Department of Pharmacology, College of Medicine, Pusan National
University, Pusan, Korea (K.W.H., K.Y.K., J.H.L., H.K.S.); Chonbuk
National University, Chonbuk, Korea (Y.G.K.); Central Research
Institute, Dongbu Hannong Chemical Co. Daejon, Korea (S.-O.K., H.L.);
and Research Institute of Chemical Technology, Daejon, Korea (S.-E.Y.)
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Introduction |
Increasing
evidence has shown that transient focal ischemia initiates a cascade of
detrimental events, including the accumulation of intracellular
calcium, and the formation of free radicals and cytokines, including
tumor necrosis factor-
, which lead to disruption of cellular
homeostasis and structural damage of ischemic brain tissue (Kochanek
and Hallenbeck, 1992
; Feuerstein et al., 1994). Reactive oxygen
species, such as superoxide, hydrogen peroxide, and hydroxyl radical,
have been demonstrated to have a role in the mediation of neuronal
death in numerous disorders, including cerebral ischemia-reperfusion
injury (Chan, 1996). Apoptosis is implicated in ischemic brain injury
(Linnik et al., 1995). Free radicals are demonstrated to induce lipid
peroxidation and DNA damage (Dirnagl et al., 1999), and to produce
apoptosis (Kluck et al., 1997). The Bcl-2 family, consisting of
antiapoptotic (e.g., Bcl-2 and Bcl-XL) and
proapoptotic (e.g., Bax and Bad) members, plays important roles in the
regulation of cell death (Oltvai et al., 1993; Hockenbery, 1995). The
expression of Bcl-2 protein in the mitochondrial outer membrane acts to
inhibit cytochrome c translocation to cytosol (Kluck et al.,
1997; Gross et al., 1999; Shimizu and Tsujimoto, 2000) and its
over-expression prevents superoxide anion production, and blocks
cytochrome c release (Cai and Jones, 1998), whereas Bax
protein induces release of cytochrome c from mitochondria
and activation of caspases, which are critical steps in the apoptotic
processes (Jürgensmeier et al., 1998). Therefore, it might be
expected that increasing the levels of antioxidant enzyme (Keller et
al., 1998) or treatment with antioxidants will effectively suppress
neuronal damage (Huh et al., 2000).
In the pilot study, KR-31378
[(2S,3S,4R)-N"-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N'-benzylguanidine] showed the properties necessary to scavenge the intracellular ROS and
peroxyl radicals. In our previous in vitro results, KR-31378 effectively protected human umbilical vein endothelial cells from lipopolysaccharide-induced cell death in association with reduction in
tumor necrosis factor- production and inhibition of
oligonucleosomal DNA fragmentation (Kim et al., 2002). We recently
studied the effect of KR-31378, a benzopyran analog, on the action
potential characteristics in isolated rat ventricular myocytes in
comparison with pinacidil. KR-31378 showed little effect on the
KATP channel opening of rat ventricular myocytes.
However, KR-31378 showed the opening of a large-conductance
calcium-activated K+ current in rat basilar
arterial smooth muscle cells, which was reversibly blocked by
iberiotoxin, a large-conductance calcium-activated K+ channel blocker (10-100 nM), but not by
glibenclamide, a selective ATP-sensitive K+
channel blocker.
From the viewpoint that KR-31378 exerts antioxidant and maxi-K channel
opening actions, we attempted to identify new therapeutic targets for
the treatment of focal ischemic damage. We examined the neuroprotective
effects of KR-31378 on the cerebral infarct size and volume that
occurred after subjecting the rats to the 2-h occlusion of MCA and 24-h
reperfusion. To verify the mechanism(s) by which KR-31378 ameliorates
the cerebral ischemic damage, the effect of KR-31378 was determined by
TUNEL staining technique and DNA fragmentation assay on the tissue
sections corresponding to the penumbral zone of the injured rat brains.
The focus was to identify the ischemia-reperfusion-induced alterations
in Bcl-2, Bax proteins, and cytochrome c release from
mitochondria under treatment with and without KR-31378.
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Materials and Methods |
Preparation of Animals.
All animal studies carefully
conformed to the guidelines outlined in the Guide for Animal
Experiments edited by the Korean Academy of Medical Sciences and were
approved by the Animal Experimental Committee of the Pusan National
University, College of Medicine.
Male Sprague-Dawley rats weighing 280 to 320 g were anesthetized
with pentobarbital sodium (60 mg/kg, intraperitoneally). Core body
temperature was monitored continuously by a rectal thermistor probe and
maintained at 37 ± 0.5°C by placing the animals on a heating
pad (Homeothermic Blanket System; Harvard Apparatus, South Natick, MA).
Mean arterial blood pressure was monitored through the femoral artery
with a Statham P23D pressure transducer (Gould, Cleveland, OH) and
physiological variables, including blood gas, were checked before and
after MCA occlusion (STAT Profile 3; Nova Biomedicals, Boston, MA). The
mean partial pressure of CO2 was monitored
with an end tidal CO2 analyzer (CapStar-100; IITC
Life Science, Woodland Hills, CA).
Occlusion of the MCA was induced by the procedure of Longa et al.
(1989) with minor modifications. Briefly, surgical nylon suture thread
(3-0 in size) with a round tip was advanced from the external carotid
artery into the lumen of the internal carotid artery to occlude the
origin of the MCA. The wound was closed, and the animals were allowed
to recover over a 120-min period of occlusion. Two hours after MCA
occlusion, reperfusion was accomplished by pulling the suture thread
back to the bifurcation until the tip cleared the internal carotid
artery. Rats received 10, 30, or 50 mg/kg of KR-31378 dissolved in
dimethyl sulfoxide intraperitoneally three times (100 µl in volume)
at 5 min, 4 h, and 8 h after the completion of 2-h MCA
occlusion. Sham-operated control rats were subjected to neck incision
to expose the left common, external, and internal carotid arteries,
without insertion of suture thread into the artery. Other procedures
were identical to those in the MCA occlusion rats except for injection
of vehicle (100 µl of dimethyl sulfoxide solution intraperitoneally).
Analysis of Cerebral Infarct.
At 24 h of reperfusion
after 2 h of MCA occlusion, rats were given an overdose of
thiopental sodium and decapitated. The brain was then quickly removed,
frozen by suspension over liquid nitrogen, and cut into 2-mm thick
coronal block slices. The slices were immersed in a 2% solution of
2,3,5-triphenyltetrazolium chloride in normal saline at 37°C for 30 min and then fixed in 10% phosphate-buffered formalin at 4°C. The
2,3,5-triphenyltetrazolium chloride-stained brain slices were
photographed using a charge-coupled device video camera. The size of
infarct was calculated with an image analysis system (Image-Pro Plus;
Media Cybernetics, Silver Spring, MD) and expressed as the percentage
of infarcted tissue in reference to the ipsilateral hemisphere.
TUNEL Staining.
At 24 h after reperfusion, the animals
were deeply anesthetized with thiopental sodium and decapitated. The
brains were quickly removed, frozen in OCT compound, an
embedding medium for frozen tissue specimens (Sakura Finetek, Inc.,
Torrance, CA) by immersion in liquid nitrogen-equilibrated isopentane,
and then stored at 
70°C. The distances of the infarct rims from the
midline were determined on the brain slices from the coronal block of
samples. Brains were sectioned at bregma levels +4.7, +2.7, +0.7,
1.3, 3.3, 5.3, and 7.3 mm (seven sections).
The section obtained at the level of bregma +0.7 to 1.3 mm (section
3) was subjected to TUNEL staining. The distances of the infarct rims
from the midline were determined on the slices of the coronal blocks of
samples dissected for measurement of infarct areas. The region of
lateral cortex between 2- and 4-mm distance from the midline of the
coronal section, which falls within the border of infarction in
vehicle-treated animals, was spared in KR-31378-treated animals and can
be considered part of the ischemic penumbra. Coronal sections of 5-µm
thickness of the samples (2 × 2-mm2 region)
from the ipsilateral side were cut on a cryostat at 20°C. TUNEL
study was performed using a DNA fragmentation detection kit (QIA33;
Oncogene, Boston, MA). The sections were counterstained with
hematoxylin. TUNEL-positive cells were counted under a 100× objective.
To count cells in the penumbral zone, a 1 × 1 mm2 grid was placed under the slide and the
TUNEL-positive cells, estimated to be typical features of apoptosis
showing membrane disrupture, blebbing, and chromatin condensation, were
counted in each grid square and summed by an investigator blinded to
treatment. Necrotic cells showing diffuse cytoplasmic staining and lack
of nuclear condensation were not counted.
DNA Fragmentation Assay.
Sections obtained at the level of
bregma +0.7 to 1.3 mm (section 3) were subjected to determination of
laddering of DNA fragmentation. For oligonucleosomal fragmentation of
genomic DNA, cells were lysed in 1 ml of lysis buffer (10 mM Tris-HCl,
pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% sodium dodecyl sulfate, and 0.5 mg/ml proteinase K). Digestion was continued for 1 to 3 h at
55°C, followed by addition of RNase A to 0.1 mg/ml and running dye
(10 mM EDTA, 0.25% bromphenol blue, 50% glycerol). Equivalent amounts
of DNA (15-20 µg) were loaded into wells of 1.6% agarose gel and
electrophoresed in 0.5× TAE buffer (40 mM Tris-acetate, 1 mM EDTA) for
2 h at 6 V/cm. DNA was visualized by ethidium bromide staining.
Gel pictures were taken by UV transillumination with a Polaroid camera
(Polaroid Corp., Cambridge, MA). Bands were quantified by Molecular
Analyst software using the Bio-Rad image analysis system (Bio-Rad
Laboratories, Hercules, CA).
Western Blot Analyses.
The penumbral zones of coronal
sections 4 and 5 (at bregma levels 1.3 ~ 3.3 and 3.3 ~ 5.3 mm) were used for Western blot assays. After MCA occlusion,
the samples corresponding to the penumbra zone were homogenized, and
cells were lysed in lysis buffer containing 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 100 µg/ml phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin, and 1% Triton X-100. Following
centrifugation at 12,000 rpm, 50 µg of total protein from each sample
was loaded into a 12% SDS-polyacrylamide gel electrophoresis gel, and
transferred to nitrocellulose membrane (Amersham Biosciences,
Piscataway, NJ). The blocked membranes were then incubated with the
antibody of Bcl-2 and Bax (Santa Cruz Biotechnology, Santa Cruz, CA).
Mitochondrial cytochrome c was prepared as follows. After
MCA occlusion-reperfusion, the samples were washed in ice-cold
phosphate-buffered saline and were homogenized in buffer A (20 mM
Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM
Na-EDTA, 1 mM Na-EGTA, 1 mM dithiothreitol, and 0.1 mM
phenylmethylsulfonyl fluoride) containing 250 mM sucrose, and then
centrifuged twice at 750g for 10 min at 4°C. The harvested supernatants were again centrifuged at 10,000g for 10 min at
4°C, and the resulting mitochondrial pellets were dissolved in the 1× SDS sample buffer. Western blots were performed with the antibody of cytochrome c (Santa Cruz Biotechnology). The
immunoreactive bands were visualized using chemiluminescent reagent of
the Supersignal West Dura Extended Duration substrate kit (Pierce,
Rockford, IL). The signals of the bands were quantified using a
Calibrated imaging densitometer (GS-710, Bio-Rad Laboratories). The
protein concentration of the lysate was determined using a Bio-Rad DC
assay kit (Bio-Rad Laboratories).
Drugs.
KR-31378 was generously donated by the Korea Research
Institute of Chemical Technology, Daejon, Korea.
Statistical Analysis.
Repeated measures analysis of variance
was used for comparison of the results of hemispheric infarct area.
Other data were analyzed with Student's t test. Results are
expressed as means ± S.E.M. Differences were considered to be
significant when P < 0.05.
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Results |
Mean arterial blood pressure in the rats subjected to 2 h of
MCA occlusion and 24 h of reperfusion (105.8 ± 8.7 mm
Hg, n = 20) did not significantly differ from the
sham-operated rats (110.3 ± 6.4 mm Hg, n = 5).
Intraperitoneal injection of 30 or 50 mg/kg KR-31378 caused little
change in mean arterial blood pressure.
Effect of KR-31378 on Infarct Size.
The ischemic zone was
consistently identified in the cortex and striatum of the left cerebral
hemisphere as a distinct pale-stained area in the rats subjected to
2 h of ischemia and 24 h of reperfusion, which was attenuated
by treatment with either 30 or 50 mg/kg KR-31378 (Fig.
1). The infarct area was significantly
reduced when the animals received three administrations of 30 or 50 mg/kg KR-31378 at 5 min, 4 h, and 8 h after the completion of
2 h of ischemia (Fig. 2). Treatment
with 10 mg/kg KR-31378 was without effect.
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Fig. 1.
Illustrative coronal sections showing infarct area in
the cortex and striatum of the left cerebral hemisphere as a distinct
pale-stained area in the rats subjected to 2 h of ischemia/24 h of
reperfusion (vehicle) and attenuation of infarct area by treatment with
50 mg/kg KR-31378.
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Fig. 2.
Dose-dependent inhibitory effect of KR-31378 on the
infarct area of each coronal section in rats subjected to 2 h of
ischemia/24 h of reperfusion. Animals received KR-31378 (10, 30, or 50 mg/kg, intraperitoneally) at 5 min, 4 h, and 8 h after the
completion of 2 h of ischemia. Infarct area significantly
decreased in the second, third, and fourth coronal sections by
treatment with 30 and 50 mg/kg KR-31378. Results are expressed as
means ± S.E.M. of five to eight animals. *,
P < 0.05; **, P < 0.01 versus vehicle.
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Antiapoptotic Effect.
At 24 h of reperfusion, the samples
corresponding to the penumbra zone were obtained. A strong staining for
TUNEL was present in a moderate to a large number of cells in the
vehicle-treated ischemic brain, whereas TUNEL staining was negative in
the sham-operated control (Fig. 3).
Treatment with KR-31378 (30 and 50 mg/kg) significantly reduced the
number of TUNEL-positive cells (P < 0.05 and
P < 0.01) (Fig. 3).
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Fig. 3.
Representative photomicrographs of cerebral cortex
corresponding to the penumbral zone subjected to 2 h of
ischemia/24 h of reperfusion, which was stained with TUNEL. Top left,
sham-operated control; top middle, vehicle-treated; and top right,
KR-31378 (50 mg/kg)-treated cerebral cortex. The magnification of three
photographs was 100×, and the black line box of middle upper was
magnified to 400×. Lower panel represents the analyses of
TUNEL-positive cells. Results represent means ± S.E.M. of four
animals. *, P < 0.05; **,
P < 0.01 versus vehicle.
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Oligonucleosomal DNA fragmentation is known as a hallmark of apoptosis.
Figure 4 shows the segmented DNA at 180- to 200-bp intervals, reflecting the activity of endonuclease cleavage
of DNA at internucleosomal sites. Treatment with KR-31378 (30 and 50 mg/kg, intraperitoneally) strongly suppressed the laddered feature of
DNA fragmentation by 60% (P < 0.001) and 83.7%
(P < 0.001), respectively. Furthermore, we observed a
long-term protective effect of KR-31378 on the DNA fragmentation. The
samples were obtained on 1, 3, and 7 days of reperfusion after the
completion of 2 h of MCA occlusion. KR-31378 (50 mg/kg) was
administered intraperitoneally at 5 min, 4 h, and 8 h after
2 h of ischemia. In this result, reduced DNA fragmentation by
KR-31378 was still evident in the samples of 3 and 7 days of
reperfusion, even though the degree of DNA fragmentation gradually
increased with time (Fig. 5). KR-31378
(10 mg/kg) was without effect.
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Fig. 4.
Top panel, representative findings of agarose gel
electrophoresis showing DNA laddering. At 24 h of reperfusion
after 2 h of MCA occlusion, the samples corresponding to the
penumbral zone were obtained. DNA from penumbral lesion showed signs of
laddering of internucleosomal fragmentations. Animals received KR-31378
(10, 30, and 50 mg/kg KR-31378; KR10, KR30, KR50) at 5 min, 4 h,
and 8 h after the completion of 2 h of ischemia. Sections
obtained were at the level of bregma +0.7 to 1.3 mm (section 3). M
represents 100-bp DNA ladder marker. Bottom panel, the results of
densitometric analysis representing means ± S.E.M. of four
animals. ***, P < 0.001 versus vehicle
(Veh). Cont, sham-operated control.
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Fig. 5.
Representative findings of agarose gel
electrophoretic DNA laddering showing long-term protective effect of
KR-31378 on the DNA fragmentation. The samples were obtained from the
rats on 1, 3, and 7 days of reperfusion after the completion of 2 h of MCA occlusion. KR-31378 (50 mg/kg; KR) was administered
intraperitoneally at 5 min, 4 h, and 8 h after 2 h of
ischemia. Reduced DNA fragmentation by KR-31378 was still evident in
the samples from 3 and 7 days of reperfusion. M represents 100-bp DNA
ladder marker. Veh, vehicle; Cont, sham-operated control.
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Western Blot Analyses.
Figure 6
shows Western blot for Bcl-2 of the ischemic cortex corresponding to
the penumbral zone. Samples obtained from rats subjected to 2 h of
MCA occlusion and 24 h of reperfusion showed a markedly decreased
Bcl-2 protein level (0.34 ± 0.09 relative density), whereas those
from sham-operated control rats showed a considerable amount of Bcl-2
protein. The decreased Bcl-2 level in the ipsilateral ischemic brain
was markedly and concentration dependently reversed by treatment with
KR-31378 (10, 30, or 50 mg/kg, intraperitoneally). KR-31378 (50 mg/kg),
however, showed little enhancing effect on the Bcl-2 level in the
contralateral region of the brain subjected to MCA occlusion in
comparison with the sham-operated control group.
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Fig. 6.
Top, representative findings of Western blot for
Bcl-2 protein expression in duplicate with different samples from each
animal. At 24 h of reperfusion after 2 h of MCA occlusion,
the samples corresponding to the penumbral zone were obtained. Bottom
graph shows densitometric analyses of Bcl-2 protein level. KR-31378
(10, 30, and 50 mg/kg KR-31378; KR10, KR30, KR50) was injected at 5 min, 4 h, and 8 h after 2 h of ischemia. Bcl-2 protein
level was markedly decreased in the vehicle-treated ischemic brain,
which was markedly and concentration dependently reversed in the
KR-31378-treated ischemic brains. Results are expressed as means ± S.E.M. of four animals.  , P < 0.001 versus sham-operated control (Cont); *, P < 0.05; **, P < 0.01; ***,
P < 0.001 versus vehicle (Veh).
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Figure 7 shows the results of Western
blot for Bax protein expression and its densitometric analysis.
Although the brain tissues from sham-operated control rats showed a
trace level of Bax protein expression (relative density = 1), the
samples of ipsilateral side subjected to 2 h of MCA occlusion and
24 h of reperfusion revealed markedly increased Bax protein levels
(40.1 ± 2.0 relative density), which were significantly and dose
dependently reduced to 6.4 ± 0.6 relative density
(P < 0.001) by 50 mg/kg KR-31378.
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Fig. 7.
Top, representative findings of Western blot for Bax
protein expression in duplicate with different samples from each
animal. At 24 h of reperfusion after 2 h of MCA of occlusion,
the samples corresponding to the penumbral zone were obtained. Bottom,
graph shows densitometric analyses of Bax protein level. KR-31378 (10, 30, and 50 mg/kg KR-31378; KR10, KR30, KR50) was injected at 5 min,
4 h, and 8 h after 2 h of ischemia. Bax protein level
was markedly increased in the vehicle-treated ischemic brain, which was
markedly reduced in the KR-31378-treated ischemic brains. Results are
expressed as means ± S.E.M. of four animals. ,
P < 0.001 versus sham-operated control (Cont).
*, P < 0.05; **, P < 0.01; ***P < 0.001 versus vehicle (Veh).
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Western blot for cytochrome c release and its densitometric
analysis are shown in Fig. 8. Cytosolic
fractions obtained from samples from control rats showed trace amounts
of cytochrome c level (relative density = 1). Samples
obtained from rats subjected to 2 h of MCA occlusion and 24 h
of reperfusion revealed over 4-fold increases in cytochrome
c release from mitochondria, which was dose dependently
decreased by treatment with increasing doses of KR-31378.
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Fig. 8.
Top, representative findings of Western blot for
cytochrome c expression in duplicate with different
samples from each animal. At 24 h of reperfusion after 2 h of
MCA occlusion, the samples corresponding to the penumbral zone were
obtained. Bottom graph shows densitometric analysis of cytochrome
c level. KR-31378 (10, 30, and 50 mg/kg KR-31378; KR10,
KR30, KR50) was injected at 5 min, 4 h, and 8 h after 2-h
ischemia. Cytochrome c release was markedly increased in
the vehicle-treated ischemic brain, which was markedly reduced in the
KR-31378-treated brains. Results are expressed as means ± S.E.M.
of four animals. , P < 0.001 versus
sham-operated control (Cont); **, P < 0.01;
***, P < 0.001 versus vehicle (Veh).
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Discussion |
In the current study, we demonstrate the in vivo results from the
rat cerebral cortex subjected to 2 h of occlusion of MCA followed
by 24 h of reperfusion, in that KR-31378 exerted a potent protection against cerebral infarct size in association with the suppression of abundant TUNEL-positive cells and inhibition of DNA
fragmentation. Additionally, this compound preserved profoundly increased expression of Bcl-2 protein with significantly decreased Bax
protein and cytochrome c release from mitochondria in the penumbral zone.
Li et al. (1995) and Yao et al. (2001) have documented that apoptosis
contributes to the development of ischemic infarction with DNA
fragmentation, which prominently occurs in the penumbral zone with
moderately reduced cerebral blood flow. These reports suggest that the
apoptotic dynamic processes, including TUNEL-positive cells and
laddered feature of DNA fragmentation, can be the therapeutic targets
for ischemic brain injury. In the present study, we adopted the
peri-infarct region of 2- to 4-mm distance from the midline of the
third, fourth, and fifth coronal sections as corresponding to the
penumbral zone. In the penumbral region, blood flow is reduced to a
critical level during occlusion of MCA, but reperfusion after ischemia
provides an excess of oxygen with restored blood flow, leading to not
only sustaining neuronal viability but also catalyzing numerous
enzymatic oxidative reaction to produce ROS, triggering apoptosis or
laddered DNA fragmentation (Yao et al., 2001).
The large burst of ROS produced during an ischemia-reperfusion episode
plays a major role in the ensuing cerebral damage by inducing neuronal
apoptosis (Bredesen, 1995; Chan, 1996). A role for superoxide in
producing ischemic neuronal death was suggested by Yang et al. (1994),
in that they showed that increased infarct size observed at 24 h
after MCA occlusion was significantly decreased in transgenic mice
over-expressing human copper-zinc superoxide dismutase, which was
suggestive of an important role for oxygen free radicals in ischemic
brain injury. The most intriguing result was that even postischemic
administration of KR-31378 (at 5 min, 4 h, and 8 h after the
completion of 2 h of occlusion of MCA) successfully suppressed
cerebral infarct size and volume with a significantly reduced number of
TUNEL-positive cells and by the suppression of the laddered feature of
DNA fragmentation. TUNEL technique may provide an advantage of anatomic
preciseness. However, it is rather unspecific for apoptotic DNA
fragmentation since it may stain the necrotic cells (Grasl-Kraupp et
al., 1995). Thus, gel electrophoresis was employed for detection of
oligonucleosomal DNA fragmentation to confirm apoptotic cell death.
ROS, including hydrogen peroxide and hydroxyl radical, and lipid
hydroperoxides, are all importantly implicated in the processes of
apoptosis as second messengers in cytokine (i.e., tumor necrosis factor- and interleukin-1)-induced apoptosis (Kroemer et al., 1995; Li et al., 1997). We have recently observed that KR-31378 as well
as -tocopherol
(10
8-105 M)
significantly reduced the elevated production of thiobarbituric acid-reactive substance in native low density lipoprotein when incubated with CuSO4, suggesting that KR-31378
has a role of antioxidant (data not shown). Thus, it is likely that the
KR-31378-induced reduction in cerebral infarct area correlates well
with its ability to scavenge ROS and to suppress lipid peroxidation. In
that hydroxyl and peroxyl radicals are biologically toxic radical
species and not detoxified by an enzymatic mechanism (Floyd, 1990;
Halliwell and Gutteridge, 1990), KR-31378 with a wide spectrum of
scavenging capacity may indicate its great significance in protecting
cells from oxidative stress.
The complex processes of apoptosis implicate the activation of cysteine
proteases of the caspase family, alterations in plasma membrane
phospholipids, and nuclear DNA condensation and fragmentation (Bredesen, 1995). A decrease in the immunoreactivity of Bcl-2 and
increase in Bax protein was demonstrated in neurons with ischemic cortex and thalamus, leading to neuronal apoptosis (Gillardon et al.,
1996). Farlie et al. (1995) and Martinou (1999) have suggested that
over-expression of Bcl-2 in transgenic mice protects neurons from
ischemia-induced cell death. In the present work, we did not clarify
the mechanism by which Bcl-2 exerts the protective effects against
ischemic damage. Reportedly, oxygen-derived free radicals have been
implicated in the pathogenesis of infarction caused by ischemia, and
antioxidants such as catalase and polyethylene glycol-conjugated
superoxide dismutase protected neurons from ischemic damage (Liu et
al., 1989). The antiapoptotic effects of Bcl-2 in ischemia are ascribed
to its antioxidant function and its ability to reduce the generation of
ROS (Hockenbery et al., 1993; Kane et al., 1993).
A question arises as to how KR-31378 elicits up-regulation of Bcl-2
expression. Haendeler et al. (1996) have illustrated the role of
antioxidants in the regulation of the Bcl-2 protein family in which
N-acetylcysteine and the combination of vitamins C and E (10 µM) inhibited lipopolysaccharide-induced apoptosis. This reduction of
apoptosis was paralleled by an increase in Bcl-2 and a decrease in Bax
protein levels. Thus, it is suggested that increased cell survival and
up-regulation of Bcl-2 protein expression by KR-31378 may be related to
its antioxidant effect.
Bax protein is one of the Bcl-2 family homologous to Bcl-2 (Oltvai et
al., 1993), and its activity is neutralized by binding to Bcl-2 (Sato
et al., 1994). The findings showing that KR-31378 strongly suppressed
the increased Bax levels induced by ischemic insult were consistent
with the inhibitory effect of KR-31378 on the
lipopolysaccharide-induced up-regulation of Bax protein in human
umbilical vein endothelial cells (Kim et al., 2002).
Recent studies have implicated mitochondria as an important regulatory
site of the apoptotic process (Kroemer, 1998), especially in relation
to the rise of cytochrome c release from mitochondria to
cytosol (Zhang et al., 2000). It was suggested that Bcl-2
prevents the loss of the mitochondrial membrane potential and the
release of cytochrome c to cytosol (Kluck et al., 1997;
Gross et al., 1999), and over-expression of Bcl-2 blocks cytochrome
c release by preventing superoxide production (Cai and
Jones, 1998), whereas Bax protein promotes apoptosis by triggering the
release of cytochrome c from mitochondria, thereby
promoting activation of caspase cascade (Jürgensmeier et
al., 1998). As shown in the profile of Bax, the increased cytochrome
c release was also markedly suppressed by KR-31378. It
remains, however, to be elucidated how KR-31378 up-regulates Bcl-2 and
down-regulates Bax expression and cytochrome c release and
whether these variables happen to occur together for protection by
KR-31378 in the tissues subjected to ischemia-reperfusion.
As a mechanistic study, we identified the opening of calcium-activated
K+ current by KR-31378 in rat basilar arterial
smooth muscle cells. The current was reversibly blocked by addition of
iberiotoxin, a large-conductance calcium-activated
K+ channel blocker (10-100 nM) to the bath but
was not blocked by glibenclamide, a selective ATP-sensitive
K+ channel blocker (data not shown). Our
speculation was that, at higher intracellular
Ca2+ due to ischemic damage, KR-31378 might
produce greater increases in maxi-K channel-mediated currents. Since
maxi-K channels are present in many brain regions, including the cortex
and hippocampus (Knaus et al., 1996), it is therefore predicted that
maxi-K channels, when opened, may reduce voltage-dependent
Ca2+ entry into the cells after restoration of
membrane potential (Latorre et al., 1989). At the present time, it is
undetermined whether enhancement of Bcl-2 protein level in association
with suppression of DNA fragmentation by KR-31378 is attributable to the opening of maxi-K channels and to the reduction in intracellular Ca2+ accumulation. Nevertheless, it is suggested
that the protective mechanism of KR-31378 involves antioxidant cell
protective action as well as a large-conductance
Ca2+-activated K+ channel
opening effect.
Taken together, KR-31378-induced up-regulation of Bcl-2 and
down-regulation of Bax protein and cytochrome c release
correlate well with the impressive neuroprotective effect of KR-31378
to suppress DNA fragmentation and brain infarct occurring after MCA occlusion/reperfusion. In conclusion, postischemic treatment with KR-31378 effectively decreased cerebral infarct size and laddered DNA
fragmentation, which were associated with prominent preservation of
Bcl-2 protein and significant reduction in Bax protein and cytochrome
c release.
This work was supported by a fund from the Center for Bioactive
Substances (Korea Research Institute of Chemical Technology, Daejon),
the Korea Science & Engineering Foundation, and Research Institute of
Genetic Engineering (Pusan National University, Pusan, Korea).
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Abstract
Introduction
