Polyphenols from Camellia sinenesis attenuate experimental cholestasis-induced liver fibrosis in rats
Zhi Zhong,1
Matthias Froh,2
Mark Lehnert,1
Robert Schoonhoven,3
Liu Yang,1
Henrik Lind,1
John J. Lemasters,1 and
Ronald G. Thurman2,
Departments of 1Cell and Developmental Biology, 2Pharmacology, and 3Environmental Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7090
Submitted 7 January 2003
; accepted in final form 22 May 2003
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ABSTRACT
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Accumulation of hydrophobic bile acids during cholestasis leads to generation of oxygen free radicals in the liver. Accordingly, this study investigated whether polyphenols from green tea Camellia sinenesis, which are potent free radical scavengers, decrease hepatic injury caused by experimental cholestasis. Rats were fed a standard chow or a diet containing 0.1% polyphenolic extracts from C. sinenesis starting 3 days before bile duct ligation. After bile duct ligation, serum alanine transaminase increased to 760 U/l after 1 day in rats fed a control diet. Focal necrosis and bile duct proliferation were also observed after 12 days, and fibrosis developed 23 wk after bile duct ligation. Additionally, procollagen-
1(I) mRNA increased 30-fold 3 wk after bile duct ligation, accompanied by increased expression of
-smooth muscle actin and transforming growth factor-
and the accumulation of 4-hydroxynenonal, an end product of lipid peroxidation. Polyphenol feeding blocked or blunted all of these bile duct ligation-dependent changes by 4573%. Together, the results indicate that cholestasis due to bile duct ligation causes liver injury by mechanisms involving oxidative stress. Polyphenols from C. sinenesis scavenge oxygen radicals and prevent activation of stellate cells, thereby minimizing liver fibrosis.
bile duct ligation; fibrosis; oxidative stress; transforming growth factor-
CHRONIC CHOLESTATIC LIVER diseases, including primary biliary cirrhosis, extrahepatic biliary arresia, idiopathic adulthood ductopenia, primary sclerosing cholangitis, idiopathic neonatal hepatitis, Byler's disease, and arteriohepatic dysplasia, are leading indications for liver transplantation (23, 38, 49, 57). Various drugs, total parenteral nutrition, chronic liver transplant rejection, and graft-vs.-host disease can also produce chronic cholestasis (40). How cholestasis induces liver injury and fibrosis remains unclear. Oxidative stress caused by accumulation of hydrophobic bile acids is likely involved (48). Previous studies showed that hepatic mitochondria generate reactive oxygen species when isolated hepatocytes are exposed to hydrophobic bile acids (35, 48). This mitochondrial free radical production may be an important mechanism of cholestatic liver injury. However, oxypurinol, an inhibitor of xanthine oxidase, decreases hepatocelluar injury without decreasing lipid peroxidation in mitochondria and microsomes, suggesting that mitochondrial oxidative stress does not play an important role in cholestatic liver injury (47a). Therefore, the major source of free radicals remains unclear. In biliary obstructed rats, hepatic glutathione levels and activities of antioxidant enzymes decrease, whereas 4-hydroxynonenal and malondialdehyde levels increase (34, 35, 47). These findings also support the involvement of oxidative stress in cholestatic stress.
Green tea, Camellia sinenesis, contains high levels of polyphenols, including catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, and gallocatechin gallate (see Fig. 1) (12, 17). Spin-trapping techniques show that water extracts of C. sinenesis are excellent scavengers of reactive oxygen radicals from stimulated neutrophils (60). This antioxidant effect is more potent than that of vitamins C and E. Extracts from C. sinenesis also inhibit lipid peroxidation in in vitro systems, in experimental animals, and in humans (12, 17, 20, 29, 43, 45). Epidemiological and experimental evidence suggests beneficial effects of green tea extract in reducing the risk of heart disease and cancer (19, 50, 54, 56), most likely due to the antioxidant property of the polyphenols (12, 55). Accordingly, this study was designed to test the hypothesis that polyphenols from C. sinenesis decrease oxidative stress after cholestasis and thereby decrease liver fibrosis.

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Fig. 1. Molecular structures of C. sinenesis polyphenols. Green tea extract contains 85% polyphenols by weight. Composition of polyphenols in green tea extract used is shown as %total polyphenols.
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MATERIALS AND METHODS
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Animals. Male Sprague-Dawley rats (250300 g) were fed a chow diet (Purina, St. Louis, MO) containing 0 or 0.1% of an extract of C. sinenesis (Taiyo Kagaku, Yokkaichi, Mie, Japan) containing 85% polyphenols by weight, starting 3 days before surgery. Preliminary studies showed that average daily food consumption was
90 g/kg body wt, which was not altered by addition of polyphenols. Figure 1 shows the structures and relative contents of different polyphenols in the C. sinenesis extract used. The major polyphenol,
50%, was epigallocatechin gallate. Rats underwent bile duct ligation (BDL) and transection or sham operation under ether anesthesia, as described elsewhere (53). Briefly, the common bile duct was located through a midline abdominal incision, double ligated near the liver, and transected between ligatures. Some rats were given gadolinium chloride (GdCl3; 20 mg/kg body wt iv 24 h before BDL and repeated once every 3 days afterward) to destroy Kupffer cells selectively (1). All animals received humane care in compliance with institutional guidelines. Animal protocols were approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill.
Clinical chemistry and histology. Blood samples were collected from the tail vein at times indicated in the figure legends. Serum alkaline phosphatase, alanine transaminase, and bilirubin were measured using analytic kits from Sigma (St. Louis, MO). On the day of death, each rat was anesthetized with pentobarbital sodium (75 mg/kg ip), the abdomen was opened, and the portal vein was cannulated with a 20-gauge cannula. The liver was rinsed using a syringe containing 10 ml normal saline, followed by slow infusion of 5 ml 10% buffered formaldehyde (VWR International, West Chester, PA). After 48 h in fixative, paraffin sections were prepared and stained with hematoxylin-eosin or 0.1% Sirius red (Polysciences, Warrington, PA) and Fast green FCF (Sigma) (30). Areas in sections stained for collagens by Sirius red were quantified by image analysis using a Universal Imaging Image-1/AT image acquisition and analysis system (West Chester, PA) incorporating an Axioskop 50 microscope (Carl Zeiss, Thornwood, NY) and x4 objective lens. Detection thresholds were set for the red color of stained collagen based on an intensely labeled point and a default color threshold range that was assigned. The degree of labeling in each section was determined from the area within the color range divided by the total cellular area.
Immunohistochemistry for 4-hydroxynonenal, PCNA, and
-smooth muscle actin. 4-Hydroxynonenal is a product of lipid peroxidation (10). To detect 4-hydroxynonenal adduct formation in the liver, some sections were deparaffinized with xylene and taken through a graded series of alcohol and water mixtures to rehydrate the tissue. Hydrated sections were exposed to mouse anti-4-hydroxynonenal monoclonal antibodies (Alpha Diagnostic, San Antonio, TX) at a 1:200 dilution in 0.1 M phosphate buffer-Tween for 30 min at room temperature. Peroxidase-conjugated anti-mouse IgG1 antibody (DAKO, Carpinteria, CA) was then applied, and 3,3'-diaminobenzidine chromagen was added as the peroxidase substrate. After the immunostaining procedure, a light counterstain of Meyer's hematoxylin was applied so that 4-hydroxynonenal-labeled cells could be identified easily. Immunohistochemistry for PCNA was performed with monoclonal primary antibodies against PCNA (DAKO) at a dilution of 1:100 in PBS-Tween containing 1% bovine serum albumin at room temperature for 60 min. Staining for
-smooth muscle actin (
-SMA) was performed with monoclonal primary antibodies against
-SMA (DAKO) at a dilution of 1:200 at room temperature for 10 min.
AP-1 and NF-
B determination using EMSA. Measurement of AP-1 and NF-
B by EMSA was performed, as described in detail elsewhere (59). Briefly, nuclear extracts from liver tissue were incubated 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, and 0.05 mM EDTA. 32P-labeled DNA probes (2 µl) with the consensus sequences for AP-1 or NF-
B [200,000 counts per min (cpm)/µl, Cerenkov counting] containing 0.4 ng of double-stranded oligonucleotides were added with or without a 250-fold excess of the cold oligonucleotide as competitor. Mixtures were incubated for 20 min and separated through a 6% polyacrylamide (29:1 cross-linking) gel, and autoradiography was followed by visualization.
RNase protection assay for procollagen
1(I), TNF-
, and transforming growth factor-
mRNA. Total RNA was isolated from liver tissue using RNA STAT 60 (Tel-Test). RNase protection assays were performed using the RiboQuant multiprobe assay system (Pharmingen, San Diego, CA) or an individual probe. Briefly, with the use of a multiprobe template set (rCK-1) for TNF-
and transforming growth factor (TGF)-
or a single-probe template for procollagen-
(I), 32P-labeled RNA probes were transcribed with T7 polymerase followed by phenol-chloroform extraction and ethanol precipitation. Twenty micrograms of total RNA per sample were hybridized to 3.4 x 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 then resolved on 5% acrylamide-bisacrylamide (19:1) urea gels. After being dried, gels were visualized by autoradiography (15).
Statistical analysis. ANOVA and the Student-Newman-Keuls post hoc tests were used, and P < 0.05 was selected before the study to indicate significance.
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RESULTS
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Polyphenols decrease liver injury and cell proliferation after cholestasis. In untreated rats fed a standard chow diet, serum alanine transaminase (ALT) levels averaged 39 U/l (Fig. 2) and were not significantly altered by feeding of polyphenols or sham operation (data not shown). After BDL, ALT increased to 760 U/l after 1 day, indicating liver injury. ALT levels decreased afterward and reached a new steady-state level of
120 U/l after 1 wk (data not shown). When rats were fed C. sinenesis polyphenols, ALT after BDL was decreased by 45% (Fig. 2). Three weeks after BDL, ALT levels were 176 U/l in the rats fed control diet and 59 U/l in the rats fed a diet containing polyphenols (P < 0.05). These data show that polyphenols protect against hepatocellular injury at both early and late stages of cholestasis. Serum alkaline phosphatase (ALP), which mainly reflects cholangiocyte injury, also increased after BDL from
65 U/l before operation to 430 U/l 1 day afterward. Total bilirubin also increased from 0.7 mg/dl in untreated rats to 58 mg/dl 1 day after BDL. Treatment with C. sinenesis polyphenols did not significantly alter ALP release or total bilirubin levels after BDL (data not shown), indicating that polyphenols did not ameliorate cholestasis.

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Fig. 2. Polyphenols decrease alanine aminotransferase release after bile duct ligation (BDL). Rats were fed a control chow diet or chow containing 0.1% C. sinenesis polyphenols starting 3 days before BDL or sham operation. Blood was collected 1 day (d) and 3 wk (w) after surgery. Values are means ± SE (n = 45 in each group). aP < 0.05 vs. sham operation; bP < 0.05 vs. BDL rats fed a control diet.
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Histology revealed normal liver architecture after sham operation (Fig. 3). After BDL, focal necrosis developed within 2 days in livers of rats receiving a control diet. Necrotic areas accounted for 8.2% of liver sections in the bile duct-ligated group (data not shown). Polyphenols decreased necrotic areas to 2.6% (P < 0.05). Bile duct dilation and proliferation began 2 days after BDL and were well developed after 2 wk. C. sinenesis polyphenols minimized these pathological changes (Fig. 3). Immunohistochemical staining for PCNA was used to identify proliferating cells. After sham operation, total PCNA labeling was 0.3 cells/high power field (x40 objective), which increased to 14.3 cells/high power field after BDL (Fig. 4). Dietary polyphenols blunted cell proliferation after BDL to 4.5 cells/high power field, a 68% decrease (Fig. 4). Proliferating cells were predominantly cholangiocytes, which increased to 10.5 cells/high power field from a basal level of 0.05 cells/high power field. BDL also caused proliferation of parenchymal and nonparenchymal cells. Proliferation of parenchymal cells increased 14-fold, and proliferation of cells other than parenchymal cells and cholangiocytes increased 52-fold (Fig. 4). Dietary polyphenols blunted proliferation of cholangiocytes by 78%, and proliferation of cells other than cholangiocytes and parenchymal cells was blunted by 77% (Fig. 4). Dietary polyphenols tended to decrease proliferation of parenchymal cells, but the difference was not statistically significant (Fig. 4).

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Fig. 3. Polyphenols decrease histopathological changes after bile duct ligation. Livers were harvested 3 wk after BDL, and hematoxylin and eosin staining was performed to evaluate pathological changes. Shown are representative images: top, sham operation with control diet; middle, BDL with control diet; bottom, BDL with polyphenol-containing diet. Arrows identify necrotic areas. Bar is 200 µm.
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Fig. 4. Polyphenols inhibit cell proliferation after BDL. PCNA was detected by immunohistochemical staining, as described in MATERIALS AND METHODS. Ten microscope fields (x40 objective) were selected randomly, and PCNA-positive cells per field were counted. Values are means ± SE (n = 4 in each group). aP < 0.05 vs. sham operation; bP < 0.05 vs. BDL rats fed a control diet.
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Polyphenols decrease hepatic fibrosis after BDL. To evaluate the effects of polyphenols on liver fibrosis after BDL, liver sections were stained with Sirius red for collagen. No fibrosis was observed in livers from sham-operated rats (Fig. 5, top left). In rats fed a control diet, hepatic fibrosis developed within 2 wk after BDL (data not shown) and was severe after 3 wk (Fig. 5, top right). When rats fed C. sinenesis polyphenols were subjected to BDL, histology revealed decreased fibrosis (Fig. 5, bottom left). Image analysis revealed that Sirius red stained an area of 1% of liver sections from sham-operated rats (Fig. 6). Sirius red staining increased to 15.0% after 3 wk following BDL (P < 0.05). Treatment with C. sinenesis polyphenols suppressed this increase in Sirius red staining after BDL to 4% of the measured areas (Fig. 6; P < 0.05 compared with standard chow diet).

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Fig. 5. Polyphenols minimize hepatic fibrosis after BDL. Livers were harvested 3 wk after BDL, and Sirius red staining of sections was performed. Shown are representative images: top left, sham operation with control diet; top right, BDL with control diet; bottom left, BDL with C. sinenesis polyphenol-containing diet; bottom right, BDL with gadolinium chloride (GdCl3) treatment. Bar is 200 µm.
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Fig. 6. Quantification of hepatic fibrosis. Liver sections were stained with Sirius red, as described in Fig. 5. Five microscope fields (x4 objective) were selected randomly, and fractional areas that were Sirius red positive were determined by image analysis, as described in MATERIALS AND METHODS. Values are means ± SE (n = 45 in each group). aP < 0.05 vs. sham operation; bP < 0.05 vs. BDL with control diet.
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Collagen gene expression was evaluated by RNase protection assay for procollagen-
1(I) mRNA. Procollagen-
1(I) mRNA was barely detectable in livers from sham-operated rats (Fig. 7A). Three weeks after BDL, however, procollagen-
1(I) mRNA increased 30-fold (Fig. 7, A and B). Treatment with C. sinenesis polyphenols blocked the increase in procollagen-
1(I) mRNA expression by 76% (Fig. 7, A and B, P < 0.05).
Role of Kupffer cells in cholestatic liver injury. A previous study (41) showed that destruction of Kupffer cells attenuated liver fibrosis caused by carbon tetrachloride. To investigate whether Kupffer cells play an important role in cholestatic liver injury, rats were treated with GdCl3, a selective toxicant for Kupffer cells, before BDL. Suppression of Kupffer cells with GdCl3 neither blunted ALT release (data not shown) nor attenuated liver fibrosis after BDL (Fig. 5, bottom right, and Fig. 6).
Polyphenols decrease 4-hydroxynonenal adduct formation after BDL. To investigate whether cholestasis causes oxidative stress to the liver, formation of 4-hydroxynonenal adducts, a product of lipid peroxidation (10), was detected immunohistochemically in liver sections. 4-Hydroxynonenal staining was barely detectable in livers from sham-operated rats, as expected (Fig. 8, top). By contrast, after 3 wk following BDL, 4-hydroxynonenal adducts accumulated in the liver (Fig. 8, middle). 4-Hydroxynonenal-positive areas in liver sections increased to 17% (Fig. 9; P < 0.01). Treatment with C. sinenesis polyphenols decreased 4-hydroxynonenal staining to 3.5% of the measured liver area (Fig. 8 and 9; P < 0.01).

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Fig. 8. Polyphenols inhibit 4-hydroxynonenal formation after BDL. Livers were harvested 3 wk after BDL, and sections were stained immunohistochemically to assess 4-hydroxynonenal adduct formation. Shown are representative images: top, sham operation with control diet; middle, BDL with control diet; bottom, BDL with C. sinenesis polyphenol-containing diet. Bar is 100 µm.
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Fig. 9. Quantification of 4-hydroxynonenal formation after BDL. Livers were harvested 3 wk after BDL, and sections were stained immunohistochemically to assess 4-hydroxynonenal adduct formation. Ten microscope fields (x10 objective) were selected randomly, and the %stained area was estimated as described in MATEIRALS AND METHODS. aP < 0.05 vs. sham operation; bP < 0.05 vs. BDL with control diet.
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C. sinenesis polyphenols suppress
-smooth muscle actin formation after BDL. Activated stellate cells are the major source of matrix proteins in diseased liver (14). Accordingly, we evaluated
-SMA, an indicator of stellate cell activation, by immunohistochemical staining after BDL. In livers from sham-operated rats, small amounts of
-SMA were detected in the smooth muscle and endothelium of blood vessels. After BDL,
-SMA increased markedly in perisinusoidal cells (Fig. 10, middle).
-SMA-positive areas, estimated by image analysis, increased from 0.3 to 13.9%, consistent with stellate cell activation in cholestatic livers. Feeding of C. sinenesis polyphenols suppressed this increase in
-SMA by 80% (P < 0.01), indicating inhibition of stellate cell activation (Fig. 10, bottom).
Effects of experimental cholestasis and C. sinenesis polyphenols on NF-
B, activating protein-1, TNF-
, and TGF-
. Activation of transcription factors activating protein-1 (AP-1) and NF-
B and expression of cytokines TGF-
and TNF-
are important in the fibrotic response (13). Accordingly, we examined the effects of polyphenols on these profibrotic responses to cholestasis. NF-
B/DNA complexes were barely detectable in livers from sham-operated rats but increased 3.7-fold after BDL, consistent with our previous study (62). Polyphenols suppressed this activation (data not shown). A small amount of AP-1/DNA complex was detected in livers from sham-operated rats (Fig. 11A). Three weeks after BDL, however, AP-1/DNA complex levels increased 1.8-fold, indicating activation of AP-1 (Fig. 11A). Polyphenols largely suppressed this activation (Fig. 11A).
Minimal amounts of TGF-
1 and -
3 mRNA but no TGF-
2 mRNA were detected in livers from rats after sham operation. After BDL, TGF-
1, -2, and -3 mRNA increased by fourfold or more. Polyphenol treatment suppressed these increases in TGF-
expression (Fig. 11, B and C). Similarly, TNF-
mRNA was barely detectable in livers from sham-operated rats (Fig. 11B) but was increased after BDL (Fig. 11B). Polyphenol treatment suppressed this increase in TNF-
mRNA expression (Fig. 11B).
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DISCUSSION
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Plant-derived polyphenols minimize cholestasis-induced liver injury. Current therapy for cholestasis, such as ursodeoxycholic acid, does not prevent fibrosis (46). Therefore, new strategies to prevent cholestasis-induced liver injury and fibrosis are needed. Previous studies suggest that oxidative stress occurs during cholestasis and likely plays a role in cholestasis-induced liver injury (3, 35, 48). Accordingly, antioxidant therapy represents a potential strategy to prevent liver injury and fibrosis. Previous studies show that antioxidants, including N-acetylcysteine, vitamin E, silymarin, and quercetin, decrease lipid peroxidation and partially ameliorate liver injury after BDL, but the effects of antioxidants on fibrosis remain controversial (4, 33, 35, 36, 47).
Leafy plant tissues exist in a prooxidant environment of high oxygen and bright light, with consequent formation of singlet oxygen and other reactive oxygen species. Not surprisingly, plants such as C. sinenesis (Chinese green tea) contain high levels of polyphenols (Fig. 1), which are excellent scavengers of reactive oxygen species (12, 17). These polyphenols are more potent antioxidants than vitamins C and E (60). Polyphenol-rich extracts from C. sinenesis inhibit lipid peroxidation in vitro, in experimental animals, and in humans (12, 17, 20, 29, 43, 45). Accordingly, we assessed the effect of C. sinenesis polyphenols in a rat model of cholestasis induced by BDL.
In confirmation of previous work from several laboratories (34, 47, 53), BDL caused hepatic ALT release (Fig. 2), cell necrosis (Fig. 3), cholangiocyte proliferation (Fig. 4), and fibrosis (Figs. 5 and 6). Polyphenols substantially decreased liver injury, as reflected by ALT release and necrosis, and dramatically ameliorated fibrosis caused by cholestasis, as shown by Sirius red staining and procollagen-
1(I) mRNA expression (Figs. 5 and 6). Polyphenols exist in many plants and are especially abundant in C. sinenesis, whose dried leaves are used worldwide to brew tea. Green tea and polyphenol-enriched green tea extracts have no known toxicity. Thus polyphenols from C. sinenesis and possibly other plant sources represent a promising potential strategy to decrease fibrosis in human cholestatic diseases.
Oxygen radical scavenging and polyphenol protection against fibrosis. Polyphenols may protect against fibrosis by a variety of mechanisms. However, polyphenol treatment did not decrease total serum bilirubin levels in bile duct-ligated rats. Thus polyphenols do not ameliorate fibrosis by decreasing cholestasis. An alternative mechanism for protection against fibrosis is polyphenol-mediated scavenging of free radicals produced during cholestasis. In support of this hypothesis, polyphenols decreased the accumulation of 4-hydroxynonenal adducts in rat livers after BDL (Figs. 8 and 9), indicating decreased lipid peroxidation (10). Oxidant-induced cell death may also cause exaggerated repair leading to fibrosis. Therefore, prevention of cell death may decrease subsequent fibrosis. In support of this hypothesis, polyphenols decreased ALT release and necrosis of parenchymal cells (Fig. 2), as well as minimizing fibrosis (Figs. 5 and 6). However, fibrosis also occurred in the areas where focal necrosis was not evident, suggesting that other profibrotic mechanisms may be involved.
Another possibility is that polyphenols prevent activation of Kupffer cells, thus decreasing formation of inflammatory and fibrogenic mediators. The role of Kupffer cells in fibrosis is controversial. Destruction of Kupffer cells attenuated liver fibrosis caused by carbon tetrachloride (41). By contrast, in a rat model of reversible biliary obstruction, inactivation of Kupffer cells impaired collagen metabolism and inhibited the resolution of fibrosis (42). Kupffer cells release many mediators that activate stellate cells, including TNF-
, TGF-
, human growth factor, PDGF, and reactive oxygen species (2, 13). PDGF and TNF-
are mitogenic factors for stellate cells (13, 37), although TNF-
can stimulate apoptosis of fibrogenic cells and thus inhibit fibrosis (13). TNF-
production and NF-
B activation increase during cholestasis (7, 11). Activation of NF-
B, probably due to oxidative stress, could lead to expression of TNF-
. A recent report from this laboratory (25) shows that hydrophobic bile acids can activate Kupffer cells and increase production of TNF-
. In addition, we showed here that activation of NF-
B and expression of TNF-
and TGF-
were increased by cholestasis and that these effects were blocked by polyphenols (Fig. 11). However, suppression of Kupffer cell function with GdCl3, a treatment that blocks carbon tetrachloride-induced fibrosis (41), did not attenuate fibrosis caused by cholestasis (Figs. 5 and 6). This finding indicates that Kupffer cells likely do not play a prominent role in cholestasis-induced fibrosis in vivo and that polyphenols do not work by inhibiting Kupffer cell activation.
Cholangiocytes may play an important role in cholestasis-induced fibrosis and cirrhosis. Cholangiocytes synthesize and secrete a number of proinflammatory and fibrosis-related mediators, including IL-6, endothelin-1, monocyte chemotatic protein-1, PDGF, TNF-
, and reactive oxygen species (8, 31, 32, 51, 58). These mediators enable the cholangiocytes to communicate extensively with other liver cells, including hepatic stellate cells, inflammatory cells, and portal fi-broblasts. Ductular reaction to liver injury is considered the pacemaker of portal fibrosis (9). In a variety of cells, including cholangiocytes, reactive oxygen species stimulate proliferation (5, 28, 39, 52). By scavenging oxygen radicals, polyphenols may inhibit proliferation of cholangiocytes and their production of fibrogenic and inflammatory mediators. In support of this hypothesis, cholangiocytes proliferated dramatically after BDL as expected, and this effect was attenuated by polyphenols (Fig. 4).
Activation of hepatic stellate cells appears to be a critical step in hepatic fibrogenesis that is regulated by several factors, including cytokines and oxidative stress (14, 21, 24). Previously, the antioxidant N-acetylcysteine was shown to inhibit stellate cell activation (22). Therefore, polyphenols may prevent fibrosis by inhibiting oxidant-dependent activation and proliferation of stellate cells. In support of this hypothesis,
-SMA, a marker of stellate cell activation, dramatically increased after BDL (Fig. 10), an effect that was largely blocked by polyphenols. Consistent with this observation, a recent study (44) shows that epigallocatechin gallate, one of the major polyphenols from C. sinenesis, inhibits proliferation of the human hepatic stellate cell line LI90, probably by suppressing MEK and Akt phosphorylation. Activated stellate cells also produce TGF-
, which, in turn, causes further proliferation of stellate cells and strongly stimulates production of collagens (13, 18). Oxidative stress not only increases production of TGF-
but also activates latent TGF-
(6, 27). Additionally, H2O2 activates transcriptional factors such as AP-1 and NF-
B (16) that are involved in stellate cell activation and synthesis of TGF-
(16, 24). Consistent with these earlier findings, accumulation of 4-hydroxynonenal adducts, activation of AP-1, and increases in TGF-
mRNA were all observed in the present study after BDL (Figs. 8 and 11), supporting the hypothesis that oxidative stress stimulates TGF-
production, leading to collagen gene and protein expression (Fig. 7). Moreover, polyphenol treatment largely prevented accumulation of 4-hydroxynonenal adducts, blocked activation of AP-1 and production of TGF-
, and prevented procollagen-
1(I) mRNA and
-SMA protein expression (Figs. 8 and 11). Together, our data indicate that polyphenols suppress fibrosis after BDL, at least in part, by scavenging reactive oxygen species, thus inhibiting hepatocellular necrosis, cholangiocyte proliferation, stellate cell activation, and fibrogenic cytokine TGF-
formation.
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DISCLOSURES
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This work was supported, in part, by National Institutes of Health (NIH) Grants DK-62089, DK-37034, and AA-09156. Imaging facilities were supported, in part, by NIH Center Grants 5-P30-DK-34987 and 1-P50-AA-11605.
Portions of this work were presented at the 52nd Annual Meeting of American Association for the Study of Liver Disease, October 2001, Dallas, Texas (61).
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FOOTNOTES
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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 27599-7090 (E-mail: zzhong{at}med.unc.edu).
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.
Deceased 14 July, 2001. 
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