Prevention of hepatic ischemia-reperfusion injury by
green tea extract
Zhi
Zhong1,2,
Matthias
Froh2,
Henry D.
Connor2,
Xiangli
Li2,
Lars
O.
Conzelmann2,
Ronald P.
Mason3,
John J.
Lemasters1, and
Ronald G.
Thurman2,
Departments of 1 Cell and Developmental Biology
and 2 Pharmacology, University of North
Carolina at Chapel Hill 27599; and 3 Laboratory Of
Pharmacology and Chemistry, National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina 27709
 |
ABSTRACT |
These experiments were designed to determine
whether green tea extract (GTE), which contains polyphenolic free
radical scavengers, prevents ischemia-reperfusion injury to the
liver. Rats were fed a powdered diet containing 0-0.3% GTE
starting 5 days before hepatic warm ischemia and reperfusion.
Free radicals in bile were trapped with the spin-trapping reagent
-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) and measured using electron spin resonance spectroscopy. Hepatic ischemia-reperfusion increased transaminase
release and caused pathological changes including focal necrosis and
hepatic leukocyte infiltration in the liver. Transaminase release was diminished by over 85% and pathological changes were almost totally blocked by 0.1% dietary GTE. Ischemia-reperfusion increased
4-POBN/radical adducts in bile nearly twofold, an effect largely
blocked by GTE. Epicatechin, one of the major green tea polyphenols,
gave similar protection as GTE. In addition, hepatic
ischemia-reperfusion activated NF-
B and increased TNF-
mRNA and protein expression. These effects were all blocked by GTE.
Taken together, these results demonstrate that GTE scavenges free
radicals in the liver after ischemiareoxygenation, thus
preventing formation of toxic cytokines. Therefore, GTE could prove to
be effective in decreasing hepatic injury in disease states where
ischemia-reperfusion occurs.
ischemia; free radicals; liver; nuclear factor-kappaB; cytokines
 |
INTRODUCTION |
ISCHEMIA-REPERFUSION
INJURY to the liver occurs in trauma and hemorrhagic shock and
after hepatic surgery, including tumor resection and transplantation.
Reactive oxygen species produced on reperfusion play a critical
role in the injury caused by ischemia-reperfusion (13, 30, 32). Accumulation of purine derivatives due to ATP degradation and reduction of mitochondrial ubiquinone produce superoxide radicals on reoxygenation (5, 25, 30, 32). In
addition, macrophages and neutrophils in previously ischemic tissue are activated and produce oxygen radicals via NADPH oxidase (4). Indeed, superoxide production by Kupffer cells and
neutrophil accumulation increases over fivefold after hepatic
ischemia-reperfusion (24). Moreover, gadolinium
chloride, a drug that selectively destroys and/or inactivates Kupffer
cells, minimizes liver injury after ischemia-reoxygenation
(6). Reactive radical species not only directly
damage cell membranes, DNA, and protein, they also trigger formation of
toxic cytokines and increase adhesion molecules leading to an
inflammatory response, tissue damage, and multiple organ failure
(1, 13, 22).
Green tea (Camellia sinenesis) contains high levels of
polyphenols including (+)-catechin, (
)-epicatechin,
(+)-gallocatechin, (
)-epigallocatechin, (
)-epicatechin
gallate, and (
)-gallocatechin gallate (15, 21).
Polyphenols from green tea are efficient free radical and singlet
oxygen scavengers (39, 47), and green tea extract (GTE)
inhibits lipid peroxidation in in vitro systems, in experimental
animals, and in humans (15, 21, 34, 36). Considerable
epidemiological and experimental evidence shows beneficial effects of
GTE in reducing the risk of heart disease and cancer (27, 41,
43), most likely due to the antioxidant property of polyphenols
(15). Accordingly, this study is designed to test the
hypothesis that GTE blocks free radical formation after ischemia-reperfusion, thus preventing liver injury.
 |
METHODS |
Animals.
Male Sprague-Dawley rats (200-250 g) were fed a chow diet (Purina)
containing 0-0.3% GTE (Taiyo Kagaku, Yokkaichi, Mie, Japan), which contained 85% of polyphenols by weight, for 5 days and were fasted overnight before surgery. Detailed polyphenolic compositions are
shown on Table 1. Some rats were fed a
diet containing 0.085% epicatechin that had a polyphenol content equal
to 0.1% GTE diet. Rats were anesthetized with pentobarbital (50 mg/kg
ip), and body temperature was maintained at 37°C with warming lamps.
The upper abdomen was opened with a vertical midline incision, and
hepatic ischemia was induced by clamping the artery and portal
vein to the upper three lobes of the liver (i.e., ~70% of total
liver). One hour later, the ischemic liver was reperfused by
opening the vascular clamp and the wound was closed with a running
suture (3-0). Rats were killed 1.5-24 h later
for histology and assessment of cytokine mRNA. All animals were given
humane care in compliance with institutional guidelines.
Serum transaminase, TNF-
, and histology.
Blood samples were collected from the tail vein at times indicated in
the figures. Serum was obtained by centrifugation and stored at
20°C. Aspartate aminotransferase (AST) activity was determined
using commercially available analytical kits from Sigma (St. Louis, MO).
In some rats, 200 µl of blood was collected using a heparinized
syringe into 75 µl aprotinin, and plasma was collected and analyzed
for TNF-
using an ultrasensitive, enzyme-linked immunosorbent assay
kit (Biosource). Data were corrected for dilution.
On the day animals were killed, animals were anesthetized with
pentobarbital (70 mg/kg ip), the abdomen was opened, and the portal
vein was cannulated with a 20-gauge intravenous catheter. The liver was
rinsed with 10 ml normal saline, perfusion fixed with 10% buffered
formaldehyde, and processed for histology. Sections were stained with
hematoxylin-eosin and analyzed microscopically. In addition, myeloid
cell antigen ED-1, a marker of monocytes/macrophages, and
myeloperoxidase, a marker of neutrophils, were detected in some
sections by immunohistochemical staining. Sections were deparaffinized in xylene, rehydrated in a series of graded alcohol concentrations, and
placed in phosphate-buffered saline with 1% Tween-20.
Immunohistochemistry was performed with a monoclonal primary antibody
against ED-1 (Serotek, Raleigh, NC) at the concentration of 1:250 and
incubated for 1 h at room temperature or with a primary rabbit
antibody against myeloperoxidase (DAKO, Carpinteria, CA) at the
concentration of 1:200 and incubated for 30 min. The immunostaining was
visualized using the DAKO EnVision kit (DAKO) following the
instructions of the manufacturer, and the slides were counterstained
with hematoxylin.
Measurement of myeloperoxidase in liver homogenates.
Liver tissue (200 mg) was homogenized in 2 ml of 20 mM potassium
phosphate buffer (pH 7.4) and centrifuged at 1,700 g at
4°C for 20 min. The pellet was resuspended in potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethyl ammonium bromide and 10 mM EDTA. Activity of myeloperoxidase in the suspension was
detected as described elsewhere (38).
Detection of free radical adducts.
To assess free radical formation by the liver, the spin-trapping
reagent
-(4-pyridyl 1-oxide)-N-tert-butylnitrone (4-POBN; 1 g/kg body wt) was dissolved in 0.5 ml normal saline and injected slowly into the tail vein after opening the vascular clamps. A polyethylene cannula (PE-50) was placed in the common bile duct, and
bile was collected for 1 h into 50 µl of transition metal chelator solution (30 mM dipyridyl and 30 mM bathocuproine) on ice to
prevent ex vivo free radical formation. Samples were stored at
80°C
until analysis. Bile samples were thawed, placed in an electron spin
resonance (ESR) flat cell and bubbled with oxygen in the presence of an
ascorbate oxidase paddle to oxidize ascorbate to ascorbate dione, which
is no longer a free radical. Free radical adducts were detected with a
Bruker ELEXSYS ESR spectrometer. Instrument conditions were as follows:
20-mW microwave power, 1.0-G modulation amplitude, and 80-G scan range.
Spectral data were stored on an IBM compatible computer and were
analyzed for ESR hyperfine coupling constants by computer simulation
(12). Quantification of free radical adducts was achieved
by double integration of ESR spectra using the calculation function of
the ESR program and normalized to bile volume collected in 1 h
(12).
NF-
B determination using electrophoretic mobility shift
assays.
Measurement of NF-
B by EMSA was performed as described in detail
elsewhere (46). Briefly, nuclear extracts from liver
tissue were preincubated 10 min on ice with 1 µg poly(dI-dC) in a
buffer containing 1 mM HEPES (pH 7.6), 40 mM MgCl2, 0.1 M
NaCl, 8% glycerol, 0.1 mM DTT, 0.05 mM EDTA, and 10 mg/ml bovine serum
albumin. A 32P-labeled DNA probe with the consensus
sequence for NF-
B (200,000 counts/min/µl, Cerenkov
counting) containing 0.4 ng of double-stranded oligonucleotide was
added with or without a 250-fold excess of cold oligonucleotide as
competitor. Mixtures were incubated 20 min and separated on a 5%
polyacrylamide (29:1 cross-linking) gel (18).
RNase protection assay for TNF-
mRNA.
Livers were rinsed with 10 ml normal saline and frozen in liquid
nitrogen. Total RNA was isolated from liver tissue using RNA STAT 60 (Tel-Test), and RNase protection assays were performed using the
RiboQuant multiprobe assay system (Pharmingen). Briefly, using the
multiprobe template set rCK-1(9), 32P-labeled
RNA probes were transcribed with T7 polymerase followed by
phenol:chloroform extraction and ethanol precipitation. Ten micrograms
of total RNA per sample was hybridized to 5 × 105 cpm
of probe overnight at 56°C and digested with RNase followed by
proteinase K treatment, phenol/chloroform extraction, and ethanol and
ammonium acetate precipitation. Samples were resolved on a 5%
acrylamide/bisacrylamide (19:1) urea gel, which was visualized by
autoradiography after drying (16).
Statistical analysis.
All groups were compared using ANOVA plus Student-Newman-Keuls post hoc
test as appropriate. Differences were considered significant at
P < 0.05.
 |
RESULTS |
Effects of GTE on release of transaminase and liver pathology.
AST (Fig. 1A), an indicator of
liver injury, averaged ~50 U/l before ischemia-reperfusion.
AST release was not significantly altered by sham operation; however,
transaminase increased gradually after reperfusion, peaked at 3 h,
and decreased slowly afterward. AST increased ~25-fold 3 h after
reperfusion (Fig. 1A). Importantly, GTE blunted the increase
of transaminase caused by ischemia-reperfusion in a
dose-dependent manner (Fig. 1B). When rats were fed 0.1% GTE or 0.085% epicatechin, serum AST levels at 3 h after
ischemia-reperfusion were only 20 and 34% of values in rats
that were fed control diet (Fig. 2).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1.
Transaminase release after hepatic
ischemia-reperfusion (I/R). Rats were fed regular chow diets
with 0-0.3% green tea extract (GTE) for 5 days and were fasted
overnight before surgery. Hepatic ischemia was induced by
clamping the artery and portal vein to the upper three lobes (i.e.,
~70%) of the liver. One hour later, ischemic livers were
reperfused by opening the vascular clamp as described in
METHODS. Blood samples were collected postoperatively from
the tail vein at times indicated, and aspartate aminotransferase (AST)
activities were determined. Values are means ± SE. A:
time course of AST release (0.1% GTE, n = 4 in each
group). aP < 0.05 compared with rats
receiving control diet and sham operation;
bP < 0.05 compared with rats receiving
control diet and I/R. B: dose-dependency of the effect of
GTE on AST releases. *P < 0.05 compared with rats
receiving control diet and subjected to I/R.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
Protection by GTE and epicatechin (EC) against
transaminase release after I/R. Conditions were the same as Fig. 1
except that rats were fed regular chow diets containing 0.1% GTE or
0.085% EC. Blood samples were collected 3 h after reperfusion,
and AST activity was determined. Values are means ± SE
(n = 4-6 in each group).
aP < 0.05 compared with rats receiving
control diet and sham operation; bP < 0.05 compared with rats receiving control diet and I/R by ANOVA with
Student-Newman-Keuls post hoc test.
|
|
No pathological changes were observed in liver tissue 24 h after
sham operation (Fig. 3A). In
contrast, liver specimens from rats after ischemia-reperfusion
exhibited focal necrosis and infiltration of leukocytes as expected
(Fig. 3B). Dietary GTE (0.1%) and epicatechin (0.085%)
significantly decreased these pathological changes (Fig. 3,
C and D). To confirm infiltration of leukocytes
after ischemia-reperfusion, immunohistochemical staining was
performed to detect myeloid cell antigen ED-1, a marker of
monocytes/macrophages, as well as myeloperoxidase, an indicator of
neutrophils, in some slides. A small number of ED-1 positive cells were
observed in livers from rats given sham operation (Fig.
4A). Twenty-four hours after
ischemia-reperfusion, the number of ED-1 positive cells
increased dramatically (Fig. 4B), and this effect was
largely blocked by GTE as well as by epicatechin (Fig. 4, C
and D). Similar changes were observed for myeloperoxidase-positive cells (data not shown). These results show
that green tea polyphenols prevented white blood cell infiltration after ischemia-reperfusion.

View larger version (183K):
[in this window]
[in a new window]
|
Fig. 3.
Protection by GTE and EC against hepatic pathological changes
caused by I/R. Conditions were the same as Fig. 2. Twenty-four hours
after reperfusion, livers were perfused with 10 ml normal saline to
remove blood and fixed with 10% buffered formaldehyde. Fixed tissue
was embedded in paraffin and processed for light microscopy. Sections
were stained with hematoxylin and eosin. Arrows identify necrotic
areas. Representative images (original magnification = ×40) are
shown from at least 4 sections from each group. A:
sham-operated rats; B: 24 h after I/R;
C: 0.1% GTE + I/R; D: 0.085% EC + I/R.
|
|

View larger version (185K):
[in this window]
[in a new window]
|
Fig. 4.
Protection by GTE and EC against increases in monocytes/macrophages
caused by I/R. Conditions were the same as Fig. 3. ED-1, a marker of
monocytes/macrophages, was detected in sections by immunohistochemical
staining. Representative images are shown (original magnification = ×200) from at least 4 sections from each group. A:
sham-operated rats; B: 24 h after I/R; C:
0.1% GTE + I/R; D: 0.085% EC + I/R.
|
|
Effects of GTE on myeloperoxidase activity in liver
homogenates.
Activity of myeloperoxidase, an indicator of neutrophil infiltration,
was ~24 U/g liver from sham-operated animals. Enzyme activity
gradually increased after ischemia-reperfusion, doubling 1.5 h after reperfusion and increasing about threefold at 3 and 24 h. GTE blocked the increases in myeloperoxidase activity in liver homogenates caused by ischemia-reperfusion almost
completely (Fig. 5).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
Protection by GTE against increased myeloperoxidase
activity in the liver after I/R. Rats were fed regular chow diets with
0.1% GTE for 5 days, and livers were harvested 1.5-24 h after
I/R. Each liver was perfused with 10 ml normal saline to remove blood
and was then frozen in liquid nitrogen. Myeloperoxidase in liver
homogenates was determined as described in METHODS. Values
are means ± SE. (n = 4 samples in each group).
aP < 0.05 compared with rats receiving
control diet and sham operation; bP < 0.05 compared with rats receiving control diet and I/R by ANOVA with
Student-Newman-Keuls post hoc test.
|
|
Effects of GTE on free radical production after
ischemia-reperfusion.
To assess oxidative stress, free radicals were trapped with a
spin-trapping agent and detected using ESR spectrometry. Free radical
adduct signals were barely detectable in sham-operated rats fed with or
without GTE (Fig. 6, A and
B). However, a six-line ESR signal was increased
significantly in the bile 1 h after reperfusion (Fig.
6C). Computer simulation of the spectrum (Fig.
6D) was accomplished using hyperfine coupling constants of
aN =15.70 G and aH = 2.62 G for a single radical species. Such coupling constants are
characteristic of carbon-centered 4-POBN radical adducts and closely
match values (aN = 15.63 G;
aH = 2.73 G) obtained from bile of rats
given spin trap and oxidized polyunsaturated fatty acids
(8). Subsequent mass spectral analysis of a lipid
metabolizing linoleic acid/lipoxidase system that produced the same ESR
spectral parameters confirmed that the free radical adducts contained
lipid fragments, such as pentyl (23). Pentyl free radicals
are formed in vivo on the
-scission of arachidonic or linoleic
acid-derived alkoxyl radicals (28). Free radical adduct
signals were minimal in sham-operated rats but increased about twofold
during ischemia-reperfusion (Fig.
7). Importantly, GTE and epicatechin
almost completely prevented free radical adduct production due to
ischemia-reperfusion (Figs. 6, E and F,
and 7).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 6.
Protection by GTE and EC against increased free radical
adducts in bile detected by electron spin resonance signal intensity
after I/R. Conditions were the same as Fig. 2. The spin-trapping
reagent -(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN;
1 g/kg) was injected into the tail vein on opening the vascular clamps.
Bile excreted during the first hour after reperfusion was collected via
a polyethalene cannula (PE-50) placed in the common bile duct into 50 µl of 30 mM dipyridyl and 30 mM bathocuproine on ice to prevent ex
vivo radical formation and stored at 80°C until analysis. Shown are
typical ESR spectra: A: sham-operated; B:
sham-operated + GTE; C: I/R; D: simulation
of ESR spectra of C; E: 0.1% GTE + I/R;
and F: 0.085% EC + I/R.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 7.
Quantification of free radical formation after I/R.
Conditions were the same as Fig. 6. Free radical adducts in bile after
sham operation, I/R, I/R + GTE, and I/R + EC were quantified
by double integration of ESR spectra as described in
METHODS and normalized to the bile volume collected in
1 h. Values are means ± SE (n = 4-5
samples in each group). aP < 0.05 compared
with sham operation; bP < 0.05 compared
with I/R with control diet by ANOVA with Student-Newman-Keuls post hoc
test.
|
|
Effects of GTE on NF-
B activation after
ischemia-reperfusion.
NF-
B, a transcription factor controlling synthesis of a variety of
proinflammatory cytokines (10, 37), can be activated by
oxidative stress (35). NF-
B/DNA complexes are barely
detectable in livers from sham-operated rats (Fig.
8A). Three hours after reperfusion, however, NF-
B was increased about twofold (Fig. 8,
A and B). GTE largely inhibited NF-
B
activation after ischemia-reperfusion (Fig. 8).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 8.
Protection by GTE against NF- B activation after I/R.
Rats were fed regular chow diets with 0.1% GTE for 5 days, and livers
were harvested 3 h after reperfusion. Each liver was perfused with
10 ml normal saline to remove blood and frozen in liquid nitrogen.
NF- B in nuclear extracts from liver tissue was measured by EMSA, as
described in METHODS. A: representative gel;
B, average densitometric analysis of the NF-kB/DNA
complexes. Values are means ± SE; n = 4 samples
in each group. aP < 0.05 vs. sham
operation; bP < 0.05 vs. I/R by ANOVA with
Student-Newman-Keuls post hoc test.
|
|
Effects of GTE on cytokine production.
TNF-
is an important mediator of the inflammatory process
(7). TNF-
mRNA increased about fourfold after
ischemia-reperfusion, and this effect was largely blocked by
GTE (Fig. 9A). Plasma TNF-
levels were also increased ~2.3-fold above basal levels 3 h
after reperfusion (Fig. 9B), and gradually decreased to
basal values 24 h later (data not shown). GTE blunted increases in
plasma TNF-
totally (Fig. 9B).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 9.
Protection by GTE against increased TNF- formation
after I/R. Rats were fed regular chow diets with 0.1% GTE for 5 days,
and livers were harvested 3 h after reperfusion. A:
TNF- mRNA was assessed by a RNase protection assay, as described in
the METHODS. B: blood (200 µl) was collected,
by using a heparinized syringe, into 75 µl aprotinin, and plasma was
analyzed for TNF- using an enzyme-linked immunosorbent assay. Values
are means ± SE; n = 4-6 samples in each
group. aP < 0.05 vs. sham operation;
bP < 0.05 vs. I/R by ANOVA with
Student-Newman-Keuls post hoc test.
|
|
 |
DISCUSSION |
Free radicals modulate NF-
B activation and toxic cytokine
formation after ischemia-reperfusion of the liver.
Accumulation of purine derivatives and reduced mitochondrial ubiquinone
during ischemia causes superoxide radical production on
reoxygenation (5, 25, 30, 32). In addition, activation of
macrophages and neutrophils also produces oxygen radicals via NADPH
oxidase (4, 19, 24); therefore, reactive oxygen species are produced on reperfusion. Indeed, free radical formation
increased significantly after ischemia-reperfusion of the
liver (Fig. 6 and 7). Reactive radical species can directly damage cell
membranes and macromolecules. Considerable evidence shows that oxygen
radicals cause lipid peroxidation, thus directly damaging cell
membranes (13). In addition, oxygen radicals can damage
other biologically important macromolecules, such as DNA and protein
molecules, leading to cell injury (13, 22). In support of
this hypothesis, increased free radical formation is associated with
tissue injury (AST release and necrosis) after hepatic
ischemia-reperfusion (Figs. 1-3).
In addition to its direct damage on cell components, reactive oxygen
species activate phospholipase A2 (17), which
increases production of lipid-derived vasoactive and chemotactic
mediators, such as eicosanoids and platelet-activating factor.
Oxidative stress also activates NF-
B (35), a
transcription factor that consists of p65 and p50 subunits. NF-
B
translocates from the cytosol to the nucleus when it is activated
(3), leading to synthesis of proinflammatory cytokines and
cell adhesion molecules (10, 26, 37). Several studies show
that reactive oxygen species directly induce NF-
B activation and
cytokine production (11, 20, 35), and reactive oxygen and
nitrogen species are well recognized as important signaling molecules
regulating expression of cytokines and enzymes (44). Redox
status in the cytosol also regulates the phosphorylation and
degradation of I
B, an inhibitor of NF-
B (14).
Ischemia-reperfusion injury to the liver occurs in trauma and
hemorrhagic shock and after hepatic surgery, including tumor resection
and transplantation. Under these situations, endotoxemia frequently
occurs. Reactive oxygen production also mediates endotoxin-induced NF-
B activation and TNF-
production (20, 40).
Therefore, oxidative stress may contribute to an inflammatory
response induced by endotoxemia after hepatic
ischemia-reperfusion. In support of this hypothesis, NF-
B
was activated and the proinflammatory cytokine TNF-
increased
after ischemia-reperfusion of the liver (Fig. 8 and 9).
Infiltration of white blood cells was also observed (Fig. 4 and 5).
Taken together, our data confirm that liver
ischemia-reperfusion increases oxidative stress, an effect that
not only produces direct tissue damage (necrosis, AST release) but also
modulates production of toxic cytokines leading to inflammation and
leukocyte infiltration, consistent with previous studies
(13).
GTE prevents free radical and TNF-
formation.
Previous studies showed that GTE inhibits lipid peroxidation in in
vitro systems, experimental animals, and humans (15, 21, 34,
36). GTE also decreases malondialdehyde and lipid hydroperoxide
levels in blood and increases total antioxidant capacity in animals and
humans (36, 42). Considerable epidemiological and
experimental evidence shows that green tea decreases oxidation of LDL
and increases the ratio of HDL-to-LDL, thus decreasing the risk of
heart disease (31, 36, 42). Beneficial effects of green
tea are most likely due to polyphenols, which are efficient free
radical and singlet oxygen scavengers (39, 47). Because liver ischemia-reperfusion causes free radical production and green tea polyphenols are effective free radical scavengers, this study
was designed to test the hypothesis that GTE will block free radical
formation, thus preventing injury. Indeed, GTE and one of its major
polyphenolic components, epicatechin, significantly reduced liver
injury after ischemia-reperfusion (Figs. 2-4). Protection by GTE and epicatechin was associated with decreased free radical formation (Figs. 6 and 7). In addition, GTE prevented NF-
B
activation (Fig. 8) and proinflammatory cytokine formation (Fig. 9).
Consistent with these observations, a previous report showed that green
tea polyphenols blunted endotoxin-induced NF-
B activation and
TNF-
production (45). Based on the present data, we
cannot rule out the possibility that GTE inhibits NF-
B activation by
nonantioxidant mechanism. However, a variety of structurally diverse
antioxidants and antioxidative enzymes has been shown previously to
inhibit NF-
B-mediated cytokine production stimulated by endotoxin,
consistent with prooxidant stimulation of NF-
B activation and
cytokine synthesis (2, 20, 29, 33, 40, 44, 45). Therefore,
GTE also likely inhibits NF-
B activation by scavenging free
radicals. Taken together, the present study demonstrates that GTE
effectively decreases oxidative stress after hepatic
ischemia-reperfusion and thus might be useful in disease status
where liver reperfusion injury plays a role.
Inhibition of TNF-
production and leukocyte infiltration after
ischemia-reperfusion also raises the theoretical possibility that GTE might increase the risk of septic complications. There is no
report from epidemiological or clinical studies regarding this issue.
However, trauma, hemorrhage/resuscitation and
ischemia-reperfusion are all associated with prooxidant states
and an increased risk of sepsis (1). Further studies will
be needed to determine whether the antioxidant effect of GTE increases
or decreases the risk for sepsis.
 |
ACKNOWLEDGEMENTS |
The authors thank Julia Vorobiov, Department of Gastrointestinal
Biology and Disease, University of North Carolina for her assistance
with TNF-
measurement.
 |
FOOTNOTES |
Deceased 14 July 2001.
This study was supported, in part, by National Institutes of Health grants.
Present address for H. Connor: Dept. of Chemistry, Kentucky Wesleyan
College, Owensboro, KY 42301.
Address for reprint requests and other correspondence: Z. Zhong,
Dept. of Cell and Developmental Biology, CB# 7090, Univ. of North
Carolina at Chapel Hill, Chapel Hill, NC 27599.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 12, 2002;10.1152/ajpgi.00216.2001
Received 22 May 2001; accepted in final form 5 June 2002.
 |
REFERENCES |
1.
Abello, PA,
Buchman TG,
and
Bulkley GB.
Shock and multiple organ failure.
Adv Exp Med Biol
366:
253-268,
1994[Medline].
2.
Anderson, MT,
Staal FJ,
Gitler C,
Herzenberg LA,
and
Herzenberg LA.
Separation of oxidant-initiated and redox-regulated steps in the NF-kappa B signal transduction pathway.
Proc Natl Acad Sci USA
91:
11527-11531,
1994[Abstract/Free Full Text].
3.
Baeuerle, PA,
and
Baltimore D.
A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB.
Genes Dev
3:
1689-1698,
1989[Abstract].
4.
Bellavite, P.
The superoxide-forming enzymatic system of phagocytes.
Free Radic Biol Med
4:
225-261,
1988[ISI][Medline].
5.
Boveris, A,
and
Chance B.
The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen.
Biochem J
134:
707-716,
1973[ISI][Medline].
6.
Bremer, C,
Bradford BU,
Hunt KJ,
Knecht KT,
Connor HD,
Mason RP,
and
Thurman RG.
Role of Kupffer cells in the pathogenesis of hepatic reperfusion injury.
Am J Physiol Gastrointest Liver Physiol
267:
G630-G636,
1994[Abstract/Free Full Text].
7.
Carrico, CJ,
Meakins JL,
Marshall JC,
Fry D,
and
Maier RV.
Multiple-organ failure syndrome.
Arch Surg
121:
196-208,
1986[ISI][Medline].
8.
Chamulitrat, W,
Jordan SJ,
and
Mason RP.
Fatty acid radical formation in rats administered oxidized fatty acids: in vivo spin trapping investigation.
Arch Biochem Biophys
299:
361-367,
1992[ISI][Medline].
9.
Cogoli, JM,
and
Dobson JG.
An easy and rapid method for the measurement of [
-32P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with 32Pi.
Anal Biochem
110:
331-337,
1981[ISI][Medline].
10.
Collart, MA,
Baeuerle P,
and
Vassalli P.
Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B.
Mol Cell Biol
10:
1498-1506,
1990[ISI][Medline].
11.
Dudek, EJ,
Shang F,
and
Taylor A.
H2O2-mediated oxidative stress activates NF-kappa B in lens epithelial cells.
Free Radic Biol Med
31:
651-658,
2001[ISI][Medline].
12.
Duling, DR.
Simulation on multiple isotropic spin-trap EPR spectra.
J Magnetic Resonance
104:
105-110,
1994[ISI].
13.
Farber, JL,
Kyle ME,
and
Coleman JB.
Mechanisms of cell injury by activated oxygen species.
Lab Invest
62:
670-679,
1990[ISI][Medline].
14.
Flohe, L,
Brigelius-Flohe R,
Saliou C,
Traber MG,
and
Packer L.
Redox regulation of NF-kappa B activation.
Free Radic Biol Med
22:
1115-1126,
1997[ISI][Medline].
15.
Frankel, EN.
Natural phenolic antioxidants and their impact on health.
In: Antioxdant Food Supplements in Human Health, edited by Lester P,
Hiramastu M,
and Yoshikawa T.. San Diego, CA: Academic, 1999, p. 385-392.
16.
Gilman, M.
Ribonuclease protection assay.
In: Current Protocols in Molecular Biology, edited by Ausubel FM,
Brent R,
Kingston RE,
Moore DD,
Seiman JG,
Smith JA,
and Stuhl K.. New York: Wiley, 1999, p. 4.7.1-4.7.8.
17.
Goldman, R,
Ferber E,
and
Zor U.
Involvement of reactive oxygen species in phospholipase A2 activation: inhibition of protein tyrosine phosphatases and activation of protein kinases.
Adv Exp Med Biol
400A:
25-30,
1997[ISI].
18.
Goto, M,
Yamada K,
Katayama K,
and
Tanaka I.
Inhibitory effect of E3330, a novel quinone derivative able to suppress tumor necrosis factor
generation, on activation of nuclear factor-kappa B.
Mol Pharmacol
49:
860-873,
1996[Abstract].
19.
Granger, DN,
Benoit JN,
Suzuki M,
and
Grisham MB.
Leukocyte adherence to venular endothelium during ischemia-reperfusion.
Am J Physiol
20:
G683-G688,
1989.
20.
Han, YJ,
Kwon YG,
Chung HT,
Lee SK,
Simmons RL,
Billiar TR,
and
Kim YM.
Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-kappaB activation: role of H2O2 and nitric oxide in inducible nitric oxide synthase expression in macrophages.
Nitric Oxide
5:
504-513,
2001[ISI][Medline].
21.
Hara, Y.
Antioxidative action of tea polyphenols: Part 1.
Am Biotechnol Lab
12:
48,
1994.
22.
Hensley, K,
Robinson KA,
Gabbita SP,
Salsman S,
and
Floyd RA.
Reactive oxygen species, cell signaling, and cell injury.
Free Radic Biol Med
28:
1456-1462,
2000[ISI][Medline].
23.
Iwahashi, H,
Deterding LJ,
Parker CE,
Mason RP,
and
Tomer KB.
Identification of radical adducts formed in the reactions of unsaturated fatty acids with soybean lipoxygenase using continuous flow fast atom bombardment with tandem mass spectrometry.
Free Radic Res
25:
255-274,
1996[ISI][Medline].
24.
Jaeschke, H,
Bautista AP,
Spolarics Z,
and
Spitzer JJ.
Superoxide generation by neutrophils and Kupffer cells during in vivo reperfusion after hepatic ischemia in rats.
J Leukoc Biol
52:
377-382,
1992[Abstract].
25.
Jaeschke, H,
and
Mitchell JR.
Mitochondria and xanthine oxidase both generate reactive oxygen species in isolated perfused rat liver after hypoxic injury.
Biochem Biophys Res Commun
160:
140-147,
1989[ISI][Medline].
26.
Jobin, C,
Hellerbrand C,
Licato LL,
Brenner DA,
and
Sartor RB.
Mediation by NF-kappa B of cytokine induced expression of intercellular adhesion molecule 1 (ICAM-1) in an intestinal epithelial cell line, a process blocked by proteasome inhibitors.
Gut
42:
779-787,
1998[Abstract/Free Full Text].
27.
Kada, T,
Kaneko K,
Matsuzaki S,
Matsuzaki T,
and
Hara Y.
Detection and chemical identification of natural bio-antimutagens. A case of the green tea factor.
Mutat Res
150:
127-132,
1985[ISI][Medline].
28.
Kadiiska, M,
Morrow JD,
Awad JA,
Roberts LJ,
and
Mason RP.
Identification of free radical formation and F2-isoprostanes in vivo by acute Cr(VI) poisoning.
Chem Res Toxicol
11:
1516-1520,
1998[ISI][Medline].
29.
Lakshminarayanan, V,
Lewallen M,
Frangogiannis NG,
Evans AJ,
Wedin KE,
Michael LH,
and
Entman ML.
Reactive oxygen intermediates induce monocyte chemotactic protein-1 in vascular endothelium after brief ischemia.
Am J Pathol
159:
1301-1311,
2001[Abstract/Free Full Text].
30.
McCord, JM.
Oxygen-derived radicals: a link between reperfusion injury and inflammation.
Fed Proc
46:
2402-2406,
1987[ISI][Medline].
31.
Muramatsu, K,
Fukuyo M,
and
Hara Y.
Effect of green tea catechins on plasma cholesterol level in cholesterol-fed rats.
J Nutr Sci Vitaminol (Tokyo)
32:
613-622,
1986[ISI][Medline].
32.
Parks, DA,
Bulkley GB,
Granger DN,
Hamilton SR,
and
McCord JM.
Ischemic injury in the cat small intestine: Role of superoxide radicals.
Gastroenterology
82:
9-15,
1982[ISI][Medline].
33.
Pinkus, R,
Weiner LM,
and
Daniel V.
Role of oxidants and antioxidants in the induction of AP-1, NF-kappaB, and glutathione S-transferase gene expression.
J Biol Chem
271:
13422-13429,
1996[Abstract/Free Full Text].
34.
Ruch, RJ,
Cheng SJ,
and
Klaunig JE.
Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea.
Carcinogenesis
10:
1003-1008,
1989[Abstract].
35.
Schreck, R,
Rieber P,
and
Baeuerle PA.
Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-
B transcription factor and HIV-1.
EMBO J
10:
2247-2258,
1991[Abstract].
36.
Serafini, M,
Ghiselli A,
and
Ferro-Luzzi A.
In vivo antioxidant effect of green and black tea in man.
Eur J Clin Nutr
50:
28-32,
1996[ISI][Medline].
37.
Shakhov, AN,
Collart MA,
Vassalli P,
Nedospasov SA,
and
Jongeneel CV.
Kappa B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor alpha gene in primary macrophages.
J Exp Med
171:
35-47,
1990[Abstract].
38.
Sirsjo, A,
Lewis DH,
and
Nylander G.
The accumulation of polymorphonuclear leukocytes in post-ischemic skeletal muscle in the rat, measured by quantitating tissue myeloperoxidase.
Int J Microcirc Clin Exp
9:
163-173,
1990[ISI][Medline].
39.
Slater TF. Free radical scavengers. In: (+)-Cyanidanol-3 in
Diseases of the Liver, International Workshop, edited by Conn HO.
Orlando, FL: Academic 1981, p. 11-15.
40.
Torrie, LJ,
MacKenzie CJ,
Paul A,
and
Plevin R.
Hydrogen peroxide-mediated inhibition of lipopolysaccharide-stimulated inhibitory kappa B kinase activity in rat aortic smooth muscle cells.
Br J Pharmacol
134:
393-401,
2001[Abstract/Free Full Text].
41.
Vinson, JA.
Black and green tea and heart disease: a review.
Biofactors
13:
127-132,
2000[ISI][Medline].
42.
Vinson, JA,
and
Dabbagh YA.
Effect of green and black tea supplementation on lipids, lipid oxidation and fibrinogen in the hamster: mechanisms for the epidemiological benefits of tea drinking.
FEBS Lett
433:
44-46,
1998[ISI][Medline].
43.
Wang, ZY,
Khan WA,
Bickers DR,
and
Mukhtar H.
Protection against polycyclic aromatic hydrocarbon-induced skin tumor initiation in mice by green tea polyphenols.
Carcinogenesis
10:
411-415,
1989[Abstract].
44.
Wolin, MS.
Interactions of oxidants with vascular signaling systems.
Arterioscler Thromb Vasc Biol
20:
1430-1442,
2000[Abstract/Free Full Text].
45.
Yang, F,
de Villiers WJ,
McClain CJ,
and
Varilek GW.
Green tea polyphenols block endotoxin-induced tumor necrosis factor-production and lethality in a murine model.
J Nutr
128:
2334-2340,
1998[Abstract/Free Full Text].
46.
Zabel, U,
Schreck R,
and
Baeuerle PA.
DNA binding of purified transcription factor NF-
B. Affinity, specificity, Zn2+ dependence, and differential half-site recognition.
J Biol Chem
266:
252-260,
1991[Abstract/Free Full Text].
47.
Zhao, BL,
Li XJ,
He RG,
Cheng SJ,
and
Xin WJ.
Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals.
Cell Biophys
14:
175-185,
1989[ISI][Medline].
Am J Physiol Gastrointest Liver Physiol 283(4):G957-G964
0193-1857/02 $5.00
Copyright © 2002 the American Physiological Society