nm and
the enzyme activity was expressed in units (1 U=50%
inhibition of the oxidation of oxymine by the
xanthine-xanthine-oxidase system). Cu/Zn SOD and MnSOD activity were
distinguished by the inhibition of the former by 2 mmol/L of
KCN added to the assay cocktail. Cu/Zn SOD activity was
determined by subtracting MnSOD activity from the total
SOD activity. Glutathione (GSH), a tripeptide antioxidant,
plays multiple biological functions. It is involved in the
detoxification of harmful molecules, such as reactive oxygen
species (hydrogen peroxide and hydroperoxides) through
glutathione peroxidase. The activity of GSH-Px was
determined by quantifying the rate of
H2O2-induced oxidation of reduced GSH to oxidized glutathione. A yellow product which
had absorbance at 412 nm could be formed as GSH reacted
with the dithiobisnitrobenzoic acid. One unit of GSH-Px was
defined as the amount that reduced the level of GSH by 1
µmol/L in 1 min per mg of protein. The assay of CAT activity
was based on its ability to decompose
H2O2. The absorbance of the supernatant at 254 nm changed when the
H2O2 solution was injected into the cuvette. The disappearance
of H2O2 was measured at 240 nm for 60 s at 1 min intervals.
The change of the absorbance reflected the CAT activity.
Influence of inhibitors of receptors on the
neuropro-tective effects of stearic
acid[9] MK886 is an indole compound that was originally identified as a potent inhibitor of
the 5-lipoxygenase (5-LOX)-activating protein (FLAP).
Recently, it has been found to inhibit PPARα via a
non-competitive mechanism as shown by its effects on the
binding of fatty acids to the PPARα
protein[12]. Cortical neurons were treated with 5 µmol/L MK886 or 100 µmol/L BADGE (a
PPARγ antagonist) in the presence or absence of stearic acid
to determine whether antagonists of PPAR could block the
neuroprotection provided by 30 µmol/L stearic acid. In
addition, the influence of the protein blocker, cycloheximide
(30 µmol/L), on the neuroprotective effects of stearic acid
(10 µmol/L) was also investigated at the same time. Cell
viability was measured by using the MTT method.
Immunoblotting of PPARγ after treatment of stearic
acid Harvested cortical cells were homogenized in a buffer
containing 20 mmol/L HEPES, pH 7.4, 2 mmol/L EGTA, 10 mmol/L
Tris-HCl, 1 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L
dithioerythreitol (DTE), and 10 µg/mL aprotinin. The cells
were sonicated to disrupt the cell membranes, and the soluble
(cytoplasmic) and pellet fractions were separated by
centri-fugation. The homogenate was centrifuged at
100 000×g, and the soluble fraction was collected. Samples were
adjusted by the addition or dilution to 0.05% Triton X-100.
Equal amounts of proteins, as determined by the BCA
method, were separated by SDS-PAGE and transferred to a
polyvinylidene fluoride membrane in a Bio-Rad (Hercules,
CA, USA) transblot electrophoresis apparatus at 100 V for 2
h using Towbin's buffer (25 mmol/L Tris, pH 8.3, 192 mmol/L
glycine, and 20% methanol). The membranes containing
immobilized proteins were blocked with 5% non-fat milk in
Tris buffer (20 mmol/L Tris, pH 7.5, and 0.5 mol/L NaCl) for
1 h. A polyclonal PPARγ that cross-reacts with both human
and mouse PPAR-γ was prepared in the TS buffer and added
to the transblots for overnight incubation. After washing,
the membranes were incubated with a goat anti-rabbit
immunoglobulin G horseradish peroxidase-conjugated antibody
as the secondary antibody in the TS buffer. Immunoreactive
bands were visualized by a standard enhanced
chemiluminescence procedure. Sp1 levels were used as loading
controls for nuclear protein
expression[13].
Statistical analysis Cerebral cortexes from 10 rat fetuses
were used for each experimental group, and data collected
from 3_6 independent experiments were used to calculate
means, which are expressed as mean±SD. SPSS statistical
software 10.0 for Windows was used and statistical
significance was evaluated by using one-way ANOVA and the SNK
test. Statistical significance was assumed if
P<0.05.
Results
Effects of stearic acid on the viability of cortical
neurons We used a NSE antibody to immunostain primary
cultured cortical neurons. The healthy cortical neurons had
long axons and dendrites. We could see that non-neuronal
cells were very few and the purity of cultured cortical
neurons was about 92%, indicating that stearic acid exhibited its
effect directly, but not indirectly via non-neuronal cells
(Figure 1). Stearic acid (1_30 µmol/L) did not exhibit
significant cell toxicity after 24 h incubation. However, it
significantly decreased cell viability after 24 h incubation when its
concentrations were above 100 µmol/L. Based on our
screening results, we chose concentrations of reagents that did
not produce significant effects on cell viability for the
experiments described below (Figure 2).
Effects of pretreatment with stearic acid on 3 different
insults in cortical neurons The neuroprotective effects of
stearic acid on glutamate, NaN3, or
H2O2-induced neurotoxicity were investigated. The cell viability was assessed by
using MTT reduction and LDH release assays. The cells
were incubated with different concentrations of stearic acid
for 24 h, and then exposed to 100 µmol/L glutamate, 50
µmol/L H2O2, or 20 µmol/L
NaN3 for another 24 h. We found that
treatment with 100 µmol/L glutamate resulted in significantly
decreased cell viability (MTT 44.3%±6.4%; LDH, 78.8%±6.2%). There was a statistically significant difference
between the glutamate and control groups (P<0.01). Results of
both MTT reduction and LDH release assays showed that
pretreatment of cortical neurons with stearic acid (3_30
µmol/L) dose-dependently prevented glutamate insult (Figure 3).
After the cells were subjected to 50 µmol/L
H2O2 for 24 h, cell viability was reduced by approximately 52%.
H2O2 induced an increase of LDH leakage by 2.1 times than that of the
control. There was a statistically significant difference
between the H2O2 and control groups
(P<0.01). Similarly, SA (3_30 µmol/L) protected cortical neurons against
H2O2 insult in a dose-dependent manner (Figure 4). After subjected to
20 µmol/L of NaN3, cell viability was reduced by
approximately 45%. NaN3 induced an increase of LDH leakage by 2.4 times than that of the control. There was a statistically
significant difference between the NaN3 and control groups
(P<0.01). SA (3_30 µmol/L) did not protect cortical neurons
against NaN3 insult (P>0.05; Figure 5).
Effects of pre-incubation time on stearic acid
neuroprotection of cortical neurons from glutamate or
H2O2 insult We determined the minimum pre-incubation time with
stearic acid needed to achieve neuroprotection. Cortical
neurons required exposure to stearic acid (10 µmol/L) for 4_8 h prior to the administration of glutamate or
H2O2 to achieve significant neuroprotection. Stearic acid
time-dependently protected neurons against glutamate and
H2O2 insults during the incubation time from 4 h to 12 h prior to the
administration of insults (Figure 6).
Lipid peroxidation The treatment of cells with glutamate
or H2O2 caused an obvious elevation of TBARS [(200±10
nmol/g protein or 220±6 nmol/g protein),
P<0.01 compared with the control group (10±4) nmol/g protein]. The elevation
was inhibited by stearic acid (3_30 µmol/L; Figure 7).
Effects of stearic acid on glutamate
uptake The pretreatment of primary cortical neurons with 3_30 µmol/L of
stearic acid for 24 h produced a concentration-dependent
facilitation of the glutamate uptake in these cells, and reached
a maximal increase of 148%±12% at 30 µmol/L. These results
suggest that the effects of stearic acid on the promotion of
glutamate uptake may partially contribute to its
neuropro-tection (Figure 8).
Effect of stearic acid on antioxidant
enzymes Neural damage or death occurs if the increase in reactive oxygen
species produced in the brain during exposure to hypoxia is
not counterbalanced by an increase in the cells' antioxidant
defense systems. After incubation for 24 h, we evaluated
and compared the antioxidant enzyme activity (SOD,
GSH-Px, and CAT) in stearic acid groups with the corresponding
control groups. Stearic acid was able to dose-dependently
induce the defenses against oxidative stress through an
increase in the activity of Cu/Zn SOD and CAT in cortical
neurons during 24 h of incubation (Table 1). After the cells
were exposed to glutamate or
H2O2 insult for 24 h, obvious
decreases of antioxidant enzymes, SOD, CAT, and GSH-Px
were observed (P<0.01, compared with the corresponding
control groups). Stearic acid dose-dependently attenuated
the decrease of SOD, CAT, and GSH-Px activity (Table 2).
Influence of inhibitors on the neuroprotective effects of
stearic acid It is known that the 5-LOX inhibitor can protect
against ischemic-like injury in PC12 cells in
vitro by modulating p38 MAP kinase
activity[12]. Therefore, we tested whether MK886 could directly protect cortical neurons
against glutamate or H2O2 insult. MK886 (5 µmol/L) at a
concentration of 10 µmol/L significantly improved the
activity in tissues insulted by glutamate and
H2O2 (P<0.05), whereas BADGE at a concentration of 100 µmol/L produced
no significant effect on cell viability insulted by glutamate or
H2O2 (P>0.05). These results suggest that the inhibitor of
5-LOX directly protects tissues against oxidative stress. When
neurons were treated with 5 µmol/L of MK886 during
pre-incubation, the protection against glutamate or
H2O2 insult afforded by stearic acid was not abolished. In contrast, 100
µmol/L BADGE blocked its protective effect completely. In
addition, cycloheximide (30 µmol/L) was able to completely
abolish the neuroprotective effects of stearic acid after
pretreatment with cortical neurons, which suggests that the
neuroprotection of stearic acid was mediated by new protein
synthesis (Figure 9).
Expression of the PPARγ protein in cortical
neurons It has been reported that cortical neurons expressed the
PPARγ protein. Here, we investigated the effects of stearic acid on
the expression of the PPARγ protein in cortical neurons
during oxidative stress. Cortical neurons showed a 48 kDa band
that reacted with an antibody directed at PPARγ. Our results
show that the H2O2 slightly increased the expression of
PPARγ after incubation for 4 h (P>0.05). However,
H2O2 significantly decreased the expression of
PPARγ (P<0.05) after incubation with neurons for 24 h, and the inhibitory effect of
H2O2 on the expression of
PPARγ can be attenuated by 30 µmol/L of stearic acid (Figure 10).
Discussion
Brain ischemia induces neuronal loss which is in part
caused by excitotoxicity and free radical
formation[14]. Glutamate is the primary excitatory neurotransmitter in the
central nervous system. In the ischemic brain, extracellular
glutamate is elevated rapidly after the onset of ischemia,
which results in excessive activation of glutamate receptors
and mediated neuronal injury[15]. It is known that the
presence of 0.5 mmol/L glutamate in the extracellular space can
reverse glutamate uptake and decrease the synthesis of
glutathione in neurons[16,17].
Excitotoxic cell death is due, at least in part, to the
excessive activation of ion type glutamate receptors, and hence,
excessive Ca2+ influx through the receptors' associated ion
channel[17]. Glutamate uptake from the extracellular space is
essential to shaping excitatory postsynaptic currents and
for preventing excitotoxic death due to overstimulation of
glutamate receptors[15]. Stearic acid dose-dependently
promoted the glutamate uptake in cerebral neurons. However,
we did not find any promotion effects of stearic acid on
glutamate uptake in astrocytes after incubation for 24 h (data
not shown). Since stearic acid increased extracellular
glutamate uptake, this property undoubtedly would help
stearic acid to serve as a potent neuroprotector against
glutamate toxicity in the early stages of ischemia.
Persistent activation of glutamate receptors can
significantly decrease the level of GSH. Reactive oxygen species
(ROS) will accumulate strikingly in a very short time as the
decreasing of GSH[18]. Mitochondria are not only a source
of main intracellular generators of ROS, but also the targets
of ROS attack[19]. Although defenses against damage
produced by ROS are extensive, including enzymatic and small
molecule antioxidants as well as repair enzymes, an increased
production of ROS or a poor antioxidant defense network
can lead to a progressive damage in the cell with a decline in
physiological function[20]. Therefore, oxidative
stress-induced neuron death has been implicated in acute cerebral
ischemia and chronic neurodegeneration
diseases[21,22].
H2O2, a by-product of oxidative stress, has been implicated in
triggering apoptosis and/or necrosis in various cell types,
including cultured neurons. Exogenous hydrogen peroxide, a
low molecular weight compound, can easily penetrate the
lipid membrane, and cause lipid, protein, and DNA peroxidation. Here, we tested the hypothesis that stearic
acid may also provide protection against oxidative stress.
We found that the cortical neurons were significantly
protected by stearic acid against glutamate and
H2O2 insults, which suggest that neuroprotective effects of stearic acid
are mediated, at least in part, by the promotion of an
antioxidant mechanism. MK801, a highly potent, selective, and
non-competitive NMDA receptor antagonist, can
significantly protect hippocampal slices against 3 different insults
(P<0.05), which suggests that persistent activation of NMDA
more or less contributes to neuronal damage induced by 3
different insults.
Mitochondria are the powerhouse of the cell; their
primary physiological function is to generate ATP through
oxidative phosphorylation via the electron transport chain.
NaN3, an inhibitor of cytochrome c oxidase and CAT,
significantly decreased the viability of cerebral neurons. Although
SA can dose-dependently increase the activity of CAT after
24 h incubation, SA acid did not protect cerebral neurons
against NaN3 insult. These results suggest that the
inhibitory effect of NaN3 on CAT activity is not counterbalanced
by the promotion effects of stearic acid, and the
neuropro-tection of stearic acid mainly acts on the membrane and cytoplasm instead of mitochondria during oxidative stress.
Reactive oxygen-induced free radicals play an important
role as mediators of tissue injury associated with
inflammatory and ischemic states. Several enzymes (SOD, GSH-Px
and CAT) are important in the antioxidant defense system
because they metabolize either free radicals or reactive
oxygen intermediates to non-radical products. For example, SOD
constitute the major enzymatic mechanism for
O2_· degradation and catalyze the conversion of
O2_· into
H2O2. Hydrogen peroxide is in turn converted to water and molecular
oxygen by CAT or GSH-Px. GSH-Px utilizes reduces GSH as
the hydrogen donor. Under the same condition, the
improvement of antioxidant enzymes activity closely associates with
new protein synthesis or modification of their
stereochemical configuration. The Cu/Zn SOD can be significantly
induced by acute stressors, but not by chronic or combined
stress conditions, while MnSOD can be induced under
various oxidative stress and inflammatory
conditions[23,24]. We precisely evaluated the effects of SA on the activity of
crucial antioxidant enzymes, and found that treatment with 10
µmol/L stearic acid for 24 h significantly promoted the
activity of Cu/Zn SOD in cortical neurons. In addition, we did not
find any direct effective of stearic acid on GSH-Px activity
after 24 h incubation. It has been reported that under
oxidative stress, the activity of antioxidant enzymes increased. In
our study, we found that the activity of antioxidant enzymes
(SOD, CAT, and GSH-Px) in cultured cortical neurons were
reduced remarkably by 24 h treatment with
H2O2. The inhibitory effect of
H2O2 on antioxidant enzymes could be
attenuated by stearic acid, which suggests that the ability of stearic
acid to preserve the antioxidant enzyme activity may
contribute to its neuroprotective effect. It is known that the
cytosolic form of Cu/Zn SOD appears specialized to remove
superoxide produced as a result of injury. Therefore, stearic
acid can only protect brain tissue viability against
H2O2 insult, but
NaN3 insult at a given range of concentrations and its
modulatory effects on the enzyme catalytic rates are very
specific and significant for each antioxidant enzyme.
In some experiments, different ligands have been used
according to the different isotypes of
PPAR[25_27], and these agonists all play a role in neuroprotection to different extents.
Several fatty acids bind to all 3 PPAR isoforms, although
there is a preference of PPARα for polyunsaturated fatty
acids. We have reported that the inhibitor of
PPARγ significantly blocked the neuroprotective effects of stearic acid on
brain slices. Here, we determined whether inhibitors of PPAR
also blocked the neuroprotection of SA on cerebral neurons.
MK886 and BADGE are 2 important antagonists of PPAR.
We found that BADGE completely blocked the
neuropro-tective effect provided by stearic acid, whereas MK886 did
not abolish its protective effect. MK886 can directly protect
cortical neurons against glutamate insults. Although MK886
did not abolish the effects of stearic acid, we can not
conclude that PPARα is not involved in the process of
neuropro-tection. BADGE can completely block the neuroprotective
effect of stearic acid, which suggests that PPARγ may play a
more important role in the protective effect because BADGE
has no significant effect on injured cortical neurons by itself.
It has been reported that the activation of PPARγ can
significantly improve Cu/Zn SOD and catalase expression in liver
cells and blood vessel walls[28,29]. Our results confirm that
stearic acid significantly improves antioxidant enzymes
activity in cortical neurons, and its neuroprotective effects
against oxidative stress have been completely blocked by
the protein synthesis inhibitor after pretreatment with
cycloheximide (CHX). These results strongly suggest that stearic
acid significantly enhances the antioxidative enzymes
activity via the activation of PPARγ and synthesis of new
proteins in neural cells.
Growing evidence suggests that the expression of
PPARγ in the brain is mainly involved in inflammation and
neurodegeneration. Western blotting and RT-PCR results
show that PPARα, PPARβ, and PPARγ are expressed in
embryonic cortical neurons in vitro and that their expression is
differentially modulated during their in
vitro maturation. The expression of PPARα and
PPARβ gradually increased during neuronal maturation, whereas the expression of
PPARγ gradually decreased correspondingly during neuronal
maturation[30]. A decrease in PPARγ levels might cause the
development or exacerbation of
inflammation[31]. Our results suggest that the ability of stearic acid to preserve the expression
of PPARγ may contribute to its neuroprotective effect as
well.
Stearic acid, one of the most common fatty acids in brain
phospholipid, originated in the circulation and was
sequestered from the blood by the brain alone with precursor fatty
acids. The high concentration of stearic acid in brain gray
matter suggests that this fatty acid has an important role in
neural function. Very preterm babies are born with minimal
fat stores and suboptimal circulating levels of these nutrients.
Children with neurodevelopmental handicaps receiving adequate nourishment are generally calmer and appear more
normal than those who are undernourished. Patients with
less severe disabilities have an increased functional status
with improved nutrition[32]. Therefore, stearic acid may
effectively protect against some central nervous system
injuries in preterm infants. The adult brain is typically
resistant to changes in fatty acid composition; the developing
brain, by contrast, is much more plastic.
Newborn animals are more tolerant than adults of
cerebral hypoxia/ischemia insult, and their blood-brain barriers
are easier to pass. Our results suggest that effective
methods of treatment for brain ischemia may keep stearic acid at
the given range of concentrations.
In conclusion, stearic acid can effectively protect
cortical neurons against oxidative stress at a given range of
concentrations. Its neuroprotective effect is possibly mainly
mediated by the activation of PPARγ and synthesis of a new
protein in cortical neurons.
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