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Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan
2To whom correspondence should be addressed. E-mail: acksugi{at}agr.shizuoka.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the cytokine that is thought to play a pivotal role
in the pathogenesis of LPS-induced liver injury, although it
significantly suppressed plasma concentrations of interleukin
(IL)-1ß, IL-2, IL-4, IL-6, IL-10 and interferon (IFN)-
. TNF- +
GalN–induced liver injury and apoptosis were also suppressed by
dietary green tea. In contrast, dietary caffeine significantly
suppressed LPS-induced enhancement not only of plasma IL-1ß,
IL-6, IL-10 and IFN- concentrations, but also of TNF-
concentration. The results suggest that green tea might suppress LPS +
GalN–induced liver injury mainly through the inhibition of
TNF-–induced apoptosis of hepatocytes, rather than through the
suppression of TNF- production, although the suppressed production
of TNF- may be associated with the hepatoprotective effect of
caffeine.
KEY WORDS: • green tea • liver injury • caffeine • lipopolysaccharide • apoptosis • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), suggesting that these
beverages might warrant investigation from the viewpoint of nutrition.
To date, a number of studies have shown that green tea or its
constituents possess a variety of effects, including antioxidation
(Matsuzaki and Hara 1985
), antimutation (Kada et al. 1985
), anticarcinogenesis (Fujiki et al. 1996
), antibiotic action (Toda et al. 1989
),
antihypercholesterolemia (Muramatsu et al. 1986
),
antihypertension (Hara and Tonooka 1990
),
antihyperglycemia (Shimizu et al. 1988
) and
anti-inflammatory action (Sagesaka et al. 1996
). In
addition, we demonstrated that green tea could suppress
D-galactosamine
(GalN)3
-induced liver injury in rats (Sugiyama et al. 1998
). Our
recent studies showed that the protective effect of green tea against
GalN-induced liver injury was ascribed mainly to flavonoid
glycosides, theanine and soluble dietary fibers, whereas the effect of
tea catechins (tea polyphenols), characteristic constituents of teas,
was relatively weak (He et al. 2000
, Sugiyama et al. 1999
, Wada et al. 1999
). In contrast,
Yang et al. (1998)
reported that tea catechins could
inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor
(TNF)- production in both mouse peritoneal macrophages and mice in
vivo. LPS is a component of the outer membrane of gram-negative
bacteria, which have been used frequently in combination with GalN to
induce liver injury in rodents. These results suggest that tea
catechins may be effective in suppressing liver injury induced by LPS
because LPS is known to cause liver injury through enhanced TNF-
production (Tiegs et al. 1989
). However, it is unclear
whether tea or its constituents, including tea catechins, actually
suppress LPS-induced liver injury. On the other hand, synthetic
methylxanthine derivative drugs, e.g., pentoxifylline
[3,7-dimethyl-1-(5-oxohexyl)xanthine] and A802715
[1-(5-hydroxy-5-methyl)hexyl-3-methyl-7-propylxanthine], have been
shown to inhibit TNF- production, thereby suppressing
LPS-induced liver injury (Fischer et al. 1993
,
Jilg et al. 1996
, Sinha et al. 1995
).
These findings suggest the possibility that caffeine, the most common
naturally occurring methylxanthine, may also have a suppressive effect
on LPS-induced liver injury. However, the hepatoprotective effect
of caffeine against LPS-induced liver injury has not been fully
demonstrated.
In this study, we first examined whether green tea extract could
protect rats from LPS-induced liver injury, as assessed by plasma
alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
activities in GalN-sensitized rats. Because green tea had a
suppressive effect, the effects of five fractions, which were prepared
from the green tea extract by successive extraction with different
organic solvents, were investigated to determine what type of
constituent contributes to the effect of green tea. Furthermore, we
investigated the effects of dietary green tea extract on
LPS-induced enhancement of plasma concentrations of various kinds
of cytokines, including TNF-, and on the apoptosis of liver cells in
GalN-sensitized rats to gain insight into the mechanism by which
green tea suppresses LPS-induced liver injury. The effects of green
tea extract on TNF-–induced liver injury and of caffeine on plasma
cytokine concentrations were also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The green tea (Sen-cha) we used was obtained from a market (Shizuoka,
Japan). Lipopolysaccharide from Escherichia coli 055 and
GalN hydrochloride were purchased from Wako Pure Chemical (Osaka,
Japan) and Sigma-Aldrich (St. Louis, MO), respectively. Rat
recombinant TNF- was purchased from Funakoshi (Tokyo, Japan).
Mineral and vitamin mixtures (AIN-76) were purchased from Oriental
Yeast (Tokyo, Japan).
Extraction and fractionation of green tea.
Green tea was extracted as follows. 10 volumes (v/wt) of boiling water
was added to tea, allowed to stand for 30 min at room temperature and
the extract was filtered through five sheets of gauze. The extract was
lyophilized and powdered with a mixer. The dry matter thus obtained was
23.5 g/100 g of green tea. The powder was further fractionated into
five fractions (fractions I–V) by successive extraction with
chloroform, ethyl acetate, n-butanol and 70% (v/v)
ethanol (Fig. 1
). The concentrations of caffeine and catechins in the green tea extract
and fractions I–V were measured by HPLC (Model LC 10A; Shimadzu,
Kyoto, Japan) using an octadecylsilyl column (6 x 150 mm;
Shimadzu) according to Terada et al. (1987)
.
|
Male Wistar rats, 5 wk old, weighing 90–120 g (Experiments 1, 2, 4–6)
or 6-wk-old male Wistar rats weighing 120–140 g (Experiment 3) were
obtained from Japan SLC (Hamamatsu, Japan). The rats were housed
individually in hanging stainless steel wire cages and kept in an
isolated room at a controlled temperature (23–25°C) and ambient
humidity (50–60%). Light was maintained on a 12-h light:dark cycle
(lights on 0600 to 1800 h). After rats were acclimated to the
facility for 4 or 5 d and given free access to water and a
commercial stock diet (Type MF; Oriental Yeast), they were fed the
semipurified experimental diets. Body weight and food consumption were
measured daily. The composition of the control diet was as follows
(g/kg): casein, 250; cornstarch, 402.5; sucrose, 200; corn oil, 50;
mineral mixture (AIN 1977
), 35; vitamin mixture
(AIN 1977
), 10; choline bitartrate, 2.5; and cellulose,
50. Supplements were added to the control diet at the expense of
cellulose.
In this study, six separate experiments were conducted. In Experiment 1, rats were fed either the control diet or a diet supplemented with powder of the green tea extract at 30 g/kg. In Experiment 2, rats were fed either the control diet or diets supplemented with one of the five fractions derived from the green tea extract. The addition levels of each fraction (Fr.) were as follows (g/kg diet): Fr. I, 2.15; Fr. II, 8.16; Fr. III, 4.64; Fr. IV, 10.71; and Fr. V, 4.34. In Experiment 3, rats were fed the control diet and force-fed water or a green tea extract alone by stomach tube at levels of 0.4, 0.8 and 1.2 g/kg body 1.5 h before the injection of drugs. In Experiment 4, rats were divided into two groups and fed the control diet or a green tea extract–supplemented (35 g/kg) diet. In Experiment 5, rats were divided into three groups and fed the control diet or a caffeine-supplemented (2 g/kg) diet. In Experiment 6, rats were divided into three groups and fed the control diet or a green tea-supplemented (35 g/kg) diet. Normal and control rats were fed the control diet.
After the experimental diets were fed for 10 (Experiment 3) or 14 d (Experiments 1, 2, 4 and 5), rats were injected intraperitoneally
with LPS (10 µg/kg) and GalN (250 mg/kg) on d 11 or
15. In Experiment 6, rats were injected intravenously with TNF- (8
µg/kg) via the tail vein 20 min after an
intraperitoneal injection of GalN (250 mg/kg) on d 15. Untreated rats
were injected with saline. In Experiments 1, 2 and 3, rats were killed
by decapitation 22 h after the injection of drugs. In Experiment
4, rats were killed just before the injection (time 0) and at 1, 3, 6,
12 and 24 h after injection of LPS + GalN. In Experiment 5, rats
were killed 1 and 8 h after injection of drugs. In Experiment 6,
rats were killed 9 h after injection of drugs. Blood plasma was
separated from heparinized whole blood by centrifugation at 2000
x g for 20 min at 4°C; the plasma and liver were
stored at -80°C until analyses. The experimental design of this
study was approved by the Laboratory Animal Care Committee of the
Faculty of Agriculture, Shizuoka University.
Biochemical analysis.
The activities of plasma ALT and AST, the marker enzymes of liver
injury, were measured with a kit (Transaminase C II-Test,
Wako). The enzyme activity was expressed as µmol or
mmol/(min · L plasma) at 25°C. Plasma concentrations of
interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-10, interferon (IFN)-,
macrophage inflammatory protein (MIP)-2, and TNF- were measured with
Rat IL-1ß ELISA Kit (Wako), Rat IL-2 ELISA Kit (Wako), Rat IL-4 ELISA
Kit (Wako), Rat IL-6 ELISA Kit (Wako), Rat IL-10 ELISA Kit (Wako), Rat
IFN- ELISA Kit (Wako), Rat MIP-2 ELISA Kit (Wako), and Rat TNF-
ELISA Kit (Cosmo Bio, Tokyo, Japan), respectively. Hepatic DNA
fragmentation was detected and quantified by agarose gel
electrophoresis and DNA ELISA, respectively, according to Leist et al. (1995)
and Hase et al. (1999)
with slight
modifications. In brief, frozen liver (1 g) was homogenized in 4
volumes (v/wt) of ice-cold PBS containing 10 mmol/L EDTA. The
homogenate was centrifuged at 13,000 x g for 20
min at 4°C, and total DNA was prepared from the supernatant (0.3 mL)
by extraction with an equal volume of phenol-chloroform (1:1, v/v),
precipitation in ethanol and subsequent treatment with 20 mg/L of
ribonuclease A (Sigma-Aldrich) for 30 min at 37°C. The total DNA was
electrophoresed on 1.8% agarose gel. An aliquot of the supernatant of
liver homogenate was diluted and subjected to the direct analysis of
oligonucleosome-bound DNA with an ELISA kit (Boehringer Mannheim,
Mannheim, Germany).
Statistical analysis.
Results were expressed as means ± SEM In Experiment
5, paired data at each time were analyzed by Student’s
t test. In other experiments, data were tested for
homogeneity of variance using Bartlett’s test and, if necessary, were
log-transformed to homogeneity before one-way ANOVA. The
difference between the means was tested at P < 0.05 using Duncan’s multiple range test (Duncan 1957
)
when the F-value was significant at P
< 0.05. In Experiments 1, 2, 4 and 5, the effects of body weight
or body weight gain of rats on ALT and AST activities were analyzed by
an analysis of covariance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Green tea extract (30 g/kg) slightly depressed the body weight gain and
food intake of rats compared with those fed the control diet
(Table 1
). Injection of LPS + GalN significantly increased plasma ALT and AST
activities compared with saline-injected normal rats. The enhanced
ALT and AST activities were significantly suppressed by dietary
supplementation with the green tea extract. There was no correlation
between body weight (or body weight gain) and the activities of ALT or
AST in each dietary group, suggesting that the two variables are
independent (data not shown). Similarly, no association was detected
between the final body weight or body weight gain and the enzyme
activities in the subsequent experiments (2, 4 and 5).
|
HPLC showed that the green tea extract used in this study contained
5.90 g caffeine and 28.2 g catechins/100 g, and that the main
constituents of fractions I and II were caffeine and catechins,
respectively; fraction I contained 88.5 g caffeine and a trace
amount of catechins, whereas fraction II contained 1.0 g caffeine
and 88.5 g catechins/100 g. Body weight gain was slightly
depressed by dietary supplementation only with fraction I compared with
rats fed the control diet; the values for normal rats, control rats and
rats fed the fractions I, II, III, IV and V were 65 ± 2
(n = 6), 64 ± 1 (n = 12), 48
± 2 (n = 10), 61 ± 3 (n = 10), 65 ± 3 (n = 10), 67 ± 2 (n
= 10), 64 ± 2 g/14 d (n = 10), respectively.
Food intake was also slightly depressed only by fraction I (data not
shown). These data are essentially in agreement with our previous
results (Sugiyama et al. 1999
). Although all five of the
fractions significantly suppressed the LPS + GalN–induced enhancement
of plasma ALT and AST activities, the effect of fraction I was
significantly stronger than those of the other fractions (Fig. 2
); fraction I lowered the plasma enzyme activities to about one third of
the value of control rats.
|
Force-feeding of the green tea extract alone significantly suppressed
the LPS + GalN–induced enhancement of plasma ALT and AST activities in
a dose-dependent manner (Fig. 3
). The maximum dose, 1.2 g/kg body, corresponded to one third to one
half the ingestion of green tea extract per day from the green
tea–supplemented (30 g/kg) diet.
|
Dietary supplementation with green tea extract at 35 g/kg depressed the
growth and food intake of rats compared with the control group (body
weight gain, 70.1 ± 0.8 for control group; 50.2 ± 0.8 g/14
d for + tea group, n = 50 each, P < 0.001; food intake, 197 ± 2 for control group; 177 ± 1 g/14
d for + tea group, n = 50, P < 0.001). In this experiment, time-dependent changes in plasma enzyme
activities and cytokine concentrations were measured up to 24 h
after the injection of LPS + GalN. Plasma ALT and AST activities were
increased by LPS + GalN injection in control rats with maximum values
at 24 h, and these increases were significantly suppressed in the
green tea–fed rats (Fig. 4
). Injection of LPS + GalN markedly increased plasma concentrations of
all the cytokines measured with different peak times (Figs. 5
and 6
). Green tea supplementation did not suppress the peak concentrations of
TNF- and MIP-2 1 h after the injection of LPS + GalN, although
it slightly accelerated the decline of these cytokine concentrations at
3 h or later (Fig. 5A
, B
). In contrast,
plasma concentrations of IL-6, IFN-, IL-1ß, IL-2, IL-4 and IL-10
were significantly suppressed by dietary green tea.
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). This was confirmed by the quantitative determination of DNA
fragmentation. In the control rats, injection of LPS + GalN markedly
increased the DNA fragmentation of liver cells (Fig. 8
). This time-dependent change in DNA fragmentation was apparently
faster than those of plasma ALT and AST activities shown in Figure 4
.
Increases in DNA fragmentation at 6, 12 and 24 h were
significantly suppressed by dietary supplementation with green tea
extract.
|
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Dietary supplementation with caffeine (2 g/kg) depressed growth and
food intake of rats (Table 2
). The LPS + GalN–induced increases in plasma ALT and AST activities,
measured 8 h after the injection of the drugs, were significantly
suppressed by dietary supplementation with caffeine. On the basis of
the results of Experiment 4, plasma concentrations of TNF- and MIP-2
were measured 1 h after the injection of the drugs; concentrations
of IL-1ß, IL-6, IL-10 and IFN- were measured 8 h after the
injection of drugs. Plasma concentrations of most of the cytokines
measured, except for MIP-2, were significantly lower in
caffeine-fed rats than in control rats. In contrast to the effect
of green tea observed in Experiment 4, caffeine significantly
suppressed the LPS + GalN–induced increase in plasma TNF-
concentration.
|
-induced liver
injury (Experiment 6).
Intravenous injection of TNF- significantly increased plasma ALT and
AST activities and hepatic DNA fragmentation in GalN-sensitized
rats (Table 3
). The increases in these enzyme activities and DNA fragmentation were
significantly suppressed by dietary supplementation with green tea
extract.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 1999
). The major finding of this
study was that green tea also had a protective effect against LPS
+ GalN–induced liver injury in rats when fed to rats for 14 d and when force-fed once before injection of the drugs, indicating
that the effect of green tea is elicited quickly. Of the five fractions
derived from green tea, the caffeine-containing fraction (fraction
I) had the strongest effect (Fig. 2)
and the effect of green tea was
reproduced by authentic caffeine (Table 2)
, suggesting that the
protective effect of green tea against LPS + GalN–induced liver
injury might be attributable mainly to caffeine. Because growth and
food intake of rats were depressed by dietary supplementation with
either fraction I or caffeine, the effects of green tea on these
variables could also be ascribed to caffeine. However, it is unlikely
that the protective effect of caffeine against LPS + GalN–induced
liver injury was associated with the depressions of growth and food
intake per se because there was no significant effect of final body
weight or body weight gain on the plasma enzyme activities when
analyzed by analysis of covariance. Furthermore, green tea had a strong
effect even when it was force-fed to rats once before injection of
the drugs, a situation in which the effects of growth and food intake
could be ignored. Consistent with the results presented here,
Ueda and Yamazaki (1997)
reported that green tea extract
or caffeine could suppress systemic inflammation induced by muramyl
dipeptide derivative and OK-432, a bacterial application, in mice.
These findings may be indirect evidence that green tea or caffeine
exhibits a hepatoprotective effect by suppressing inflammation. In
contrast, Yang et al. (1998)
demonstrated that the
enhanced mortality and serum TNF- concentration induced by a large
amount of LPS alone (40 mg/kg body) could be suppressed by single
force-feeding of tea polyphenols (catechins) in mice. In this study
also, fraction II, which contained tea catechins as exclusively major
constituents, slightly but significantly suppressed LPS + GalN–induced
liver injury. However, it should be noted that the effect of tea
catechins was considerably weaker than that of caffeine at least in
this LPS + GalN–induced liver injury model.
TNF- is synthesized and released by macrophages including Kupffer
cells, the resident macrophages in the liver, in response to
stimulation by LPS and plays a critical role in LPS + GalN–induced
acute liver injury or hepatitis (Bradham et al. 1998
).
Therefore, one of the objectives of this study was to determine whether
the protective effect of green tea against LPS + GalN–induced liver
injury can be accounted for by the suppression of TNF- release from
macrophages. For this purpose, we investigated the time-dependent
effects of dietary green tea on plasma concentrations of eight kinds of
cytokines, including TNF-. Of these cytokines, TNF-, IL-1ß,
IL-6 and MIP-2 (a murine functional homologue of human IL-8) are
produced mainly by macrophages, whereas IL-2, IL-4, IL-10 and IFN-
are produced mainly by T lymphocytes. Unexpectedly, green tea extract
did not suppress plasma TNF- concentration at the peak time 1 h
after the injection of LPS + GalN, although green tea suppressed
LPS-induced increases in plasma concentrations of IL-1ß,
IL-2, IL-4, IL-6, IL-10 and IFN-. Therefore, the results suggest
that the protective effect of green tea against LPS + GalN–induced
liver injury cannot be ascribed to the suppression of TNF- release
from macrophages into the circulation, at least under the conditions
used. In contrast, green tea suppressed TNF--induced liver injury in
GalN-sensitized rats (Table 3)
, suggesting that the protective
effect of green tea against LPS + GalN–induced liver injury might be
elicited through the inhibited action of TNF- rather than the
decreased release of TNF-. The results also indicate that the effect
of green tea on macrophage-derived cytokines was selective because
plasma concentrations of IL-1ß and IL-6 were significantly suppressed
by dietary green tea, whereas TNF- and MIP-2 concentrations were
unaffected. In contrast, green tea seemed to suppress the
LPS-associated activation of T lymphocytes because plasma
concentrations of T-cell–derived cytokines were all suppressed by
green tea.
Unlike green tea extract, caffeine significantly suppressed LPS +
GalN–induced enhancement of plasma TNF- concentration (Table 2)
. In
another series of experiments, we also found that LPS + GalN–induced
enhancement of plasma TNF- concentration could be suppressed
effectively by intraperitoneal injection of caffeine (100 mg/kg)
1.5 h before LPS + GalN injection; TNF- concentrations 1 h
after injection of drugs for normal, control and +caffeine rats were 7
± 2, 9900 ± 1200 and 518 ± 46 ng/L, respectively.
Therefore, it is likely that caffeine protects rats from LPS +
GalN–induced liver injury at least in part through suppressed TNF-
release. Ueda and Yamazaki (1997)
reported that the
chloroform-soluble fraction obtained from green tea, which contains
caffeine at a 90% purity, suppressed the enhancement of plasma TNF-
concentration induced by the muramyldipeptide derivative and OK-432
when the fraction was force-fed alone to mice, whereas the other
water-soluble fraction stimulated plasma TNF- concentration.
These results suggest that green tea contains both TNF-–decreasing
and TNF-–increasing constituents, and that caffeine is one of the
TNF-–decreasing constituents of green tea. The findings of this
study that green tea extract and caffeine differentially affected the
plasma TNF- concentration support the idea that green tea contains a
TNF-–increasing constituent, although the identity of the
constituent is unclear at present.
Caffeine is one of the naturally occurring methylxanthines that
exist abundantly in traditional beverages such as tea and coffee.
Hence, it might be expected that coffee also might be effective in
suppressing liver injury. Actually, we found that coffee had a
protective effect against LPS + GalN–induced liver injury when added
to the diet and fed to rats for 14 d or force-fed to rats
alone before the injection of drugs (unpublished data). Although little
information is available concerning the effects of caffeine on liver
injury and plasma concentrations of various kinds of cytokines, several
reports have shown that the methylxanthine-derivative drugs have
significant effects on liver injury and plasma cytokine concentrations.
For example, LPS + GalN–induced liver injury can be suppressed
effectively by synthetic methylxanthine-derivative drugs, such as
pentoxifylline and A802715, in mice (Fischer et al. 1993
Jilg et al. 1996
). cAMP may participate in the
hepatoprotective effect of methylxanthine-derivative drugs such as
pentoxifylline and A802715 because these drugs inhibit
phosphodiesterase activity and thereby raise cellular cAMP
concentration (Leist et al. 1996
, Semmler et al. 1993
, Sinha et al. 1995
). Actually, exogenous
dibutyryl cAMP, a stable and permeable cAMP analog, had a protective
effect against LPS-induced liver injury in Propionibacterium
acnes–treated mice (Arai et al. 1995
,
Taguchi et al. 1999
). Exogenous dibutyryl cAMP
significantly increased circulating IL-10 concentration, an
immunosuppressive cytokine, with a concomitant decrease in TNF-
concentration in LPS-treated mice, suggesting that IL-10 may be
involved in the inhibition of LPS-induced liver injury by dibutyryl
cAMP (Arai et al. 1995
). Furthermore, Jilg et al. (1996)
demonstrated that A802715 suppressed the enhancement of
plasma concentrations of TNF-, IFN-, IL-1 and granulocyte
colony–stimulating factor in mice injected with a high level of LPS
alone (5 mg/kg), whereas it inversely increased the plasma
concentrations of IL-6 and IL-10. On the basis of these results, they
also pointed out that increased IL-10 participates in the prevention of
systemic endotoxemia by the methylxanthine-derivative drug. In
contrast to those findings, this study demonstrated that green tea
extract or caffeine significantly suppressed the LPS + GalN–induced
enhancement of plasma IL-10 concentration, indicating that the
hepatoprotective effect of green tea or caffeine could not be ascribed
to an increment of circulating IL-10 in the LPS + GalN–induced
liver injury model. These contrasting results may be due to the
following different experimental conditions: 1) difference
in drugs used, a high level of LPS alone vs. a low level of LPS + GalN;
2) difference in methylxanthines used, synthetic
methylxanthine-derivative drug A802715 vs. natural methylxanthine
caffeine; or 3) difference in animal species used, mice vs.
rats.
LPS is thought to induce the apoptosis of liver cells through the
action of TNF- in GalN-sensitized mice (Leist et al. 1995
). Consistent with this, we demonstrated that TNF-
+ GalN, as well as LPS + GalN, induced the apoptosis of liver
cells in rats, as evidenced by the fact that liver DNA exhibited a
representative ladder on agarose gel electrophoresis and that increase
in DNA fragmentation preceded the increase in plasma ALT and AST
activities. It has been demonstrated that apoptosis precedes the
necrosis of liver cells (Leist et al. 1995
), and that
the prevention of liver cell apoptosis, e.g., by inhibitors for
caspases downstream of TNF receptor 1, was an effective means
suppressing LPS- or TNF-–induced liver injury in
GalN-sensitized mice or rat hepatocytes (Hamada et al. 1999
, Jaeschke et al. 1998
). Such evidence
indicates that liver cell apoptosis is an essential process for liver
injury, at least LPS- or TNF-–induced liver injury. Unlike
necrosis, apoptosis has been considered to be a noninflammatory death
of cells. Also, in liver injury induced by LPS, the apoptosis of liver
cells per se is not the direct cause of liver injury; rather,
infiltrating neutrophils (polymorphonuclear leukocytes) are thought to
initiate parenchymal cell injury and necrosis (Jaeschke et al. 1991
, Leist et al. 1995
). Recent reports
have shown that excessive apoptosis of hepatic parenchymal cells
represents an important signal for the transmigration of primed
neutrophils sequestered in sinusoids during LPS-induced liver
injury (Lawson et al. 1998
). The important finding of
this study is that green tea suppressed the apoptosis of liver cells
induced either by LPS or TNF- in GalN-sensitized rats. Another
part of our study showed that caffeine could effectively prevent LPS-
or TNF-–induced apoptosis of liver cells in rats and mice
(unpublished data), suggesting that the suppressive effect of green tea
on liver cell apoptosis is attributable mainly to caffeine. Thus, it is
likely that caffeine exerts its protective effect against LPS +
GalN–induced liver injury by both the inhibition of TNF- release
from macrophages and the inhibition of TNF-–induced apoptosis of
liver cells, although the latter mechanism seems to be the
predominant effect of green tea. In humans, liver injury or
hepatitis is caused by viruses, chemicals, alcohol and autoimmune
diseases. It has been demonstrated that TNF- participates in liver
injury induced by virus (Ando et al. 1997
), alcohol
(Yin et al. 1999
) and immune disease (Gantner et al. 1995
). Therefore, it may be possible that
caffeine-containing beverages prevent or alleviate these types of
liver injury, although further evidence is required to confirm this.
The detailed mechanism by which caffeine inhibits the apoptosis of
liver cells also remains to be elucidated.
|
FOOTNOTES |
|---|
3 Abbreviations used: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GalN, D-galactosamine; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; MIP, macrophage inflammatory protein; TNF, tumor necrosis factor.
Manuscript received September 1, 2000. Initial review completed October 9, 2000. Revision accepted February 6, 2001.
|
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