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,dagger

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 alpha -(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-kappa B and increased TNF-alpha 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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.

                              
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Table 1.   Composition of polyphenols in green tea extract

Serum transaminase, TNF-alpha , 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-alpha 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 alpha -(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-kappa B determination using electrophoretic mobility shift assays. Measurement of NF-kappa 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-kappa 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-alpha 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
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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).


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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.



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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.


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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.



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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).


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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 beta -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).


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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 alpha -(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.



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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-kappa B activation after ischemia-reperfusion. NF-kappa B, a transcription factor controlling synthesis of a variety of proinflammatory cytokines (10, 37), can be activated by oxidative stress (35). NF-kappa B/DNA complexes are barely detectable in livers from sham-operated rats (Fig. 8A). Three hours after reperfusion, however, NF-kappa B was increased about twofold (Fig. 8, A and B). GTE largely inhibited NF-kappa B activation after ischemia-reperfusion (Fig. 8).


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Fig. 8.   Protection by GTE against NF-kappa 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-kappa 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-alpha is an important mediator of the inflammatory process (7). TNF-alpha mRNA increased about fourfold after ischemia-reperfusion, and this effect was largely blocked by GTE (Fig. 9A). Plasma TNF-alpha 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-alpha totally (Fig. 9B).


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Fig. 9.   Protection by GTE against increased TNF-alpha 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-alpha 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-alpha 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Free radicals modulate NF-kappa 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-kappa B (35), a transcription factor that consists of p65 and p50 subunits. NF-kappa 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-kappa 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 Ikappa B, an inhibitor of NF-kappa 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-kappa B activation and TNF-alpha 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-kappa B was activated and the proinflammatory cytokine TNF-alpha 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-alpha 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-kappa 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-kappa B activation and TNF-alpha production (45). Based on the present data, we cannot rule out the possibility that GTE inhibits NF-kappa B activation by nonantioxidant mechanism. However, a variety of structurally diverse antioxidants and antioxidative enzymes has been shown previously to inhibit NF-kappa B-mediated cytokine production stimulated by endotoxin, consistent with prooxidant stimulation of NF-kappa B activation and cytokine synthesis (2, 20, 29, 33, 40, 44, 45). Therefore, GTE also likely inhibits NF-kappa 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-alpha 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-alpha measurement.


    FOOTNOTES

dagger 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.


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DISCUSSION
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