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The antioxidant effect of green tea catechin ameliorates experimental liver injury.

ABSTRACT

Purpose: Several studies have reported green tea catechin to have both antifibrotic and anti-oxidative effects. The goal of this study was to evaluate the effect of green tea cathechin therapy in hepatic tissue injury using cholestatic rats with bile duct ligation.

Materials and methods: We performed bile duct ligation on cholestatic seven-week-old male Wistar rats and classified them into three groups according to the method of treatment. The groups comprised the SHAM group, the NT-group (no-treatment-group), and the T-group (treatment-group). The rats were orally administered green tea catechin at a dose of 50 mg/kg/day and were sacrificed on the 17th postoperative day. We subsequently investigated the levels of fibrosis and antioxidant activity associated with various clinical markers. We evaluated the serum AST and ALT levels and performed immunohistochemica] analyses for 4-hydroxynonenal (4-HNE), 8-oxo-2'deoxyguanosine (8-OHdG) and transforming growth factor-[beta]1 (TGF-[beta]l). We also evaluated the levels of activator protein-1 m-RNA (AP-1 m-RNA) and tissue inhibitor metalloproteinase-1 m-RNA (TIMP-1 m-RNA) by Real Time PCR. Finally, we performed Azan staining and immunohistochemical staining of [alpha]-smooth muscle actin ([alpha]-SMA) to evaluate the degree of fibrosis.

Results: The values of serum AST, serum ALT, AP-1 m-RNA, [alpha]-SMA, TGF-[beta]1, 4-HNE, and 8-OHdG in the T-Group were significantly lower than those in NT-Group. Therefore, the administration of green tea catechin might have suppressed the oxidative stress, controlled the stellate cell activation and consequently reduced the fibrosis.

Conclusion: Green tea catechin may reduce hepatic fibrosis by suppressing oxidative stress and controlling the transcription factor expression involved in stellate cell activation.

[C] 2010 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords: Green tea Catechin Liver fibrosis Anti-oxidant therapy

Introduction

Many patients with biliary atresia (BA) continue to suffer from postoperative liver dysfunction after undergoing Kasai's hepatic portoenterostomy. These patients can experience chronic inflammation with or without jaundice. This inflammation can involve chronic cytoinfiltration of Glisson's capsules, which has been identified in biopsied hepatic tissue specimens in postoperative BA patients.

Baskol et al. reported the contribution of oxidative stress to the pathology of adult NASH, and the excessive production of active oxygen or reactive oxygen species is considered to be a causative factor (Baskol et al. 2007). Postoperative BA patients experience severe oxidative stress even if their liver dysfunction is mild (Asakawa et al. 2009). We therefore suggest that ingestion of antioxidants is essential for the treatment of postoperative patients with BA (Tanaka et al. 2008, 2004; Tanaka 2008). Catechins, which are classified as flavonoids, are reported to be involved in physiological regulation and display anti-oxidative or anti-allergenic functions. As shown in Fig. 1, there are eight species of catechins, namely, catechin, epicatechin, epigallocatechin, gallocatechingallate, catechingallate, gallocatechin, epicatechingallate and epigallocatechingallate. Among the different types of catechins, epigallocatechin gallate is said to be the most potent antioxidant (Okubo 2000). We evaluated the presence and degree of oxidative stress in fibrotic hepatic tissues; moreover, we evaluated the anti-oxidative effects of green tea catechin mixture, and the mechanism underlying the suppression of fibrosis by catechin.

[FIGURE 1 OMITTED]

Materials and methods

We utilized seven-week-old male Wistar rats weighing 250-300 g. Three days before the operation we initiated intragastric administration of Sunphenon BG3[TM] green tea catechin mixture in powder form which was prepared from green tea leaves in Taiyo-kagaku, Co. Ltd, Mie, JAPAN, at a dose of 50 mg/kg/day. It was composed mainly of (-)-Epigallocatechin 3-O-gallate (48.8%), (-)-gallocatechin 3-O-gallate (1.2%), (-)-epicatechin 3-O-gallate (3.7%), (-)-epigallocatechin ( 21.6%), (+)-gaIlocatechin (0%), (-)-epicatechin (3.7%), (-)-catechin gallate (0%) and (+)-catechin (1.3%) (Table 1, Fig. 1). The content of each catechin in green tea extracts was determined accordingly to the HPLC analysis described in Del Rio's publication (Del Rio et al. 2004). The column used was CAPCELL PAK C18 UG120 (4.6 mm ID x 100 mm L). The injection volume was 101, and the mobile phase consisted of methanol/water/[H.sub.3][PO.sub.4] = 18/82/0.5). The elution was performed at a flow rate of 0.8 ml/min by monitoring at 280 nm. After ether anesthesia, we performed bile duct ligation and SHAM operation on the rats. The bile duct ligation was an abdominal operation by median sternotomy in which the bile ducts were doubly ligated. The SHAM operation involved only an abdominal laparotomy. There were three study groups with eight rats in each. The groups comprised the SHAM group, NT-group (no treatment group with bile duct ligation), and T-group (Sanfenon BG3[TM] treatment group). The rats were sacrificed on the 17th postoperative day. Blood was drawn from the inferior vena cava after ether anesthesia, and the livers were removed after ligation of the portal veins, the coronary arteries and the inferior vena cava. The livers were fixed in formalin and prepared as specimens, with the exception of about 2.0 g of tissue that was stored frozen at -150 [degrees]C in liquid nitrogen immediately after extraction.
Table 1
Constitution of Sunphenon BG3[TM].

Total catechin            83.0 (% (w/w))

Catechin                        1.3
Epicatechin                     6.4
Epigallocatechin               21.6
Gallocatechin gallate           1.2
Catechin gallate                N.D.
Gallocatechin                   N.D.
Epicatechin gallate             3.7
Epigallocatechin gallate       48.8


The serum AST and ALT levels were measured via a biochemical analysis. Immunohistochemical staining with 4-hydroxy-2'-none-nal (4-HNE) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) was performed to evaluate the degree of antioxidation. 4-HNE staining was performed with anti-4-hydroxy-2-nonenal monoclonal antibody (Institute of Senescence Control of Japan, 723-1, Haruoka, Fukuroi-shi, Shizuoka, JAPAN) by the ABC method (Vector Laboratories, Burlingane CA, USA). The staining rate, which reflected the rate of anti-4-HNE antibody-positive cells, was defined as the average of five fields at 400 x magnification. The measurements were based on a DI-Ruler (Harada Industry, 1018 Kamimaruko, Ueda-shi, Nagano JAPAN). 8-OHdG staining was performed with anti-8-OHdG monoclonal antibody (Institute of Senescence Control of Japan, 723-1, Haruoka, Fukuroi-shi, Shizuoka, JAPAN) by the ABC method (Vector Laboratories, Burlingane CA, USA). The staining rate was defined as the average of five fields at 400 x magnification and reflected the rate of anti-8-OHdG antibody-positive cells in the pulse regions and the lobules.

We also evaluated levels of activator protein-1 m-RNA (AP-1 m-RNA). AP-1 is a key mediator of the activation of inflammatory cytokines. We extracted RNA from about 10 mg of the liver specimen that had been stored frozen at -- 150 [degrees]C utilizing the MELT[TM] Total Nucleic Acid Isolation System (Applied Biosystems Japan). We obtained cDNA from the total RNA with the High Capacity cDNA Archive Kit (Applied Biosystems Japan) and performed relative quantifications of AP-1 m-RNA by real-time PCR with a 7500 Fast Real-Time PCR system. The probes and primers were from TaqManTM Gene Expression Assays Realtime AP-1 (Applied Biosystems Japan).

Immunohistochemical staining of transforming growth factor-[alpha]1 (TGF-[alpha]1) in the hepatic tissues was performed to evaluate the activation of stellate cells. The staining was performed with mouse anti-human transforming growth factor-[alpha]1 monoclonal antibody (Cosmo Bio Co., Ltd, Japan) by the ABC method (Vector Laboratories, Burlingane CA, USA). For each specimen, the staining rate was defined as the average of five fields at 400 x magnification and reflected the rate of anti-human transforming growth factor-[alpha]1 antibody-positive cells. The measurements were based on a DI-Ruler (Harada Industry, 1018 Kamimaruko, Ueda-shi, Nagano JAPAN).

Measurement of tissue inhibitor of metalloproteinase-1 (TIMP-1) m-RNA was performed by real-time PCR in order to evaluate the suppression of matrix metalloproteinase. We obtained relative quantifications of AP-1 m-RNA by real-time PCR with the 7500 Fast Real-Time PCR system. The probes and primers were from TaqManTM Gene Expression Assays Realtime TIMP-1 (TIMP-1: Applied Biosystems). Azan staining and immunohistochemical staining of [alpha]-smooth muscle actin ([alpha]-SMA) was performed to evaluate the degree of fibrosis. For each specimen, the staining rate of the positive cells was defined as the average of five fields at 40 x with Azan staining and at 100 x magnification with [alpha]-SMA staining. The measurements were based on a DI-Ruler (Harada Industry, 1018 Kamimaruko, Ueda-shi, Nagano JAPAN).

This animal experimental protocol was approved by the Animal Experiments Committee of Kurume University (No. 1545). The measurements were expressed as the mean + standard deviation, and comparisons between two groups were performed with the Mann-Whitney U test. A p-value below 0.05 was considered to be statistically significant.

Results

In the blood biochemical analysis, serum AST was 901.8 [+ or -] 310.5 and 558.0 [+ or -] 202.5 IU/l in the NT-group and T-group, respectively. The serum ALT was 214.8 [+ or -] 85.2 and 146.9 [+ or -]44.9 IU/l in the NT-group and T-group, respectively. The administration of "green tea catechin mixture" significantly ameliorated both serum values (Fig. 2).

[FIGURE 2 OMITTED]

In 4-Hydroxynonenal immunohistochemical staining, the rates of stained cytoplasm were 34.08 [+ or -] 6.32% and 4.19 [+ or -] 2.74% in the NT-group and T-group, respectively (Fig. 3). In 8-OHdG immunohistochemical staining, the rates of deeply stained nuclei throughout the pulse regions and the lobules were 46.05 [+ or -]14.03% and 2.24 [+ or -] 1.09% in the NT-group and T-group, respectively (Fig. 4). The relative quantifications of m-RNA of hepatic AP-1 were 46.8 [+ or -] 23.8 and 1.33 [+ or -] 1.24 times in the NT-group and the T-group, respectively, in comparison to that in the SHAM group. The administration of green tea catechin mixture significantly decreased the above mentioned parameters (Fig. 5).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

In the immunohistochemical staining for TGF-[alpha]1, the rates of positive cells were 22.06 [+ or -] 5.4% and 0.338 [+ or -] 0.21% in the NT-group and the T-group, respectively (Fig. 6). The relative quantifications of TIMP-1 m-RNA, which indicated the suppression of matrix metalloproteinase, were 93.16 [+ or -]47.29 times and 1.83 [+ or -] 2.14 times in the NT-group and the T-group, respectively, in comparison to that in SHAM group. Green tea catechin therefore significantly decreased these parameters (Fig. 7).

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

In [alpha]-SMA staining, the rates of positive cells were 47.53 [+ or -]16.38% and 10.93 [+ or -]7.41% in the NT-group and the T-group, respectively (Fig. 8). In Azan staining, the rates of fibrotic areas were 33.21 [+ or -] 8.50% and 13.42 [+ or -] 5.36% in the NT-group and the T-group, respectively. The former group showed a high degree of bridging fibrosis that was absent in the latter (Fig. 9).

[FIGURE 8 OMITTED]

[FIGURE 9 OMITTED]

Discussion

Many studies have previously addressed oxidation and tissue injury. Chronic inflammation and excessive production of active oxygen species might lead to tissue injury and worsening of the original disease. Using necrotic models of hepatic cells in animal experiments, Rosser and Gores (1995) demonstrated the relationship between advancing hepatic dysfunction and lipoperoxide reaction (Rosser and Gores 1995). Albano and Denk et al. reported the relationship between oxidative stress and Malory body formation in hepatic fibrosis caused by alcoholic hepatic dysfunction (Albano 2007; Denk et al. 2005). Cotler et al. reported the association between oxidative stress injury and liver fibrosis by 8-OHdG scoring in the hepatic tissues of the patients with diabetes after liver transplantation (Cotler et al. 2007). Using protein analysis, Diamond et al. evaluated the relationship between oxidative stress injury and hepatic mitochondrial dysfunction in the onset of hepatitis C (Diamond et al. 2007). Geetha et al. measured serum oxidative stress markers in patients with liver cirrhosis (Geetha et al. 2007). As demonstrated above, active oxygen species are involved in the onset and advance of many hepatic diseases.

Many postoperative biliary atresia (BA) patients suffer from liver dysfunction and chronic inflammation even without jaundice after Kasai's hepatic portoenterostomy. Postoperative treatments, involving ursodeoxycholic acid or phenobarbital administration and steroid pulse therapy, have been proposed to obtain choleretic or antiinflammatory effects (Muraji et al. 2004); however, there is no consensus regarding the appropriate postoperative therapy. Many patients eventually must undergo liver transplantation. Lemonnier et al. reported that the serum lipoperoxide level of postoperative BA patients was twice as high as that of healthy subjects (Lemonnier et al. 1987). At our institution, Asakawa reported that postoperative BA patients were under severe oxidative stress even if they experienced only mild liver dysfunction without jaundice (Asakawa et al. 2009). Furthermore, Trevisani et al. reported that liver transplant patients were under mild oxidative stress after surgery (Trevisani et al. 2002). These reports further demonstrate that the excessive production of active oxygen species may be a causative factor in the advancing hepatic dysfunction of BA patients with chronic diseases even after Kasai's portoenterostomy. The enzymes Mn-SOD and Cu/ Zn-SOD reduce oxidative cellular dysfunction by disequilibrating the electron transport chains or the active oxygen species. However, in postoperative BA patients, the active oxygen species that are excessively produced in the liver outstrip the anti-oxidative capacity of these anti-oxidative enzymes so that more injurious free radicals (such as hydroxyl radicals) are produced, thus advancing liver dysfunction. Postoperative anti-oxidant therapy might ameliorate this pathology, and our department utilizes coenzymeQ10 for this purpose (Tanaka et al. 2008, 2004; Tanaka 2008).

According to Broide, Britton and Lee's reports, the mechanism of advancing hepatic fibrosis involves the process in which oxidative stress activates stellate cells, promotes the production of TGF-[alpha]1 and consequently disrupts the balance between production and reduction in the extracellular matrix (Broide et al. 2000; Britton and Bacon 1994; Lee et al. 1995). As shown in Fig. 10, the pathologic mechanism of the progression from cholestasis to fibrosis might be summarized as a extracellular matrix chemical unbalance caused by the activation of transcription factors and hepatic stellate cells. Continuous inflammation increases the expression of adhesion factors, which results in the accumulation of immune cells in the liver. The subsequent accumulation of hydrophobic bile acid leads to the activation of Kupffer cells as well as the excessive production of active oxygen species and consequent lipoperoxidation.

[FIGURE 10 OMITTED]

Green tea contains a high level of polyphenol compounds termed catechins. Polyphenol generally refers to a compound with more than two phenolic hydroxyl groups in the same molecule, and catechin is classified as a flavonoid. Flavonoid compounds are involved in physiological regulation, and have anti-oxidative and anti-allergenic functions. The anti-oxidative functions of catechins are reported to be due to the hydroxyl groups on the benzene ring, and to be the strongest in epigallocatechingallate (Sugiura and Okubo 2000). Yokozawa et al. reported a decrease of uremia after the administration of green tea polyphenol in rat models of chronic renal failure and renal hypertension (Yokozawa et al. 1996, 1994). Fujise et al. reported a decrease in anti-oxidative activation after the administration of polyphenols, anti-oxidative vitamins and chemically intensified anti-oxidative liquid foods in a rat model with invasive surgery (Fujise et al. 2005).

Recently, we performed bile duct ligation on cholestatic Wistar rats and evaluated the degree of liver dysfunction (fibrosis). We investigated the presence and degree of oxidative stress in the hepatic tissues, the anti-oxidative effects of green tea catechin, and the mechanism underlying the suppression of fibrosis by catechin. Bile duct ligation caused hepatic dysfunction in the rats by oxidative stress. Administration of green tea catechin suppressed the degree of fibrosis. After the administration of green tea catechin, we observed decreased rates of staining for 4-HNE, an indicator of hepatic lipoperoxidation, and 8-OHdG, an indicator of oxidative injuries of deoxyguanosine or a DNA constituent. The staining for 8-OHdG showed a lower number of deeply stained nuclei after catechin administration, thus indicating that the level of oxidative stress had been mitigated. Moreover, there were significant decreases in the following markers: AP-1 m-RNA, TGF-[alpha]1 (a key mediator of inflammatory cytokine activation), [alpha]-SMA (an indicator of stellate cell activation) and TIMP-1 m-RNA (an indicator of stellate cell activation and fibrosis).

Therefore, green tea catechin mixture may have systematically suppressed oxidative stress, transcription factor expression, and stellate cells activation; furthermore, catechin appeared to suppress the accumulation of extracellular matrix by controlling the inhibitors of matrix metalloproteinase, thereby reducing fibrosis. In conclusion, green tea catechin mixture administration might reduce fibrosis by suppressing oxidative stress, transcription factor expression, and stellate cell activation.

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H. Kobayashi (a), *, Y. Tanaka (a), K. Asagiri (a), T. Asakawa (a), K. Tanikawa (b), M. Kage (b), Minoru Yagi (b)

(a) Department of Pediatric Surgery, Kurume University School of Medicine, 67 Asahimachi, Kurume-city, 830-0011 Fukuoka, Japan

(b) Department of Pathology, Kurume University School of Medicine, 67 Asahimachi, Kurume-city, 830-0011 Fukuoka, Japan

* Corresponding author. Tel.: +81 942 31 7631; fax: +81 942 31 7705. E-mail address: hidefumi@juno.dti.ne.jp (H. Kobayashi).

0944-7113/$-see front matter (c) 2010 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2009.12.006
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Author:Kobayashi, H.; Tanaka, Y.; Asagiri, K.; Asakawa, T.; Tanikawa, K.; Kage, M.; Yagi, Minoru
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Clinical report
Date:Mar 1, 2010
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