Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Propofol (2,6-diisopropylphenol), the active ingredient of the common general anesthetic Diprivan, has shown direct
antioxidant activity conferred by the phenolic hydroxyl group in its structure. This moiety scavenges free radicals and
inhibits lipid peroxidation[1]. Since oxidative stress plays an important role in the brain ischemic
injury[2], the antioxidant property of propofol suggests that it may serve as a good agent to combat ischemia. Certain hypotheses have
been proposed, such as a reduction in cerebral
metabolism[3], potentiation of γ-aminobutyric
acid-mediated inhibition[4], and restoration of the glutamate uptake impaired during
injury[5,6]. Our previous study showed that propofol protected cerebral cortical
and hippocampal slices against hydrogen peroxide injury at low and mid
concentrations[7], but its intracellular mechanism is
still worth further investigation.
Reactive oxygen species (ROS), including hydrogen peroxide
(H2O2), superoxide radical, hydroxyl radical, and peroxynitrite
increased, and probably would be involved in the pathogenesis of cerebral ischemic and reperfusion
injury[8,9]. ROS-induced cellular events have been implicated, at least in part, in the activation of mitogen-activated protein
(MAP) kinases[9,10]. Three subfamilies of MAP kinases
sensitive to ROS have been identified: extracellular-signal
regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK),
and the p38 kinase. The kinase of each subfamily modulated
specific cell functions via certain signaling pathways.
However, the hypothesis that ERK1/2 may be a protective
signal and JNK-p38 a pro-apoptotic signal would not always
hold true, and their effect may depend on the nature of the
cell type, death stimulus, duration of its activation, and
probably above all, the activity of other signaling
pathways[11,12]. Therefore, the mechanism of these signal transduction
pathways involved in ROS-mediated cell damage remains to be
elucidated.
The concentration of H2O2 in healthy individuals is
normally quite low. The elevation of
H2O2 concentration upon cerebral ischemic and reperfusion injury could act as a
significant signal[13].
H2O2 has been used frequently as an
oxidative stimulus to identify redox-sensitive processes. Rat
pheochromocytoma cell line PC12 is useful for studying the
intracellular signaling mechanisms and are regarded as
a model for catecholamine-containing
neurons[14]. In the present study, we used PC12 cells injured by
H2O2 as an oxidative stress model
in vitro to examine the neuroprotective effect of propofol and its intracellular mechanism, especially
focusing on the roles of MAP kinases in the events.
Materials and methods
Chemicals Propofol and
H2O2 were purchased from Sigma Chemicals (St Louis, MO, USA). Anti-pan- and
phospho-ERK1/2
(Thr202/Tyr204), p38
(Thr180/Tyr182), and JNK
(Thr183/Tyr185) antibodies and SB203580
[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1
H-imidazole] were from Cell Signaling Technology (Beverly, MA, USA). All
other chemicals were commercial products of reagent grade
except where indicated.
Cell cultures and determination of cell viability with
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide- thiazolyl blue (MTT)reduction The PC12 cells were grown
in Dulbecco's Modified Eagle's medium (DMEM)
supplemented with 10% (v/v) donor horse serum, 5%
(v/v) newborn calf serum, and antibiotics (100 units/mL penicillin and 100
mg/mL streptomycin) in flasks precoated with collagen. The
cultures were maintained in a humidified atmosphere
containing 5% CO2 at 37 °C. The cells were grown to 70%_80%
confluence in 60 and 100 mm dishes and the growth was
arrested by incubation in serum-free DMEM for 24 h prior to
use. All experiments were performed with growth-arrested
cells to minimize basal MAP kinase activity. The
neuropro-tective effect of propofol on PC12 cell death induced by
H2O2 was investigated by using
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidethiazolyl blue] (MTT)
assay (Sigma, USA). The cells were treated with
H2O2 (200 µmol/L) for 24 h with or without propofol, and the cellular
MTT reduction was measured as described
previously[15].
Determination of apoptotic cell death with Hoechst 33258
staining and a fluorescence-activated cell sorter
To further evaluate the effect of propofol on the
H2O2-induced apoptosis of PC12 cells, we examined apoptotic nuclei staining and
DNA fragmentation in H2O2-administered PC12 cells. After
treatment with H2O2 (200 µmol/L) for 24 h with or without
propofol, the cells were stained with Hoechst 33258
(Molecular Probes, Eugene, OR, USA) and visualized by
fluorescence microscopy to evaluate morphological changes of
the nuclei as a measure of apoptosis. In addition, separate
groups of cells were also harvested and stained with Annexin
V and PI (propidium iodide) (BD Biosciences, San Jose, CA,
USA) to evaluate the percentage of apoptotic cells using
flow cytometry.
Measurement of caspase-3 activity The PC12 cells in
serum-free DMEM (60 mm collagen-coated dishes) were
pre-incubated with different concentrations of propofol for 30
min, then incubated in the presence of
H2O2 (200 µmol/L) for 4 h. The assay was performed according to the
manufac-turer's protocol. In brief, the lysates were centrifuged at
25 000×g for 3 min at 4 °C to precipitate cell debris. 50 µL of
2×reaction buffer/Dithiothreitol mix was added to the
supernatant, and finally, 5 µL of 1 mmol/L caspase-3
substrate Asp-Glu-Val-Asp (DEVD) conjugated to 7-amino-4-
trifluoromethylcoumarin (AFC) was added to each tube and
incubated for 1 h at 37 °C. After transferring the samples and
standard solution to a 96-well microplate, we measured
fluorescent activity using a fluorimeter with a 400 nm excitation
filter and a 505 nm emission filter.
Measurement of the p38 MAP kinase, JNK, and
ERK1/2 phosphorylations in PC12 cells MAP kinase
phosphorylation induced by
H2O2 in PC12 cells were
performed by Western blotting. ERK1/2, JNK, and p38 activations in the
cells were determined by using phospho-ERK1/2, phospho-p38
and phospho-JNK antibodies. Total ERK1/2, p38 and JNK
protein expressions were measured in the presence of
pan-ERK1/2, p38, and JNK antibodies. The PC12 cells in
serum-free DMEM were treated with or without propofol and then
incubated for 30 min. After incubation with
H2O2 for 10 min, the cells were collected and
lysed for the immunoblot analysis. Immunoreactive bands were visualized using
enhanced chemiluminescence (ECL, Pierce, Rockford, IL,
USA) and were quantified by densitometry in the linear
range of film exposure using a UMAX Astra 2200 scanner
and NIH Image Version 1.60 software (NIH Division of
Computer Research and Technology).
Statistical analysis Data were expressed as mean±SD.
Differences were analyzed for significance by Student's
t-test. The results were considered significant
at P<0.05.
Results
Inhibition of propofol on
H2O2-induced PC12 cell death
First, we examined the effect of propofol on
H2O2-induced PC12 cell death by measuring MTT reduction. As shown in
Figure 1, the application of
H2O2 (200 µmol/L) to PC12 cell for
24 h resulted in about 35% death. Pretreatment of cells with
propofol resulted in inhibition of
H2O2-induced cell death in a concentration-dependent manner. No effect of propofol
on cell viability was observed (data not shown).
Effect of propofol on
H2O2-induced PC12 cell apoptosis
To evaluate the effect of propofol on the apoptosis of PC12
cells, we examined the staining of apoptotic nuclei and DNA
fragmentation in H2O2-administered PC12 cells with or
without propofol pretreatment. As shown in Figure 2A,
staining with Hoechst 33258 revealed that PC12 cells showed
apoptotic nuclei after treatment with
H2O2 (200 µmol/L) for 24 h. Pretreatment with propofol decreases the rate of
apoptotic nuclei. As shown in Figure 2B, in the
determination of apoptotic cell death by a fluorescence-activated cell
sorter (FACS), 22% of cells showed apoptosis. Pretreatment
of PC12 cells with propofol (3 µmol/L) resulted in the
inhibition of H2O2-induced apoptosis to 14% of total cells. The
observation was consistent with that of the MTT test and
the results suggested that propofol attenuates
H2O2-induced PC12 cell death, including apoptosis.
Effects of propofol on
H2O2-induced caspase-3 activation
in PC12 cells Since caspase-3 has been shown to be an
important regulator of apoptotic cell death, we next
examined the effect of H2O2 (200 µmol/L) on caspase-3 activity in
PC12 cells. As shown in Figure 3, the incubation of PC12
cells with H2O2 (200 µmol/L) for 4 h significantly increased
caspases-3 activity. Pre-incubation with different
concentrations of propofol for 30 min significantly inhibited
H2O2-induced caspase-3 activation in a concentration-dependent
manner (Figure 3).
Effect of propofol on
H2O2-induced ERK1/2, JNK, and
p38 activation in PC12 cells To clarify the effect of propofol
on H2O2-induced MAP kinase phosphorylation, the PC12
cells were incubated in the presence of
H2O2 (200 µmol/L) for 10 min following pretreatment with indicated concentra-
tions of propofol for 30 min. We found that
H2O2-induced
phosphorylation of p38 instead of JNK and ERK1/2 was
inhibited by the treatment of propofol (Figure 4), which
indicated that the H2O2-induced p38 MAP kinase
phosphorylation in PC12 cells would be specifically sensitive to propofol.
Discussion
Oxidative stress has been implicated as a potential
contributor to the pathogenesis of acute central nervous
system injury. After brain injury by ischemic or hemorrhagic
stroke or trauma, the increased ROS production may lead to
tissue damage via different cellular molecular pathways. ROS
can cause damage to cardinal cellular components, such as
lipids, proteins, and DNA, resulting in subsequent cell death
by necrosis or apoptosis. In the present study, the
examined concentrations of
H2O2 were in the range of those
reached under pathophysiological conditions, such as
during transient cerebral ischemia[16]. Propofol has been shown
to cause protective action on
neurons[7], including antio-xidative
effects[17], inhibitory effects on lipid peroxida-
tion[18], and direct anti-excitotoxic
properties[19]. Other studies showed that propofol had a potential to enhance
neurological outcome and decrease the infarct size in experimental
animal models of stroke[20,21]. However, no evidence has
been reported concerning the direct effect of propofol on
neuronal-like PC12 cells and their effect on MAP kinase
activity. For the first time, this study showed that propofol
protected PC12 cells from
H2O2-induced cell death,
including apoptosis, by measuring MTT reduction, apoptotic
nuclei staining, FACS, and caspase-3 activity. We also
observed that propofol specifically inhibited
H2O2-induced p38 activation, but not ERK1/2 and JNK activation in PC12
cells.
Caspases are a family of specific cysteine proteases
whose activation is critical for the intracellular execution of
apoptotic death[22]. Among the 10+ caspases that have been
identified, caspase-3 is a common effecter to which several
procaspases and caspases converge, and therefore, can serve
as an apoptotic marker[23] induced by a variety of stimuli. It
has been shown that caspase-3 can be activated by
H2O2 exposure as a final effecter in apoptotic death
in vitro[24,25]. In agreement with this view, our results showed that
H2O2 markedly increased caspase-3 activity in PC12 cells.
More-over, we investigated the effect of propofol on the
H2O2-induced effect, where we found that propofol inhibited
H2O2-induced caspase-3 activation, as shown in Figure 3. However,
since the inhibitory effect of 10 µmol/L propofol on
H2O2-induced caspase-3 activation was incomplete (Figure 3), the
involvement of other mechanisms of propofol protective
effects against H2O2-induced PC12 cells death can not be
denied. Further studies are required to define the exact role
of caspase-3 in the mechanism of
H2O2-induced PC12 cell death.
Cellular signaling pathways are regulated by the
intracellular redox state of the cell. ROS may play an important role
as a second messenger in signal transduction cascades and
may lead to the activation of MAP
kinases[26]. Previous studies have shown that
H2O2 rapidly activated ERK and
p38 proteins in PC12 cells, and the mediation of cell death by
ROS may be closely related to MAP kinase
signaling[27,28]. In line with these studies, we found in the present study that 3
MAP kinases were rapidly and significantly activated by
oxidative stress in cultured PC12 cells (Figure 3). The
phosphorylation of the 3 MAP kinases almost simultaneously
reached their maximum at 10 min, and then the activity of the
3 MAP kinases decreased gradually (data not shown).
There-fore, we investigated the effect of propofol on the activity of
the 3 MAP kinases at 10 min. We found that propofol
suppressed p38 activation, but not JNK nor ERK1/2
phosphorylations (Figure 4), indicating that p38 is specifically sensitive
to propofol, which may contribute to its cellular protective
effect under oxidative stress. Previous
studies showed that the activation of the p38 MAP kinase induced apoptotic
death in neuroblastoma cells[29], while inhibitors of the p38
MAP kinases promoted neuronal survival in
vitro and
reduced cerebral infarction in
vivo[30,31]. In line with these studies, we also found that SB203580, a specific p38 inhibitor,
attenuated H2O2-induced PC12 cell death and caspase-3
activity (data not shown), which further verified the role of
p38 in oxidative stress-mediated cell death.
In conclusion, this study confirmed a neuroprotective
effect of propofol, at clinically relevant concentrations, on
H2O2-induced PC12 cell death, including apoptosis. It could
be assumed that this beneficial effect of propofol on PC12
cell viability is mediated, at least in part, by an inhibition of
the p38 pathway, but not that of ERK1/2 or JNK, which are
dramatically activated by
H2O2. The results of the present
study may shed light on the pharmacological basis for the
clinical application of some anesthetic compounds, such as
propofol in cerebral ischemic and/or reperfusion injury.
References
1 Mickalad M, Hans P, Deby-Dupont G, Hoebeke M, Deby C,
Lamy M. Propofol reacts with peroxynitrite to form a phenoxyl
radical: demonstration by electron spin resonance. Biochem
Biophys Res Commun 1998; 249: 833_7.
2 Saito A, Maier CM, Narasimhan P, Nishi T, SongYS, Yu
F, et al. Oxidative stress and neuronal death/survival signaling in cerebral
ischemia. Mol Neurobiol 2005; 31: 105_16.
3 Ergun R, Akdemir G, Sen S, Tasci A, Ergungor F. Neuroprotective
effects of propofol following global cerebral ischemia in rats.
Neurosurg Rev 2002; 25: 95_8.
4 Ito H, Watanabe Y, Isshiki A, Uchino H. Neuroprotective
properties of propofol and midazolam, but not pentobarbital, on
neuronal damage induced by forebrain ischemia, based on the GABA
receptors. Acta Anaesthesiol Scand 1999; 43: 153_62.
5 Daskalopoulos R, Korcok J, Farhangkhgoee P, Karmazyn M,
Gelb AW, Wilson JX. Propofol protection of sodium-hydrogen
exchange activity sustains glutamate uptake during oxidative
stress. Anesth Analg 2001; 93: 1199_204.
6 Lionel JV, Benjamin AG, Frederique MM, André LN, Nicolas J,
François MG, et al. Neuroprotective effects of propofol in a
model of ischemic cortical cell cultures: role of glutamate and its
transporters. Anesthesiology 2003; 99: 368_75.
7 Yu BW, Xue QS, Xia M, Wang ZJ, Chen HZ. The influences of
propofol on different kinds of brain injuries in rat brain
slices. Zhonghua Yi Xue Za Zhi 2003; 83: 1176_9.
8 Christophe M, Nicolas S. Mitochondria: a target for
neuropro-tective interventions in cerebral ischemia-reperfusion. Curr
Pharm Des 2006; 12: 739_57.
9 Chu CT, Levinthal DJ, Kulich SM, Chalovich EM, DeFranco DB.
Oxidative neuronal injury. The dark side of ERK1/2. Eur J
Biochem 2004; 271: 2060_6.
10 Kuhn K, Shikhman AR, Lotz M. Role of nitric oxide, reactive
oxygen species, and p38 MAP kinase in the regulation of human
chondrocyte apoptosis. J Cell Physiol 2003; 197: 379_87.
11 Ballif BA, Blenis J. Molecular mechanisms mediating
mammalian mitogen-activated protein kinase (MAPK) kinase
(MEK)-MAPK cell survival signals. Cell Growth Differ 2001; 12:
397_408.
12 Ono K, Han J. The p38 signal transduction pathway: activation
and function. Cell Signal 2000; 12: 1_13.
13 Kitagawa K, Matsumoto M, Hori M, Yanagihara T.
Neuropro-tective effect of apolipoprotein E against ischemia. Ann N Y
Acad Sci 2002; 977: 468_75.
14 Spicer Z, Millhorn DE. Oxygen sensing in neuroendocrine cell
and other cell types: pheochromocytoma (PC12) cell as an
experimental model. Endocr Pathol 2003; 14: 277_91.
15 Lee SJ, JinY, Yoon HY, Choi BO, Kim HC, Oh YK,
et al. Ciclo-pirox protects mitochondria from hydrogen peroxide toxicity.
Br J Pharmacol 2005; 145: 469_76.
16 Hyslop PA, Zhang Z, Pearson DV, Phebus LA. Measurement of
striatal H2O2 by microdialysis following global forebrain ischemia
and reperfusion in the rat: correlation with the cytotoxic
potential of H2O2 in
vitro. Brain Res 1995; 671: 181_6.
17 Zhang SH, Wang SY, Yao SL. Antioxidative effect of propofol
during cardiopulmonary bypass in adults. Acta Pharmacol Sin
2004; 25: 334_40.
18 Lee H, Jang YH, Lee SR. Protective effect of propofol against
kainic acid-induced lipid peroxidation in mouse brain
homo-genates: comparison with trolox and melatonin. J Neurosurg
Anesthesiol 2005; 17: 144_8.
19 Hans P, Bonhomme V, Collette J, Albert A, Moonen G. Propofol
protects cultured rat hippocampal neurons against
N-methyl-D-aspartate receptor-mediated glutamate toxicity.
J Neurosurg Anesthesiol 1994; 6: 249_53.
20 Young Y, Menon DK, Tisavipat N, Matta BF, Jones JG. Propofol
neuroprotection in a rat model of ischaemia reperfusion injury.
Eur J Anaesthesiol 1997; 14: 320_6.
21 Yamasaki T, Nakakimura K, Matsumoto M, Xiong L, Ishikawa
T, Sakabe T. Effects of graded suppression of the EEG with
propofol on the neurological outcome following incomplete
cerebral ischaemia in rats. Eur J Anaesthesiol 1999;
16: 320_9.
22 Garrido C, Galluzzi L, Brunet M, Puig PE, Didelot C, Kroemer G.
Mechanisms of cytochrome c release from mitochondria. Cell
Death Differ 2006; 13: 1423_33.
23 Iwai K, Kondo T, Watanabe M, Yabu T, Kitano T, Taguchi Y,
et al. Ceramide increases oxidative damage due to inhibition of
catalase by caspase-3-dependent proteolysis in HL-60 cell
apopto-sis. J Biol Chem 2003; 278: 9813_22.
24 Demelash A, Karlsson JO, Nilsson M, Björkman U. Selenium has
a protective role in caspase-3-dependent apoptosis induced by
H2O2 in primary cultured pig thyrocytes. Eur J Endocrinol 2004;
150: 841_9.
25 Song JH, Slot AJ, Ryan RW, Ross GM. Dopamine-induced death
of PC12 cells is prevented by a substituted tetrahydronaphthalene.
Neuropharmacology 2004; 46: 984_93.
26 Eguchi M, Monden K, Miwa N. Role of MAPK phosphorylation
in cytoprotection by pro-vitamin C against oxidative stress-
induced injuries in cultured cardiomyoblasts and perfused rat heart.
J Cell Biochem 2003; 90: 219_26.
27 Spicer Z, Millhorn DE. Oxygen sensing in neuroendocrine cell
and other cell types: pheochromocytoma (PC12) cell as an
experimental model. Endocr Pathol 2003; 14: 277_91.
28 Suzaki Y, Yoshizumi M, Kagami S, Koyama AH, Taketani Y,
Houchi H, et al. Hydrogen peroxide stimulates c-Src-mediated
big mitogen-activated protein kinase 1 (BMK1) and the MEF2C
signaling pathway in PC12 cell: potential role in cell survival
following oxidative insults. J Biol Chem 2002; 277: 9614_21.
29 Natzker S, Heinemann T, Figueroa-Perez S, Schnieders B, Schmidt
RR, Sandhoff K, et al. Cis-4-methylsphingosine phosphate
induces apoptosis in neuroblastoma cell by opposite effects on
p38 and ERK mitogen-activated protein kinases. J Biol Chem
2002; 383: 1885_94.
30 Horstman S, Kahle PJ, Borasio GD. Inhibitors of p38
mitogen-activated protein kinase promote neuronal survival
in vitro. J Neurosci Res 1998; 52: 483_90.
31 Piao CS, Kim JB, Han PL, Lee JK. Administration of the p38
MAPK inhibitor SB203580 affords brain protection with a wide
therapeutic window against focal ischemic insult. J Neurosci Res
2003; 73: 537_44.
|
