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Epigallocatechin-3-gallate ameliorates rats colitis induced by acetic acid
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Biomedicine & Pharmacotherapy 62 (2008) 189e196 www.elsevier.com/locate/biopha
Original article
Epigallocatechin-3-gallate ameliorates rats colitis induced by acetic acid Zhi Hua Ran*, Chi Chen, Shu Dong Xiao Department of Gastroenterology, Renji Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai Institute of Digestive Disease, 145 Shan Dong Zhong Road, Shanghai 200001, PR China Received 17 February 2007; accepted 7 February 2008 Available online 20 February 2008
Abstract Background: Epigallocatechin-3-gallate (EGCG) has been recently proved to possess anti-inflammatory effects. Aims: To investigate the effect and mechanism of epigallocatechin-3-gallate (EGCG) treatment in rats with acetic acid-induced colitis. Methods: Sixty male rats were randomly assigned into 4 groups: normal control (n ¼ 10), model placebo (n ¼ 20), EGCG (n ¼ 15), and SASP (n ¼ 15). The normal group was treated with regular feeding, while the other 3 groups were treated orally with saline 2 ml/d, EGCG 50 mg/kg/d, and SASP 0.25 g/kg/d, respectively, for 7 days using an established colitis model induced by 8% acetic acid. The disease activity index (DAI) and the therapeutic effects were evaluated. Colon mucosa damage index (CMDI) and histological score were determined. The activities of nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-a (TNF-a) and interferon-g (IFN-g) and tissue expression of nuclear factor-kBp65 (NF-kBp65) were measured. Results: EGCG notably improved the DAI (1.1 0.9), CMDI (1.5 0.9) and histological scores (4.6 3.1) compared with the placebo (3.9 0.4, p < 0.01; 3.3 0.6, p < 0.05; 9.3 2.8, p < 0.01) and SASP groups (3.0 1.2, p < 0.01; 2.3 0.9, p < 0.05; 7.9 4.0, p < 0.05). Compared with the placebo and SASP groups, the levels of NO (9.1 5.6 mmol/g prot), MDA (0.9 0.6 nmol/g prot), TNFa (24.4 1.6 PG/ml), IFN-g (33.3 0.9 PG/ml), and NF-kBp65 (28.0 2.8 cells/mm3) in EGCG-treated group were significantly reduced ( p < 0.05 or p < 0.01), while that of SOD (185.4 24.6 U/mg prot) was increased remarkably ( p < 0.05). Conclusion: EGCG exerts its antioxidant activity via decreasing NO, MDA, and increasing SOD. It ameliorates mucosal inflammation by inhibiting the production of TNF-a, IFN-g and NF-kBp65 and may be a potential therapeutic agent in colitis. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Epigallocatechin-3-gallate; Colitis; Effect; Mechanism
Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) characterized by chronic inflammation of the colon. The exact pathogenesis of UC remains uncertain [1]. Oxidative stress and aberrant immune response to the various stimulations have been proposed to be involved in the initiation and propagation of the chronic inflammation [2]. Previous studies have suggested that IBD patients rank high among users of complementary and alternative medicine because of unsatisfactory responses to conventional medicine and potential side effects [3]. Growing attention has been * Corresponding author. Tel.: þ86 21 63200874; fax: þ86 21 63266027. E-mail address: [email protected] (Z.H. Ran). 0753-3322/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2008.02.002
focused on dietary and natural medicines that inhibit, reverse, or retard this disease [4e6]. Fresh tea leaves are rich in tea polyphenols known as catechins [7,8]. The principal catechins found in tea are epicatechin (EC), epigallocatechin (EGC), epicatechingallate (ECG) and epigallocatechin-3-gallate (EGCG) [9,10]. EGCG is the most abundant catechin in green tea. In vitro and animal studies provide strong evidence that EGCG may possess antioxidant and anti-inflammatory properties to affect the pathogenesis of several chronic diseases, especially cancer and cardiovascular disease [11e15]. In the present study, we examined the therapeutic effects of EGCG compared with the conventional medicine sulfasalazine (SASP) in acetic acid-treated rats, a model of colitis in which
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oxidative stress and inappropriate cytokine productions are intimately linked. We measured the oxidative damage and antioxidant activity, detected the levels of tumor necrosis factor-a (TNF-a), interferon-g (IFN-g) and nuclear factor-kBp65 (NF-kBp65) to determine the role of EGCG. The aim of this study was to find whether EGCG may be of therapeutic benefit as a treatment regimen for UC. 1. Materials and methods 1.1. Animals Sixty male SpragueeDawley rats (initial weight 235 4 g) were purchased from Chinese Academy of Science, Shanghai Experimental Animal Center, Shanghai, China. The animals were housed under a 12-h light/dark cycle at a constant ambient temperature (20e27 C) and humidity (40e70%), fed with purified water and whole value rat chow irradiated by Co60, in accordance with approved ethical guidelines. 1.2. Reagents and drugs Reagents and drugs were obtained from the following sources: EGCG was from Roche (purity 99%); SASP was from Shanghai Sanwei Pharmaceutical Company, China (0.25 g/ tablet); nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD) kits were from Nanjing Jianchen Biochemical Engineering Company, China; TNF-a, and IFN-g ELISA kits were from R&D Systems, USA; anti-NF-kBp65 (Rel A) antibody was from Shanghai Kangchen Bio-tech Company, China; and SP-histostain-Plus kit was from Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd, China. 1.3. Induction and treatment of colitis All rats were weighed and randomly assigned into 4 groups by computer and fasted 24 h prior to the experimental procedure. The normal control (n ¼ 10) group was administered with saline by intrarectal route. The other rats of the model placebo group (n ¼ 20), EGCG group (n ¼ 15), and SASP group (n ¼ 15) received acetic acid via intrarectal administration. Rats were put under ether anesthesia, and a soft polyethylene catheter of 3 mm in diameter was introduced into the anus; then 1.5e2.0 ml of 8% acetic acid was carefully administered and remained in the colon for 15 s. Before catheter withdrawal, 3e5 ml saline was applied in order to spread the acetic acid completely in the colon [16]. Treatment began 24 h later. The normal group was treated with regular feed, while the other 3 groups were treated orally in solution via gavage with saline 2 ml/d, EGCG 50 mg/kg/d, and SASP 0.25 g/ kg/d, respectively, for 7 days. All rats were sacrificed at day 8 by cervical decapitation. The colon was removed and opened longitudinally, washed by the cold saline, and macroscopic damage was assessed immediately. Tissue adjacent to the ulcer or hyperemic areas was taken (5 mm 10 mm), fixed in 10% formalin, embedded in paraffin and stained with hematoxylin and eosin for subsequent histological examination. Additional
colon and serum samples were frozen in the liquid nitrogen until use. 1.4. Determination of disease activity index (DAI), colon mucosa damage index (CMDI), and histological score The general condition, food intake, weight, and stool consistency were observed daily, as well as occult blood stool or the presence of gross blood stool. DAI score was assessed independently by 2 investigators blinded to the protocol [17]. Briefly, no weight loss was scored as 0 point, weight loss of 1e5% as 1 point, 5e10% as 2 points, 10e15% as 3 points, and more than 15% as 4 points. For stool consistency, 0 point was given for well-formed pellets, 2 points for pasty and semiformed stools that did not stick to the anus, and 4 points for liquid stools that remained adhesive to the anus. Rectal bleeding was scored 0 point for no blood in hemoccult, 2 points for positive hemoccult, and 4 points for gross bleeding from the rectum. These scores were added and divided by 3, resulting in a total clinical score ranging from 0 (healthy) to 4 (maximal activity of colitis). CMDI score indicated mucosal injury (macroscopically) and was assessed in a blinded fashion. No damage (score 0); localized hyperemia but no erosion and ulcer (score 1); granular mucosa with hyperemia, edema, and erosion (score 2); worse mucosa injury such as putrescence and ulcer extending <1 cm alone the length of colon (score 3); and 2 or more sites of ulceration or ulcer extending >1 cm (score 4) [18]. The histological examination was processed by a gastrointestinal pathologist (blinded) and scored as the sum of ulcer, inflammation severity, epithelia dysplasia (0 ¼ none, 1 ¼ mild, 2 ¼ moderate, 3 ¼ severe) and extent (0 ¼ none, 1 ¼ mucosal, 2 ¼ submucosal and deeper extension, 3 ¼ transmural) to get an average of 15 visual fields per slide. All live animals of the treatment groups were included in the scores evaluation. Rats that died during the treatment period in the treatment model were scored as maximally ill, as they showed high clinical activity scores the day before they died. Concerning the DAI score, these animals were counted as score 4 (equally maximally ill) on the day of death. Nevertheless, these animals had to be excluded from the evaluation of post-mortem and ex vivo parameters. As the definite cause of death in the dose pilot study was not always clearly related to colitis, all rats that died before the end of study were excluded from statistical analysis. 1.5. Nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD) assay The levels of oxidative injury and antioxidant activity in tissue samples were assessed by measuring nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD). Nitric acid deoxidase, thiobarbituric acid (TBA) and xanthine oxidase method were used, respectively, according to the instructions provided by the kit. Tissue samples kept in liquid nitrogen were homogenized (10%) then the supernatant was mixed with the reagents in the kits. The mixture absorbance
Z.H. Ran et al. / Biomedicine & Pharmacotherapy 62 (2008) 189e196
was determined at 550 nm, 532 nm, and 550 nm, respectively. All measurements were carried out in duplicates. 1.6. Cytokines detection Serum samples were obtained from the rats and stored in liquid nitrogen for tumor necrosis factor-a (TNF-a) and interferon-g (IFN-g) measurements by ABC-ELISA following manufacturer’s instructions. TNF-a and IFN-g contents were determined on the basis of the OD value obtained from the micro plate reader (Model 550, BioRad) and standard curve. The linear regression of the standard curve was 0.998. Each assay was repeated 3 times, and the average result was shown. 1.7. Expression of nuclear factor-kBp65 (NF-kBp65) Expression of NF-kBp65 in the colon mucosa was measured by SP immunohistochemistry method. Four-micrometer-thick sections were prepared. Mouse anti-rat NF-kBp65 (Rel A) monoclonal antibody was diluted into 1:50 and was incubated with the sections overnight, followed by addition of a biotinylated rabbit anti-mouse IgG as the secondary antibody; DAB was used for color development. PBS was used as the alternative of the first antibody for the negative control. Positive staining is represented by the brown coloration in the cytoplasm and karyotheca. For enumeration, 10 visual fields per slide (magnification 400) were randomly chosen and positive cells were counted by using the 16D ocular micro counting net. The average was taken (cells/mm2). 1.8. Statistical analysis Data are presented as means SD. Statistical significance was determined by factorial ANOVA analysis and t-test. Differences were considered statistically significant for p < 0.05. Statistical analysis was performed using SAS 6.12 software. 2. Results
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show any signs of improvement throughout the entire experiment. Oral treatment with EGCG and SASP reduced disease progression from day 3 or 4 until the end of the study (Fig. 1). At day 7, rats in the placebo group had severe colitis, with a DAI score of 3.9 0.4 (n ¼ 11), whereas colitis was significantly blunted in the rats of EGCG-treated group (DAI 1.1 0.9; n ¼ 12) and SASP-treated group (DAI 3.0 1.2; n ¼ 14). EGCG notably improved the DAI score compared with the placebo and SASP group ( p < 0.01). 2.3. CMDI score and histological score Mucosa of rats in the normal group was smooth and intact. All acetic acid-treated rats showed injury of different extents, which consisted of multifocal lesions, such as thickening and shortening of the colon, haematose or edema hemorrhagic areas, and ulceration of the proximal and distal colon (Fig. 2). The rats in the placebo group had the most severe macroscopic damage of mucosa erosion and putrescence, multiulceration with diameter >1.0 cm. Whilst, rats in the EGCG and SASP groups revealed improved macroscopical lesion with edema, haematose mucosa, small and superficial ulcers. When CMDI scores of the colitis groups were compared, the lowest CMDI was observed in EGCG-treated rats (1.5 0.9). The difference between EGCG and placebo group (3.3 0.6, p < 0.01) and that between EGCG and SASP group (2.3 0.9, p < 0.05) was statistically significant (Table 1). Histological sections from the colitis groups displayed a loss of epithelia cells, reduction in the goblet cells, collapse of crypts, ulcerations, submucosal edema, transmural distribution, epithelia dysplasia, and mixed inflammatory cells infiltration (macrophages, lymphocytes, and neutrophils) (Fig. 3). The placebo group had the highest histological score (9.3 2.8) which was characterized by severe disintegration of tissue architecture, massive inflammatory infiltration with large areas of ulceration. Conversely, the inflammation and damage in animals treated with EGCG (4.6 3.1) and SASP (7.9 4.0) were markedly improved. The histological score was notably reduced by EGCG ( p < 0.01 vs. model placebo, p < 0.05 vs. SASP) (Table 1).
2.1. Clinical findings
2.2. DAI score Rats developed signs of colitis at day 1 after acetic acid administration, defined by a DAI score greater than 0.5. The inflammatory activity of rats in model placebo group did not
DAI changing trends 5.0
DAI score
After 24 h of administration of acetic acid, animals developed hematochezia, diarrhea and progressively body weight loss with weakness and decreased food intake. All these symptoms began to be blunted in the EGCG and SASP groups at day 3 or 4. At the end of the experiment, 9 rats from the model placebo group died, 4 for colon perforation, 3 for bleeding, 2 for megacolon; 3 rats from the EGCG group died, 2 for colon perforation, 1 for megacolon; and 1 rat died of colon perforation in the SASP group.
4.0 3.0 2.0
*# *#
1.0 0.0 model creating
1
2
3
4
5
6
7
d normal model placebo
EGCG SASP
Fig. 1. The changing trends comparison of DAI between groups. The DAI score (consisting of weight loss, stool consistency, and rectal bleeding; ranging from 0 ¼ healthy to 4 ¼ maximally ill) was assessed daily. Scores are depicted as means SEM. At day 7, DAI score in EGCG group was significantly decreased ( p < 0.01, compared with placebo and SASP group). *p < 0.01 vs. Placebo; and #p < 0.01 vs. SASP.
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Fig. 2. By comparison of macroscopic injury between 4 groups, the CMDI were made. CMDI score was assessed in a blinded fashion. No damage (score 0); localized hyperemia but no erosion and ulcer (score 1); granular mucosa with hyperemia, edema, and erosion (score 2); worse mucosa injury such as putrescence and ulcer extending <1 cm alone the length of colon (score 3); and 2 or more sites of ulceration or ulcer extending >1 cm (score 4). A: normal, B: model placebo; C: EGCG; and D: SASP.
2.4. Assay of NO, MDA, and SOD
2.5. Serum cytokines measurement
EGCG reduced the contents of NO and MDA, and elevated contents of SOD in colonic tissues. The contents of NO and MDA (9.1 5.6 mmol/g prot, 0.9 0.6 nmol/mg prot) in EGCG group were significantly decreased compared with that of in the placebo group (15.4 5.0 mmol/g prot, 1.5 0.6 nmol/mg prot; p < 0.05), although there were no differences between the EGCG and SASP groups (NO ¼ 9.8 7.7 mmol/g prot, MDA ¼ 1.4 0.7 nmol/mg prot). Interestingly, EGCG treatment dramatically increased SOD levels (185.4 34.1 U/mg prot) compared with the placebo group (82.4 24.6 U/mg prot, p < 0.01) and SASP group (76.1 25.9 U/mg prot, p < 0.01) (Fig. 4).
Serum levels of TNF-a and IFN-g were quantified by ELISA. EGCG-treated group showed the lowest level of TNF-a (24.4 1.6 pg/ml; 59.8 7.5 pg/ml in the placebo group; 38.1 3.7 pg/ml in the SASP group) and IFN-g (33.3 0.9 pg/ml; 44.5 2.6 pg/ml in the placebo group; 42.7 5.3 pg/ml in the SASP group) (Fig. 5).
Table 1 Comparison of CMDI and histological score between 4 groups
Normal (n ¼ 10) Model placebo (n ¼ 11) EGCG (n ¼ 12) SASP (n ¼ 14) Data are presented as mean SD. a p < 0.01 vs. normal. b p < 0.01 vs. model placebo. c p < 0.05 vs. SASP.
CMDI
Histological score
0.2 0.4 3.3 0.6a 1.5 0.9b,c 2.3 0.9b
0.0 0.0 9.3 2.8a 4.6 3.1b,c 7.9 4.0
2.6. Nuclear factor-kBp65 (NF-kBp65) detection The immunohistochemical staining of NF-kB in damaged colon collected on day 7 was shown in Fig. 6. Positive expression is indicated by the brown coloration in the cytoplasm and karyotheca of inflammatory cells including macrophages and neutrophils. NF-kB staining is occasionally detected in the normal control group (10.5 2.0 cells/mm2), but abundant staining can be found (Fig. 6A). However, a strong NF-kB staining in the mucosa and the edge of ulcer (60.5 4.0 cells/mm2) was noticed in model placebo group (Fig. 6B). Whilst, much less staining was observed in the EGCG group (28.0 2.8 cells/mm2) (Fig. 6C) and the SASP group (50.2 3.6 cells/mm2) (Fig. 6D). EGCG-treated animals had lowest NF-kB staining ( p < 0.01) compared with the placebo and SASP groups.
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Fig. 3. Representative histological slides from rats of normal control group (A), model placebo group (B), EGCG group (C), and SASP group (D). A: normal mucosa, and B: inflammation cells infiltrate, large ulcer, and epithelia dysplasia were observed in model placebo-treated rats. C: the histological changes were significantly improved by EGCG therapy. D: SASP also blocked acetic acid-induced changes in colon histology to some extent. Magnification 200.
3. Discussion Current therapies for patients with UC are directed toward the reduction of colonic mucosal inflammation, largely through the use of either aminosalicylates or corticosteroids. Complementary and alternative medicines are increasingly used in many patients with a variety of gastrointestinal complaints [19]. Data from other investigators suggest that individuals with chronic IBD may have even greater usage of those medicines [20]. Among them, herbal anti-inflammatory remedies, diet nutriological therapy, multivitamins, probiotics, and Chinese traditional medicines are most widely used for UC. This study is the first to demonstrate the efficacy of EGCG in rat colitis induced by acetic acid. A number of rodent models have been established to try to understand more about UC. These models have revealed some important causes of UC and provided an ideal stage for testing the therapeutic effect and mechanisms of new medicines. One of those, the acetic acid-induced colitis is a reproducible and simple model, sharing many characteristics with human colitis. The underlying pathophysiological mechanisms involved colon structure and mucosa barrier destruction by chemical stimulation, enhanced vessel permeability, increased inflammatory mediators, promotion of fibrin hydrolysis, and disturbance of cruor process. In conclusion, acetic acid-induced colitis is a stable and reliable method for testing new medicines for evaluating their clinical utility.
Previous studies have shown that EGCG is a natural herbal compound with high biochemical activity and rare side effects [21,22]. The major activities of EGCG already verified include reactive oxygen scavenging, antioxidant ability, immune modulation, bacterial inhibition, antiviral effect, cancer prevention, radiation protection, and life prolongation [23e25]. This study chose EGCG as the protective agent to examine its benefits in rat colitis. We showed that the DAI, CMDI, and histological scores of rats in EGCG-treated group were significantly improved compared with that in the placebo group, which was correlated with marked clinical symptom relief and disease remission. The effect of EGCG was also better than that of the conventional medicine SASP. The etiology of UC remains uncertain. A lot of studies have indicated that oxidative stress is one of the most important factors. Reactive oxygen metabolites are generated as by-products of cellular metabolism, primarily in the mitochondria. When cellular production of these metabolites overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, protein, and DNA may ensue. Such a state of ‘‘oxidative stress’’ is thought to contribute to the pathogenesis of a number of human diseases including UC. Over the past 10 years, the overproduction of nitric oxide (NO) has received considerable attention as an important player in the initiation and propagation of UC. NO is a weak free-radical produced from L-arginine. The role of NO in UC is still unsettled. Early in disease onset, NO both enhances and blocks chronic inflammation by
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content of NO in tissue
A NO (umol/gprot)
20.0
15.4±5.0
15.0
* 9.1±5.6
8.5±2.0
9.8±7.7
10.0
normal model placebo EGCG SASP
5.0 0.0
normal
model placebo
EGCG
SASP
content of MDA in tissue
B MDA (nmol/mgprot)
1.5±0.6 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
1.4±0.7
normal
model placebo
EGCG
SASP
content of SOD intissue
C 250.0
SOD (U/mgprot)
normal model placebo EGCG SASP
* 0.9±0.6
0.9±0.5
** ## 185.4±34.1
209.1±35.0
200.0 150.0
82.4±24.6
76.1±25.9
100.0
normal model placebo EGCG SASP
50.0 0.0
normal
model placebo
EGCG
SASP
Fig. 4. Treatment of rats with EGCG and SASP not only attenuated disease symptoms but also lower colon levels of both NO (A) and MDA (B), heighten SOD (C), whereas the levels of these 3 substance in colon showed the opposite trend in placebo group. *p < 0.05 vs. placebo; #p < 0.05 vs. SASP; **p < 0.01 vs. Placebo; and ##p < 0.01 vs. SASP.
preventing the synthesis of endothelial cell adhesion molecules and limiting leukocyte adhesion. Along with the evolution of UC, NO accumulation does not appear to protect but rather contributes to disease progression [26]. NO can react with superoxide (O 2 ) to produce peroxynitrite (OONO ), a highly toxic reactive nitrogen intermediary, which can attack adjacent fatty acids within the membranes causing a chain reaction of lipid peroxidation. The end product of lipid peroxidation, malondialdehyde (MDA) is also harmful, and may be responsible for some of the overall effect, leading to release of cell contents and cell death, causing tissue and organ damage. In addition, an association between UC and an elevated risk of colorectal cancer is well-established. The available evidence suggests that DNA damage caused by oxidative stress in the characteristic damage-regeneration cycle is a major contributor to colorectal cancer development in UC patients [27]. In healthy individuals, antioxidants protect components of the body against free-radical damage. The status of antioxidant enzymes e.g., superoxide dismutase (SOD) decides the systemic protection against inflammation. SOD restrains the lipid peroxidation in colon by eliminating free-radicals, converting superoxide into peroxide (H2O2). A significant body of research has indicated that decreasing SOD activity in the local colon tissue leads to mucosal injury because of reduced ability of oxidative
radicals scavenging [28,29]. The results of the present study showed markedly decreased level of NO and MDA, significantly increased activity of SOD in EGCG-treated rats in comparison with placebo or SASP-treated rats. The enhanced antioxidant activity of EGCG might be related to its special molecular structure appeared to be important for these actions, which includes 2 catechol groups, 3 gallate groups, and 2 hydroxyl groups [30]. These findings indicate that EGCG treatment of rats with acetic acid-induced colitis can reduce the extent of colonic mucosa injury by its antioxidant effect. UC arises from aberrant response to commonly encountered environmental stimuli and persistent infection or altered colonic microflora. It has been well-known that deregulation of immune response and inappropriate cytokines are pivotal in the inflammatory process. In this study, the serum levels of TNF-a and IFN-g in the placebo group are remarkably increased compared with that in the normal group, which a strong indication of upregulated Th1 cytokine secretion in colitis [31]. IFN-g is produced by natural killer cells and by Th1 cells. The latter are involved in the pathogenesis of chronic inflammatory bowel disease. IFN-g plays a key role in stimulating CD4þ cells converting into Th1 cells, thus enhancing the activity of macrophages. TNF-a is one of the active proinflammatory cytokines with immunomodulatory
Z.H. Ran et al. / Biomedicine & Pharmacotherapy 62 (2008) 189e196 80.0 59.8±7.5
70.0 60.0
44.5±2.6
*#
50.0 40.0
24.2±0.5
*#
26.2±1.0
42.7±5.3 38.1±3.7
33.3±0.9
24.4±1.6
30.0 20.0 10.0 0.0
normal
model placebo serum TNF– α (PG/ml)
EGCG
SASP
serum IFN– γ (PG/ml)
Fig. 5. Reduction of cytokines TNF-a and IFN-g level by treatment with EGCG. The serum cytokines content were determined by ELISA. EGCG expressed the similar level of serum cytokines as the normal group, while markedly lower than placebo and SASP group. *p < 0.01 vs. Placebo; and #p < 0.01 vs. SASP.
property. It is considered to be a critical cytokine that orchestrates the inflammatory response in UC. Moreover, TNF-a induces expression of IFN-g. This may further lead to amplified synthesis of other proinflammatory cytokines and infiltration of activated lymphocytes [18]. The increased levels of TNFa and IFN-g in the rat’s model revealed an important role
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for immune dysregulation and altered cytokines patterns that are similar to the human disease [32]. EGCG application seems capable of tilting by dampening the Th1 response. Therefore, this cytokine shift by EGCG may be the mechanism of action responsible for reversing acetic acid-induced colitis in rats. Recently, tremendous progress has been made in elucidating the role of proteins in signal transduction. NF-kB is an oxidative stress sensitive transcription factor that regulates the expression of a variety of genes important in cellular responses, including inflammation, innate immunity, and growth [33]. In unstimulated cells, NF-kB predominantly exists as a heterodimer, composed of p65 and p50 subunits, that resides in the cytoplasm in an inactive state bound to a member of the I-kB family of inhibitory proteins. NF-kB activity can be induced in most cell types upon exposure to stimuli including cytokines (TNF-a, interleukin-1), endotoxin, and oxidative stress. The liberated NF-kB then translocates to the nucleus, binds to specific sequences in the promoter region, and induces gene expression. Some studies discovered EGCG inhibited LPSmediated inflammation by decreasing the degradation of I-kB [34]. In this study, the expression of NF-kB in EGCG-treated colon tissue is much lower than that in the placebo-treated rats. The data may explain, at least in part, some of the reported anti-inflammatory effects of EGCG. In summary, the present study has demonstrated the effectiveness of EGCG as a protective therapy in an experimental
Fig. 6. EGCG reduced NF-kB induction in the inflamed colon. Obvious NF-kB staining was not seen in the normal or EGCG samples (A) but was remarkably enhanced in the mucosa of placebo- and SASP-treated animals (B and D). The increase in NF-kB staining was reduced but not eliminated by EGCG treatment (C). Magnification 200.
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model of rat colitis. It had also compared the therapeutic effects of EGCG and SASP. The current data, coupled with EGCG’s excellent safety record, suggest that the concept of testing and developing of EGCG for treatment of colitis in humans may be justified. Acknowledgment This project was supported by Shanghai Leading Academic Discipline Project, Project Number: Y0205. References [1] Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002;347:417e29. [2] Reinisch W, Dejaco C, Knoflach P, Petritsch W, Vogelsang H, Tilg H. Immunosuppressive therapy for inflammatory bowel disease: consensus by the Austrian working group on IBD. Z Gastroenterol 2004;42:1033e45. discussion 1046e1047. German. [3] Kumar S, Ghoshal UC, Aggarwal R, Saraswat VA, Choudhuri G. Severe ulcerative colitis: prospective study of parameters determining outcome. J Gastroenterol Hepatol 2004;19:1247e52. [4] Ghosh S, Van Heel D, Playford RJ. Probiotics in inflammatory bowel disease: is it all gut flora modulation? Gut 2004;53:620e2. [5] Meister D, Ghosh S. Effect of fish oil enriched enteral diet on inflammatory bowel disease tissues in organ culture: differential effects on ulcerative colitis and Crohn’s disease. World J Gastroenterol 2005;11:7466e72. [6] Stremmel W, Merle U, Zahn A, Autschbach F, Hinz U, Ehehalt R. Retarded release phosphatidylcholine benefits patients with chronic active ulcerative colitis. Gut 2005;54:966e71. [7] Wang LF, Zhang HY. A theoretical study of the different radical-scavenging activities of catechin, quercetin, and a rationally designed planar catechin. Bioorg Chem 2005;33:108e15. [8] Lee JM, Karim MM, Lee SH. Determination of catechin in aqueous solution by chemiluminescence method. J Fluoresc 2005;15:735e9. [9] Cabrera C, Artacho R, Gime´nez R. Beneficial effects of green teada review. J Am Coll Nutr 2006;25:79e99. [10] Pierre M, Onge ST. Dietary fats, teas, dairy, and nuts: potential functional foods for weight control? Am J Clin Nutr 2005;81:7e15. [11] Guo S, Yang S, Taylor C, Sonenshein GE. Green tea polyphenol epigallocatechin-3 gallate (EGCG) affects gene expression of breast cancer cells transformed by the carcinogen 7,12-dimethylbenz[a]anthracene1e3. J Nutr 2005;135:2978se86. [12] Kim J, Zhang X, Rieger-Christ KM, Summerhayes IC, Wazer DE, Paulson KE, et al. Suppression of Wnt signaling by the green tea compound ()-epigallocatechin 3-gallate (EGCG) in invasive breast cancer cells: requirement of the transcriptional repressor HBP1. J Biol Chem 2006;281:10865e75. [13] Ahmed S, Wang N, Lalonde M, Goldberg VM, Haqqi TM. Green tea polyphenol epigallocatechin-3-gallate (EGCG) differentially inhibits interleukin-1 b-induced expression of matrix metalloproteinase-1 and -13 in human chondrocytes. J Pharmacol Exp Ther 2004;308:767e73. [14] Lee YK, Bone ND, Strege AK, Shanafelt TD, Jelinek DF, Kay NE. VEGF receptor phosphorylation status and apoptosis is modulated by a green tea component, epigallocatechin-3-gallate (EGCG), in B-cell chronic lymphocytic leukemia. Blood 2004;104:788e94. [15] Hwang JT, Park IJ, Shin JI, Lee YK, Lee SK, Baik HW, et al. Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase. Biochem Biophys Res Commun 2005;338:694e9.
[16] Millar AD, Rampton DS, Chander CL, Claxson AW, Blades S, Coumbe A, et al. Evaluating the antioxidant potential of new treatments for inflammatory bowel disease using a rat model of colitis. Gut 1996;39:407e15. [17] Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin10. Science 2000;289:1352e5. [18] Myers KJ, Murthy S, Flanigan A, Witchell DR, Butler M, Murray S, et al. Antisense oligonucleotide blockade of tumor necrosis factor-a in two murine models of colitis. J Pharmacol Exp Ther 2003;304:411e24. [19] Day AS, Whitten KE, Bohane TD. Use of complementary and alternative medicines by children and adolescents with inflammatory bowel disease. J Paediatr Child Health 2004;40:681e4. [20] Langhorst J, Anthonisen IB, Steder-Neukamm U, Lu¨dtke R, Spahn G, Michalsen A, et al. Amount of systemic steroid medication is a strong predictor for the use of complementary and alternative medicine in patients with inflammatory bowel disease: results from a German national survey. Inflamm Bowel Dis 2005;11:287e95. [21] Frei B, Higdon JV. Antioxidant activity of tea polyphenols in vivo: evidence from animal studies. J Nutr 2003;133:3275Se84. [22] Katiyar SK, Challa A, McCormick TS, Cooper KD, Mukhtar H. Prevention of UVB-induced immunosuppression in mice by the green tea polyphenol ()-epigallocatechin-3-gallate may be associated with alterations in IL-10 and IL-12 production. Carcinogenesis 1999;20:2117e24. [23] Yang F, Oz HS, Barve S, de Villiers WJ, McClain CJ, Varilek GW. The green tea polyphenol ()-epigallocatechin-3-gallate blocks nuclear factor-kB activation by inhibiting I kB kinase activity in the intestinal epithelial cell line IEC-6. Mol Pharmacol 2001;60:528e33. [24] Miura Y, Chiba T, Tomita I, Koizumi H, Miura S, Umegaki K, et al. Tea catechins prevent the development of atherosclerosis in apoprotein E-deficient mice. J Nutr 2001;131:27e32. [25] Porath D, Riegger C, Drewe J, Schwager J. Epigallocatechin-3-gallate impairs chemokine production in human colon epithelial cell lines. J Pharmacol Exp Ther 2005;315:1172e80. [26] Mccafferty DM. Peroxynitrite and inflammatory bowel disease. Gut 2000;46:436e9. [27] Seril DN, Liao J, West AB, Yang GY. Oxidative stress and ulcerative colitis-associated carcinogenesis: studies in humans and animal models. Carcinogenesis 2003;24:353e62. [28] Barazzone C, White CW. Mechanisms of cell injury and death in hyperoxia. Role of cytokines and Bcl-2 family proteins. Am J Respir Cell Mol Biol 2000;22:517e9. [29] Krieglstein CF, Cerwinka WH, Laroux FS, Salter JW, Russell JM, Schuermann G, et al. Regulation of murine intestinal inflammation by reactive metabolites of oxygen and nitrogen: divergent roles of superoxide and nitric oxide. J Exp Med 2001;194:1207e18. [30] Peng W, Kuo SM. Flavonoid structure affects the inhibition of lipid peroxidation in caco-2 intestinal cells at physiological concentrations. J Nutr 2003;133:2184e7. [31] Fuss IJ, Heller F, Boirivant M, Leon F, Yoshida M, Fichtner-Feigl S, et al. Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis. J Clin Invest 2004;113:1490e7. [32] Mathew CG, Lewis CM. Genetics of inflammatory bowel disease: progress and prospects. Hum Mol Genet 2004;13:161e8. [33] Venkatraman A, Ramakrishna BS, Shaji RV, Kumar NS, Pulimood A, Patra S. Amelioration of dextran sulfate colitis by butyrate: role of heat shock protein 70 and NF-kB. Am J Physiol Gastrointest Liver Physiol 2003;285:177e84. [34] Ichikawa D, Matsui A, Imai M, Sonoda Y, Kasahara T. Effect of various catechins on the IL-12p40 production by murine peritoneal macrophages and a macrophage cell line, J774.1. Biol Pharm Bull 2004;27:1353e8.
Biomedicine & Pharmacotherapy 62 (2008) 189e196 www.elsevier.com/locate/biopha
Original article
Epigallocatechin-3-gallate ameliorates rats colitis induced by acetic acid Zhi Hua Ran*, Chi Chen, Shu Dong Xiao Department of Gastroenterology, Renji Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai Institute of Digestive Disease, 145 Shan Dong Zhong Road, Shanghai 200001, PR China Received 17 February 2007; accepted 7 February 2008 Available online 20 February 2008
Abstract Background: Epigallocatechin-3-gallate (EGCG) has been recently proved to possess anti-inflammatory effects. Aims: To investigate the effect and mechanism of epigallocatechin-3-gallate (EGCG) treatment in rats with acetic acid-induced colitis. Methods: Sixty male rats were randomly assigned into 4 groups: normal control (n ¼ 10), model placebo (n ¼ 20), EGCG (n ¼ 15), and SASP (n ¼ 15). The normal group was treated with regular feeding, while the other 3 groups were treated orally with saline 2 ml/d, EGCG 50 mg/kg/d, and SASP 0.25 g/kg/d, respectively, for 7 days using an established colitis model induced by 8% acetic acid. The disease activity index (DAI) and the therapeutic effects were evaluated. Colon mucosa damage index (CMDI) and histological score were determined. The activities of nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-a (TNF-a) and interferon-g (IFN-g) and tissue expression of nuclear factor-kBp65 (NF-kBp65) were measured. Results: EGCG notably improved the DAI (1.1 0.9), CMDI (1.5 0.9) and histological scores (4.6 3.1) compared with the placebo (3.9 0.4, p < 0.01; 3.3 0.6, p < 0.05; 9.3 2.8, p < 0.01) and SASP groups (3.0 1.2, p < 0.01; 2.3 0.9, p < 0.05; 7.9 4.0, p < 0.05). Compared with the placebo and SASP groups, the levels of NO (9.1 5.6 mmol/g prot), MDA (0.9 0.6 nmol/g prot), TNFa (24.4 1.6 PG/ml), IFN-g (33.3 0.9 PG/ml), and NF-kBp65 (28.0 2.8 cells/mm3) in EGCG-treated group were significantly reduced ( p < 0.05 or p < 0.01), while that of SOD (185.4 24.6 U/mg prot) was increased remarkably ( p < 0.05). Conclusion: EGCG exerts its antioxidant activity via decreasing NO, MDA, and increasing SOD. It ameliorates mucosal inflammation by inhibiting the production of TNF-a, IFN-g and NF-kBp65 and may be a potential therapeutic agent in colitis. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Epigallocatechin-3-gallate; Colitis; Effect; Mechanism
Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) characterized by chronic inflammation of the colon. The exact pathogenesis of UC remains uncertain [1]. Oxidative stress and aberrant immune response to the various stimulations have been proposed to be involved in the initiation and propagation of the chronic inflammation [2]. Previous studies have suggested that IBD patients rank high among users of complementary and alternative medicine because of unsatisfactory responses to conventional medicine and potential side effects [3]. Growing attention has been * Corresponding author. Tel.: þ86 21 63200874; fax: þ86 21 63266027. E-mail address: [email protected] (Z.H. Ran). 0753-3322/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2008.02.002
focused on dietary and natural medicines that inhibit, reverse, or retard this disease [4e6]. Fresh tea leaves are rich in tea polyphenols known as catechins [7,8]. The principal catechins found in tea are epicatechin (EC), epigallocatechin (EGC), epicatechingallate (ECG) and epigallocatechin-3-gallate (EGCG) [9,10]. EGCG is the most abundant catechin in green tea. In vitro and animal studies provide strong evidence that EGCG may possess antioxidant and anti-inflammatory properties to affect the pathogenesis of several chronic diseases, especially cancer and cardiovascular disease [11e15]. In the present study, we examined the therapeutic effects of EGCG compared with the conventional medicine sulfasalazine (SASP) in acetic acid-treated rats, a model of colitis in which
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oxidative stress and inappropriate cytokine productions are intimately linked. We measured the oxidative damage and antioxidant activity, detected the levels of tumor necrosis factor-a (TNF-a), interferon-g (IFN-g) and nuclear factor-kBp65 (NF-kBp65) to determine the role of EGCG. The aim of this study was to find whether EGCG may be of therapeutic benefit as a treatment regimen for UC. 1. Materials and methods 1.1. Animals Sixty male SpragueeDawley rats (initial weight 235 4 g) were purchased from Chinese Academy of Science, Shanghai Experimental Animal Center, Shanghai, China. The animals were housed under a 12-h light/dark cycle at a constant ambient temperature (20e27 C) and humidity (40e70%), fed with purified water and whole value rat chow irradiated by Co60, in accordance with approved ethical guidelines. 1.2. Reagents and drugs Reagents and drugs were obtained from the following sources: EGCG was from Roche (purity 99%); SASP was from Shanghai Sanwei Pharmaceutical Company, China (0.25 g/ tablet); nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD) kits were from Nanjing Jianchen Biochemical Engineering Company, China; TNF-a, and IFN-g ELISA kits were from R&D Systems, USA; anti-NF-kBp65 (Rel A) antibody was from Shanghai Kangchen Bio-tech Company, China; and SP-histostain-Plus kit was from Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd, China. 1.3. Induction and treatment of colitis All rats were weighed and randomly assigned into 4 groups by computer and fasted 24 h prior to the experimental procedure. The normal control (n ¼ 10) group was administered with saline by intrarectal route. The other rats of the model placebo group (n ¼ 20), EGCG group (n ¼ 15), and SASP group (n ¼ 15) received acetic acid via intrarectal administration. Rats were put under ether anesthesia, and a soft polyethylene catheter of 3 mm in diameter was introduced into the anus; then 1.5e2.0 ml of 8% acetic acid was carefully administered and remained in the colon for 15 s. Before catheter withdrawal, 3e5 ml saline was applied in order to spread the acetic acid completely in the colon [16]. Treatment began 24 h later. The normal group was treated with regular feed, while the other 3 groups were treated orally in solution via gavage with saline 2 ml/d, EGCG 50 mg/kg/d, and SASP 0.25 g/ kg/d, respectively, for 7 days. All rats were sacrificed at day 8 by cervical decapitation. The colon was removed and opened longitudinally, washed by the cold saline, and macroscopic damage was assessed immediately. Tissue adjacent to the ulcer or hyperemic areas was taken (5 mm 10 mm), fixed in 10% formalin, embedded in paraffin and stained with hematoxylin and eosin for subsequent histological examination. Additional
colon and serum samples were frozen in the liquid nitrogen until use. 1.4. Determination of disease activity index (DAI), colon mucosa damage index (CMDI), and histological score The general condition, food intake, weight, and stool consistency were observed daily, as well as occult blood stool or the presence of gross blood stool. DAI score was assessed independently by 2 investigators blinded to the protocol [17]. Briefly, no weight loss was scored as 0 point, weight loss of 1e5% as 1 point, 5e10% as 2 points, 10e15% as 3 points, and more than 15% as 4 points. For stool consistency, 0 point was given for well-formed pellets, 2 points for pasty and semiformed stools that did not stick to the anus, and 4 points for liquid stools that remained adhesive to the anus. Rectal bleeding was scored 0 point for no blood in hemoccult, 2 points for positive hemoccult, and 4 points for gross bleeding from the rectum. These scores were added and divided by 3, resulting in a total clinical score ranging from 0 (healthy) to 4 (maximal activity of colitis). CMDI score indicated mucosal injury (macroscopically) and was assessed in a blinded fashion. No damage (score 0); localized hyperemia but no erosion and ulcer (score 1); granular mucosa with hyperemia, edema, and erosion (score 2); worse mucosa injury such as putrescence and ulcer extending <1 cm alone the length of colon (score 3); and 2 or more sites of ulceration or ulcer extending >1 cm (score 4) [18]. The histological examination was processed by a gastrointestinal pathologist (blinded) and scored as the sum of ulcer, inflammation severity, epithelia dysplasia (0 ¼ none, 1 ¼ mild, 2 ¼ moderate, 3 ¼ severe) and extent (0 ¼ none, 1 ¼ mucosal, 2 ¼ submucosal and deeper extension, 3 ¼ transmural) to get an average of 15 visual fields per slide. All live animals of the treatment groups were included in the scores evaluation. Rats that died during the treatment period in the treatment model were scored as maximally ill, as they showed high clinical activity scores the day before they died. Concerning the DAI score, these animals were counted as score 4 (equally maximally ill) on the day of death. Nevertheless, these animals had to be excluded from the evaluation of post-mortem and ex vivo parameters. As the definite cause of death in the dose pilot study was not always clearly related to colitis, all rats that died before the end of study were excluded from statistical analysis. 1.5. Nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD) assay The levels of oxidative injury and antioxidant activity in tissue samples were assessed by measuring nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD). Nitric acid deoxidase, thiobarbituric acid (TBA) and xanthine oxidase method were used, respectively, according to the instructions provided by the kit. Tissue samples kept in liquid nitrogen were homogenized (10%) then the supernatant was mixed with the reagents in the kits. The mixture absorbance
Z.H. Ran et al. / Biomedicine & Pharmacotherapy 62 (2008) 189e196
was determined at 550 nm, 532 nm, and 550 nm, respectively. All measurements were carried out in duplicates. 1.6. Cytokines detection Serum samples were obtained from the rats and stored in liquid nitrogen for tumor necrosis factor-a (TNF-a) and interferon-g (IFN-g) measurements by ABC-ELISA following manufacturer’s instructions. TNF-a and IFN-g contents were determined on the basis of the OD value obtained from the micro plate reader (Model 550, BioRad) and standard curve. The linear regression of the standard curve was 0.998. Each assay was repeated 3 times, and the average result was shown. 1.7. Expression of nuclear factor-kBp65 (NF-kBp65) Expression of NF-kBp65 in the colon mucosa was measured by SP immunohistochemistry method. Four-micrometer-thick sections were prepared. Mouse anti-rat NF-kBp65 (Rel A) monoclonal antibody was diluted into 1:50 and was incubated with the sections overnight, followed by addition of a biotinylated rabbit anti-mouse IgG as the secondary antibody; DAB was used for color development. PBS was used as the alternative of the first antibody for the negative control. Positive staining is represented by the brown coloration in the cytoplasm and karyotheca. For enumeration, 10 visual fields per slide (magnification 400) were randomly chosen and positive cells were counted by using the 16D ocular micro counting net. The average was taken (cells/mm2). 1.8. Statistical analysis Data are presented as means SD. Statistical significance was determined by factorial ANOVA analysis and t-test. Differences were considered statistically significant for p < 0.05. Statistical analysis was performed using SAS 6.12 software. 2. Results
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show any signs of improvement throughout the entire experiment. Oral treatment with EGCG and SASP reduced disease progression from day 3 or 4 until the end of the study (Fig. 1). At day 7, rats in the placebo group had severe colitis, with a DAI score of 3.9 0.4 (n ¼ 11), whereas colitis was significantly blunted in the rats of EGCG-treated group (DAI 1.1 0.9; n ¼ 12) and SASP-treated group (DAI 3.0 1.2; n ¼ 14). EGCG notably improved the DAI score compared with the placebo and SASP group ( p < 0.01). 2.3. CMDI score and histological score Mucosa of rats in the normal group was smooth and intact. All acetic acid-treated rats showed injury of different extents, which consisted of multifocal lesions, such as thickening and shortening of the colon, haematose or edema hemorrhagic areas, and ulceration of the proximal and distal colon (Fig. 2). The rats in the placebo group had the most severe macroscopic damage of mucosa erosion and putrescence, multiulceration with diameter >1.0 cm. Whilst, rats in the EGCG and SASP groups revealed improved macroscopical lesion with edema, haematose mucosa, small and superficial ulcers. When CMDI scores of the colitis groups were compared, the lowest CMDI was observed in EGCG-treated rats (1.5 0.9). The difference between EGCG and placebo group (3.3 0.6, p < 0.01) and that between EGCG and SASP group (2.3 0.9, p < 0.05) was statistically significant (Table 1). Histological sections from the colitis groups displayed a loss of epithelia cells, reduction in the goblet cells, collapse of crypts, ulcerations, submucosal edema, transmural distribution, epithelia dysplasia, and mixed inflammatory cells infiltration (macrophages, lymphocytes, and neutrophils) (Fig. 3). The placebo group had the highest histological score (9.3 2.8) which was characterized by severe disintegration of tissue architecture, massive inflammatory infiltration with large areas of ulceration. Conversely, the inflammation and damage in animals treated with EGCG (4.6 3.1) and SASP (7.9 4.0) were markedly improved. The histological score was notably reduced by EGCG ( p < 0.01 vs. model placebo, p < 0.05 vs. SASP) (Table 1).
2.1. Clinical findings
2.2. DAI score Rats developed signs of colitis at day 1 after acetic acid administration, defined by a DAI score greater than 0.5. The inflammatory activity of rats in model placebo group did not
DAI changing trends 5.0
DAI score
After 24 h of administration of acetic acid, animals developed hematochezia, diarrhea and progressively body weight loss with weakness and decreased food intake. All these symptoms began to be blunted in the EGCG and SASP groups at day 3 or 4. At the end of the experiment, 9 rats from the model placebo group died, 4 for colon perforation, 3 for bleeding, 2 for megacolon; 3 rats from the EGCG group died, 2 for colon perforation, 1 for megacolon; and 1 rat died of colon perforation in the SASP group.
4.0 3.0 2.0
*# *#
1.0 0.0 model creating
1
2
3
4
5
6
7
d normal model placebo
EGCG SASP
Fig. 1. The changing trends comparison of DAI between groups. The DAI score (consisting of weight loss, stool consistency, and rectal bleeding; ranging from 0 ¼ healthy to 4 ¼ maximally ill) was assessed daily. Scores are depicted as means SEM. At day 7, DAI score in EGCG group was significantly decreased ( p < 0.01, compared with placebo and SASP group). *p < 0.01 vs. Placebo; and #p < 0.01 vs. SASP.
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Fig. 2. By comparison of macroscopic injury between 4 groups, the CMDI were made. CMDI score was assessed in a blinded fashion. No damage (score 0); localized hyperemia but no erosion and ulcer (score 1); granular mucosa with hyperemia, edema, and erosion (score 2); worse mucosa injury such as putrescence and ulcer extending <1 cm alone the length of colon (score 3); and 2 or more sites of ulceration or ulcer extending >1 cm (score 4). A: normal, B: model placebo; C: EGCG; and D: SASP.
2.4. Assay of NO, MDA, and SOD
2.5. Serum cytokines measurement
EGCG reduced the contents of NO and MDA, and elevated contents of SOD in colonic tissues. The contents of NO and MDA (9.1 5.6 mmol/g prot, 0.9 0.6 nmol/mg prot) in EGCG group were significantly decreased compared with that of in the placebo group (15.4 5.0 mmol/g prot, 1.5 0.6 nmol/mg prot; p < 0.05), although there were no differences between the EGCG and SASP groups (NO ¼ 9.8 7.7 mmol/g prot, MDA ¼ 1.4 0.7 nmol/mg prot). Interestingly, EGCG treatment dramatically increased SOD levels (185.4 34.1 U/mg prot) compared with the placebo group (82.4 24.6 U/mg prot, p < 0.01) and SASP group (76.1 25.9 U/mg prot, p < 0.01) (Fig. 4).
Serum levels of TNF-a and IFN-g were quantified by ELISA. EGCG-treated group showed the lowest level of TNF-a (24.4 1.6 pg/ml; 59.8 7.5 pg/ml in the placebo group; 38.1 3.7 pg/ml in the SASP group) and IFN-g (33.3 0.9 pg/ml; 44.5 2.6 pg/ml in the placebo group; 42.7 5.3 pg/ml in the SASP group) (Fig. 5).
Table 1 Comparison of CMDI and histological score between 4 groups
Normal (n ¼ 10) Model placebo (n ¼ 11) EGCG (n ¼ 12) SASP (n ¼ 14) Data are presented as mean SD. a p < 0.01 vs. normal. b p < 0.01 vs. model placebo. c p < 0.05 vs. SASP.
CMDI
Histological score
0.2 0.4 3.3 0.6a 1.5 0.9b,c 2.3 0.9b
0.0 0.0 9.3 2.8a 4.6 3.1b,c 7.9 4.0
2.6. Nuclear factor-kBp65 (NF-kBp65) detection The immunohistochemical staining of NF-kB in damaged colon collected on day 7 was shown in Fig. 6. Positive expression is indicated by the brown coloration in the cytoplasm and karyotheca of inflammatory cells including macrophages and neutrophils. NF-kB staining is occasionally detected in the normal control group (10.5 2.0 cells/mm2), but abundant staining can be found (Fig. 6A). However, a strong NF-kB staining in the mucosa and the edge of ulcer (60.5 4.0 cells/mm2) was noticed in model placebo group (Fig. 6B). Whilst, much less staining was observed in the EGCG group (28.0 2.8 cells/mm2) (Fig. 6C) and the SASP group (50.2 3.6 cells/mm2) (Fig. 6D). EGCG-treated animals had lowest NF-kB staining ( p < 0.01) compared with the placebo and SASP groups.
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Fig. 3. Representative histological slides from rats of normal control group (A), model placebo group (B), EGCG group (C), and SASP group (D). A: normal mucosa, and B: inflammation cells infiltrate, large ulcer, and epithelia dysplasia were observed in model placebo-treated rats. C: the histological changes were significantly improved by EGCG therapy. D: SASP also blocked acetic acid-induced changes in colon histology to some extent. Magnification 200.
3. Discussion Current therapies for patients with UC are directed toward the reduction of colonic mucosal inflammation, largely through the use of either aminosalicylates or corticosteroids. Complementary and alternative medicines are increasingly used in many patients with a variety of gastrointestinal complaints [19]. Data from other investigators suggest that individuals with chronic IBD may have even greater usage of those medicines [20]. Among them, herbal anti-inflammatory remedies, diet nutriological therapy, multivitamins, probiotics, and Chinese traditional medicines are most widely used for UC. This study is the first to demonstrate the efficacy of EGCG in rat colitis induced by acetic acid. A number of rodent models have been established to try to understand more about UC. These models have revealed some important causes of UC and provided an ideal stage for testing the therapeutic effect and mechanisms of new medicines. One of those, the acetic acid-induced colitis is a reproducible and simple model, sharing many characteristics with human colitis. The underlying pathophysiological mechanisms involved colon structure and mucosa barrier destruction by chemical stimulation, enhanced vessel permeability, increased inflammatory mediators, promotion of fibrin hydrolysis, and disturbance of cruor process. In conclusion, acetic acid-induced colitis is a stable and reliable method for testing new medicines for evaluating their clinical utility.
Previous studies have shown that EGCG is a natural herbal compound with high biochemical activity and rare side effects [21,22]. The major activities of EGCG already verified include reactive oxygen scavenging, antioxidant ability, immune modulation, bacterial inhibition, antiviral effect, cancer prevention, radiation protection, and life prolongation [23e25]. This study chose EGCG as the protective agent to examine its benefits in rat colitis. We showed that the DAI, CMDI, and histological scores of rats in EGCG-treated group were significantly improved compared with that in the placebo group, which was correlated with marked clinical symptom relief and disease remission. The effect of EGCG was also better than that of the conventional medicine SASP. The etiology of UC remains uncertain. A lot of studies have indicated that oxidative stress is one of the most important factors. Reactive oxygen metabolites are generated as by-products of cellular metabolism, primarily in the mitochondria. When cellular production of these metabolites overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, protein, and DNA may ensue. Such a state of ‘‘oxidative stress’’ is thought to contribute to the pathogenesis of a number of human diseases including UC. Over the past 10 years, the overproduction of nitric oxide (NO) has received considerable attention as an important player in the initiation and propagation of UC. NO is a weak free-radical produced from L-arginine. The role of NO in UC is still unsettled. Early in disease onset, NO both enhances and blocks chronic inflammation by
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content of NO in tissue
A NO (umol/gprot)
20.0
15.4±5.0
15.0
* 9.1±5.6
8.5±2.0
9.8±7.7
10.0
normal model placebo EGCG SASP
5.0 0.0
normal
model placebo
EGCG
SASP
content of MDA in tissue
B MDA (nmol/mgprot)
1.5±0.6 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
1.4±0.7
normal
model placebo
EGCG
SASP
content of SOD intissue
C 250.0
SOD (U/mgprot)
normal model placebo EGCG SASP
* 0.9±0.6
0.9±0.5
** ## 185.4±34.1
209.1±35.0
200.0 150.0
82.4±24.6
76.1±25.9
100.0
normal model placebo EGCG SASP
50.0 0.0
normal
model placebo
EGCG
SASP
Fig. 4. Treatment of rats with EGCG and SASP not only attenuated disease symptoms but also lower colon levels of both NO (A) and MDA (B), heighten SOD (C), whereas the levels of these 3 substance in colon showed the opposite trend in placebo group. *p < 0.05 vs. placebo; #p < 0.05 vs. SASP; **p < 0.01 vs. Placebo; and ##p < 0.01 vs. SASP.
preventing the synthesis of endothelial cell adhesion molecules and limiting leukocyte adhesion. Along with the evolution of UC, NO accumulation does not appear to protect but rather contributes to disease progression [26]. NO can react with superoxide (O 2 ) to produce peroxynitrite (OONO ), a highly toxic reactive nitrogen intermediary, which can attack adjacent fatty acids within the membranes causing a chain reaction of lipid peroxidation. The end product of lipid peroxidation, malondialdehyde (MDA) is also harmful, and may be responsible for some of the overall effect, leading to release of cell contents and cell death, causing tissue and organ damage. In addition, an association between UC and an elevated risk of colorectal cancer is well-established. The available evidence suggests that DNA damage caused by oxidative stress in the characteristic damage-regeneration cycle is a major contributor to colorectal cancer development in UC patients [27]. In healthy individuals, antioxidants protect components of the body against free-radical damage. The status of antioxidant enzymes e.g., superoxide dismutase (SOD) decides the systemic protection against inflammation. SOD restrains the lipid peroxidation in colon by eliminating free-radicals, converting superoxide into peroxide (H2O2). A significant body of research has indicated that decreasing SOD activity in the local colon tissue leads to mucosal injury because of reduced ability of oxidative
radicals scavenging [28,29]. The results of the present study showed markedly decreased level of NO and MDA, significantly increased activity of SOD in EGCG-treated rats in comparison with placebo or SASP-treated rats. The enhanced antioxidant activity of EGCG might be related to its special molecular structure appeared to be important for these actions, which includes 2 catechol groups, 3 gallate groups, and 2 hydroxyl groups [30]. These findings indicate that EGCG treatment of rats with acetic acid-induced colitis can reduce the extent of colonic mucosa injury by its antioxidant effect. UC arises from aberrant response to commonly encountered environmental stimuli and persistent infection or altered colonic microflora. It has been well-known that deregulation of immune response and inappropriate cytokines are pivotal in the inflammatory process. In this study, the serum levels of TNF-a and IFN-g in the placebo group are remarkably increased compared with that in the normal group, which a strong indication of upregulated Th1 cytokine secretion in colitis [31]. IFN-g is produced by natural killer cells and by Th1 cells. The latter are involved in the pathogenesis of chronic inflammatory bowel disease. IFN-g plays a key role in stimulating CD4þ cells converting into Th1 cells, thus enhancing the activity of macrophages. TNF-a is one of the active proinflammatory cytokines with immunomodulatory
Z.H. Ran et al. / Biomedicine & Pharmacotherapy 62 (2008) 189e196 80.0 59.8±7.5
70.0 60.0
44.5±2.6
*#
50.0 40.0
24.2±0.5
*#
26.2±1.0
42.7±5.3 38.1±3.7
33.3±0.9
24.4±1.6
30.0 20.0 10.0 0.0
normal
model placebo serum TNF– α (PG/ml)
EGCG
SASP
serum IFN– γ (PG/ml)
Fig. 5. Reduction of cytokines TNF-a and IFN-g level by treatment with EGCG. The serum cytokines content were determined by ELISA. EGCG expressed the similar level of serum cytokines as the normal group, while markedly lower than placebo and SASP group. *p < 0.01 vs. Placebo; and #p < 0.01 vs. SASP.
property. It is considered to be a critical cytokine that orchestrates the inflammatory response in UC. Moreover, TNF-a induces expression of IFN-g. This may further lead to amplified synthesis of other proinflammatory cytokines and infiltration of activated lymphocytes [18]. The increased levels of TNFa and IFN-g in the rat’s model revealed an important role
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for immune dysregulation and altered cytokines patterns that are similar to the human disease [32]. EGCG application seems capable of tilting by dampening the Th1 response. Therefore, this cytokine shift by EGCG may be the mechanism of action responsible for reversing acetic acid-induced colitis in rats. Recently, tremendous progress has been made in elucidating the role of proteins in signal transduction. NF-kB is an oxidative stress sensitive transcription factor that regulates the expression of a variety of genes important in cellular responses, including inflammation, innate immunity, and growth [33]. In unstimulated cells, NF-kB predominantly exists as a heterodimer, composed of p65 and p50 subunits, that resides in the cytoplasm in an inactive state bound to a member of the I-kB family of inhibitory proteins. NF-kB activity can be induced in most cell types upon exposure to stimuli including cytokines (TNF-a, interleukin-1), endotoxin, and oxidative stress. The liberated NF-kB then translocates to the nucleus, binds to specific sequences in the promoter region, and induces gene expression. Some studies discovered EGCG inhibited LPSmediated inflammation by decreasing the degradation of I-kB [34]. In this study, the expression of NF-kB in EGCG-treated colon tissue is much lower than that in the placebo-treated rats. The data may explain, at least in part, some of the reported anti-inflammatory effects of EGCG. In summary, the present study has demonstrated the effectiveness of EGCG as a protective therapy in an experimental
Fig. 6. EGCG reduced NF-kB induction in the inflamed colon. Obvious NF-kB staining was not seen in the normal or EGCG samples (A) but was remarkably enhanced in the mucosa of placebo- and SASP-treated animals (B and D). The increase in NF-kB staining was reduced but not eliminated by EGCG treatment (C). Magnification 200.
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model of rat colitis. It had also compared the therapeutic effects of EGCG and SASP. The current data, coupled with EGCG’s excellent safety record, suggest that the concept of testing and developing of EGCG for treatment of colitis in humans may be justified. Acknowledgment This project was supported by Shanghai Leading Academic Discipline Project, Project Number: Y0205. References [1] Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002;347:417e29. [2] Reinisch W, Dejaco C, Knoflach P, Petritsch W, Vogelsang H, Tilg H. Immunosuppressive therapy for inflammatory bowel disease: consensus by the Austrian working group on IBD. Z Gastroenterol 2004;42:1033e45. discussion 1046e1047. German. [3] Kumar S, Ghoshal UC, Aggarwal R, Saraswat VA, Choudhuri G. Severe ulcerative colitis: prospective study of parameters determining outcome. J Gastroenterol Hepatol 2004;19:1247e52. [4] Ghosh S, Van Heel D, Playford RJ. Probiotics in inflammatory bowel disease: is it all gut flora modulation? Gut 2004;53:620e2. [5] Meister D, Ghosh S. Effect of fish oil enriched enteral diet on inflammatory bowel disease tissues in organ culture: differential effects on ulcerative colitis and Crohn’s disease. World J Gastroenterol 2005;11:7466e72. [6] Stremmel W, Merle U, Zahn A, Autschbach F, Hinz U, Ehehalt R. Retarded release phosphatidylcholine benefits patients with chronic active ulcerative colitis. Gut 2005;54:966e71. [7] Wang LF, Zhang HY. A theoretical study of the different radical-scavenging activities of catechin, quercetin, and a rationally designed planar catechin. Bioorg Chem 2005;33:108e15. [8] Lee JM, Karim MM, Lee SH. Determination of catechin in aqueous solution by chemiluminescence method. J Fluoresc 2005;15:735e9. [9] Cabrera C, Artacho R, Gime´nez R. Beneficial effects of green teada review. J Am Coll Nutr 2006;25:79e99. [10] Pierre M, Onge ST. Dietary fats, teas, dairy, and nuts: potential functional foods for weight control? Am J Clin Nutr 2005;81:7e15. [11] Guo S, Yang S, Taylor C, Sonenshein GE. Green tea polyphenol epigallocatechin-3 gallate (EGCG) affects gene expression of breast cancer cells transformed by the carcinogen 7,12-dimethylbenz[a]anthracene1e3. J Nutr 2005;135:2978se86. [12] Kim J, Zhang X, Rieger-Christ KM, Summerhayes IC, Wazer DE, Paulson KE, et al. Suppression of Wnt signaling by the green tea compound ()-epigallocatechin 3-gallate (EGCG) in invasive breast cancer cells: requirement of the transcriptional repressor HBP1. J Biol Chem 2006;281:10865e75. [13] Ahmed S, Wang N, Lalonde M, Goldberg VM, Haqqi TM. Green tea polyphenol epigallocatechin-3-gallate (EGCG) differentially inhibits interleukin-1 b-induced expression of matrix metalloproteinase-1 and -13 in human chondrocytes. J Pharmacol Exp Ther 2004;308:767e73. [14] Lee YK, Bone ND, Strege AK, Shanafelt TD, Jelinek DF, Kay NE. VEGF receptor phosphorylation status and apoptosis is modulated by a green tea component, epigallocatechin-3-gallate (EGCG), in B-cell chronic lymphocytic leukemia. Blood 2004;104:788e94. [15] Hwang JT, Park IJ, Shin JI, Lee YK, Lee SK, Baik HW, et al. Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase. Biochem Biophys Res Commun 2005;338:694e9.
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