Article pubs.acs.org/jnp
Inhibitory Effect of the Gallotannin Corilagin on Dextran Sulfate Sodium-Induced Murine Ulcerative Colitis Hai-Tao Xiao,† Cheng-Yuan Lin,† Derek H. H. Ho,‡,§ Jiao Peng,‡ Yan Chen,‡,§ Siu-Wai Tsang,† Michael Wong,† Xiao-Jun Zhang,† Man Zhang,† and Zhao-Xiang Bian*,† †
School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, People's Republic of China Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Island, Hong Kong, People's Republic of China § Department of Chemistry, The University of Hong Kong, Hong Kong, People's Republic of China ‡
ABSTRACT: The therapeutic effect of corilagin (1) was evaluated in an acute colitis model induced by dextran sulfate sodium (DSS) in mice, and the mechanism of action was investigated in this study. Animals were challenged with 2% DSS drinking water for 5 consecutive days and then intraperitoneally treated with 1 (7.5, 15, and 30 mg/kg) daily for 7 days. It was found that 1 significantly decreased the disease activity index, inhibited the shortening of colon length, reduced colon tissue damage, and suppressed myeloperoxidase activity. Moreover, 1 greatly suppressed the secretion of TNFα, IL-6, and IL-1β, inhibited the degradation of IκB α, and down-regulated expression of cleaved caspase-3 and cleaved caspase-9 in colon tissues of DSS-treated mice. These findings demonstrated that 1 exerts a protective effect on DSS-induced colitis, and its underlying mechanisms are associated with inhibition of the NF-κB pathway that mitigates colon inflammatory responses and apoptosis of intestinal epithelial cells.
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and acute cholestasis.8−10 One study using RAW 264.7 cells led to the observation that 1 can reduce the production of proinflammatory mediators such as TNF-α, IL-1β, and IL-6 significantly via NF-κB pathway suppression.11 Accordingly, these results suggest that 1 may be an immunomodulator in the treatment of inflammatory disease. As the therapeutic potential of 1 against ulcerative colitis has not yet been reported, the present study was aimed to evaluate the therapeutic effect of 1 and its underlying mechanisms on DSS-induced colitis in mice.
lcerative colitis, together with Crohn’s disease, is a primary component of inflammatory bowel disease (IBD), which is a recurrent and progressive inflammatory intestinal disorder characterized by colon tissue edema, increased colon epithelial permeability, and extensive infiltration of leukocytes in the colon.1 The last decades have witnessed great advances in ulcerative colitis management, but its pathogenesis is poorly understood. It is generally agreed that this condition is a multifactorial disease initiated by the accumulation of genetic, environmental, and immunological factors.2 Current medical therapies for ulcerative colitis via inducing remission and preventing relapse mainly involve administration of aminosalicylic acid, glucocorticosteroids, immunosuppressants, and antibiotics. However, these therapies are not well tolerated due to their side effects and show a high relapse rate;3,4 therefore the development of new anticolitis agents is needed. Corilagin (β-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-Dglucose, 1), a major gallotannin contained in many medicinal plants, exerts versatile biological functions such as antioxidant, hepatoprotective, antitumor, antihypertensive, antihyperglycemic, anti-inflammatory, antiatherogenic, and antimicrobial activities.5−7 Recently, 1 has gained much interest because of its potential to modulate major inflammatory pathways. It was reported that compound 1 can mitigate inflammation significantly via suppressing nuclear factor-κB (NF-κB) activation and the downstream transcription in several inflammatory diseases, including brain injury, cystic fibrosis, © 2013 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION It is well known that DSS-induced colitis is a well-established animal model with several characteristics resembling human Received: August 20, 2013 Published: November 7, 2013 2120
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Figure 1. Effects of corilagin (1) on body weight change (A), disease activity index (B), and colon length (C and D) of animals with DSS-induced colitis. Colitis was induced in all groups except the control group. Compound 1 and infliximab were administered to mice from day 6 to day 12. The change in body weight was taken as the difference between the body weight before induction of colitis and that immediately before sacrifice on day 13. The disease activity index (DAI) was determined by combining scores of (i) body weight loss, (ii) stool consistency, and (iii) stool bleeding. On day 13, the mice were sacrificed, and the colon lengths were measured. Data are expressed as means ± SEM, n = 8 (###p < 0.001, compared with the control group; *p < 0.05, **p < 0.01, and ***p < 0.001, compared with DSS model group).
inflammatory cells such as neutrophils, monocytes, and lymphocytes into the colon mucosa is an important pathological feature of inflammatory bowel disease. Previous studies have shown that neutrophil infiltration into inflamed tissue can facilitate the formation of potent cytotoxic oxidants to induce colon tissue damage through the enzyme myeloperoxidase (MPO).14,15 The present study showed that 1 suppressed MPO activity significantly in the colon of DSSinduced colitic mice (Figure 2C). Collectively, the present results indicate clearly that 1 exerts a potent therapeutic effect against DSS-induced colitis. Although the etiology of ulcerative colitis is still unknown, it is well accepted that imbalances between pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, and antiinflammatory cytokines, such as IL-4, IL-5, and IL-10, are involved in the regulation of inflammatory status.16−18 In the present study, DSS treatment caused a sharp increase of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the colon of mice, which is consistent with the similar observation in ulcerative colitis patients.17,18 Interestingly, 1 greatly reduced the production of TNF-α, IL-1β, and IL-6 in a dose-dependent manner (Figure 3), whereas no significant effect on the production of IL-10 was observed. Previous studies have demonstrated that pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 are regulated transcriptionally by NF-κB,19,20 which is a protein complex controlling the transcription of inflammatory cytokines. At resting status, inactive NF-κB complexes are sequestered in the cytoplasm by binding to its inhibitory subunit, IκB α. Upon stimulation, IκB α is rapidly
ulcerative colitis, such as weight loss, severe diarrhea, rectal bleeding, superficial ulceration, and mucosal damage,12,13 and therefore can serve as a valid model to investigate the pathogenesis of ulcerative colitis and screen for potential therapeutic interventions. In this study, mice subjected to DSS drinking water developed typical symptoms of clinical colitis, including loss of body weight, diarrhea, and rectal bleeding. As shown in Figure 1A, compared with the control group, the body weights of the DSS-treated mice decreased significantly on day 5 and the weight loss began to recover from day 9. Mice in groups treated with 1 showed an upshift recovery from day 8, and 1 at 30 mg/kg recovered the body weight loss significantly compared with that of the DSS model group after day 9 (p < 0.05). As shown in Figure 1B, in terms of the disease activity index, mice after DSS treatment showed high scores. Compound 1 at doses of 15 and 30 mg/kg decreased the disease activity index significantly, and similar results were observed in mice administrated with a positive control agent, infliximab (5 mg/kg) (p < 0.05, p < 0.01, and p < 0.001). In addition, as another important symptomatic parameter in DSSinduced colitis, shortening of the colon length, was rectified after administration with 1 at indicated concentration levels in a dose-dependent manner (p < 0.05), as shown in Figure 1C and D. In particular, 1 at a dose of 30 mg/kg exerted a comparable inhibitory effect to that of infliximab (5 mg/kg). Further, histological observation showed that 1 attenuated colon tissue damages, such as crypt destruction, lesions, and inflammatory cell infiltration, significantly in DSS-treated mice, leading to lower histological scores (Figure 2A and B). Infiltration of 2121
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Figure 2. Effects of corilagin (1) on histopathological changes and myeloperoxidase (MPO) activity in the colon of animals with DSS-induced colitis. (A) Representative images of hematoxylin/eosin (H&E) staining (a, control group; b, DSS model group; c, DSS plus infliximab group; d, DSS plus 1 7.5 mg/kg group; e, DSS plus 1 15 mg/kg group; f, DSS plus 1 30 mg/kg group; magnification, 10×). (B) Histological scores. (C) MPO activity. Colitis was induced in all groups except the control group. Compound 1 and infliximab were administered to mice from day 6 to day 12. On day 13, the mice were sacrificed, and colon tissue damage was evaluated by histopathological analysis (H&E staining). Also, MPO activity was determined in colon homogenates. Data are expressed as means ± SEM, n = 8 (###p < 0.001, compared with the control group; *p < 0.05, **p < 0.01, and ***p < 0.001, compared with DSS model group).
caspase-9, and suppress Bcl-2 expression in the colon of DSStreated mice (Figure 5). These observations are compatible with previous studies showing the induction of intestinal epithelia cell apoptosis in DSS-induced colitis.20,24 After treatment with 1, crypt destruction was attenuated observably, and the expression levels of cleaved caspase-3 and cleaved caspase-9 were reduced significantly in the colon of DSStreated mice (Figure 2A and 5), suggesting a beneficial effect of 1 in inhibiting apoptosis of intestinal epithelial cells. In summary, the present study indicates clearly that corilagin (1) is effective in alleviating the symptoms of DSS-induced colitis in mice. This beneficial effect may be caused by suppression of inflammatory responses of the colon and intestinal epithelial cell apoptosis via inhibition of NF-κB activation. It can be concluded that corilagin (1) is a promising candidate in ulcerative colitis treatment.
phosphorylated by IκB kinase, is ubiquitinated, and proceeds to proteosome-mediated degradation, therefore allowing the translocation of NF-κB to the nucleus, where it activates the transcription of inflammatory cytokine genes via binding with specific sequences located in the promoter region of corresponding genes.19,21 As shown in Figure 4, compound 1 at the doses of 15 and 30 mg/kg significantly inhibited the degradation of IκB α and, therefore, at least partially contributed to the observed reduction of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 secreted in the colon of DSStreated mice. Growing evidence has demonstrated that NF-κB activation can initiate a number of cascade reactions to induce inflammatory cytokines and trigger the activation of caspases to promote cell death, resulting in mucosal tissue damage.19,22 In ulcerative colitis, induction of apoptosis mainly contributes to loss of epithelial cells in the colon. Evidence from biopsies showed that apoptosis occurs only in the shedding of the normal colon, while the number of apoptotic bodies is substantially increased in the colon of inflammatory bowel disease patients, and the degree of lesions is related closely to the occurrence of apoptotic and necrotic bodies.23 The present results showed that DSS treatment can cause crypt destruction (Figure 2A), induce expression of cleaved caspase-3 and cleaved
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EXPERIMENTAL SECTION
General Experimental Procedures. Corilagin (1) was provided by the Natural Product Chemistry and Phytochemical Analysis Laboratory, School of Chinese Medicine, Hong Kong Baptist University (HPLC purity >98%). Infliximab (anti-TNF-α drug) was purchased from Mitsubishi Tanabe Pharma Corp. (Osaka, Japan). Dextran sulfate sodium (DSS; molecular weight: 36000 to 50000) was 2122
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Figure 3. Effects of corilagin (1) on the production of cytokines in colon tissue of animals with DSS-induced colitis. (A) TNF-α levels. (B) IL-1β levels. (C) IL-6 levels. (D) IL-10 levels. Colitis was induced in all groups except the control group. Compound 1 and infliximab were administered to mice from day 6 to day 12. On day 13, the mice were sacrificed, and the contents of cytokines were determined in colon homogenates. Data are expressed as means ± SEM, n = 8 (##p < 0.01, ###p < 0.001, compared with the control group; **p < 0.01 and ***p < 0.001, compared with DSS model group).
Figure 4. Effect of corilagin (1) on the degradation of IκB α in the colon of animals with DSS-induced colitis. (A) Representative image of Western blot. (B) Protein expression of IκB α. Colitis was induced in all groups except the control group. Compound 1 and infliximab were administered to mice from day 6 to day 12. On day 13, the mice were sacrificed, and protein expression of IκB α was determined in colon homogenates using Western blot. Data are expressed as means ± SEM, n = 5 (#p < 0.05, compared with the control group; *p < 0.05, compared with DSS model group). purchased from MP Biomedicals (Santa Ana, CA, USA). Mouse TNFα, IL-1β, IL-6, and IL-10 kits were obtained from eBioscience (San Diego, CA, USA). IκB α, cleaved caspase-3, cleaved caspase-9, and Bcl2 rabbit antibodies were supplied by Cell Signaling Technology (Beverly, MA, USA), while β-actin mouse antibody, anti-rabbit IgG, and anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Hexadecyltrimethylammonium bromide, hematoxylin, eosin, o-dianisidine dihydrochloride, protease inhibitor cocktails, and hydrogen peroxide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). ECL Chemiluminescent Substrate Reagent Kit was supplied by Life Technologies Corporation (Carlsbad, CA, USA). Animals. Seven- to eight-week-old male C57BL/6 mice weighing about 20−24 g were purchased from the Laboratory Animal Services
Center, The Chinese University of Hong Kong. The animals were fed with a standard rodent diet with free access to drinking water and kept in rooms maintained at 22 ± 1 °C with a 12 h light/dark cycle following international recommendations. All experimental protocols (protocol nos. FRG1011-027 and IRMC1112-02) were approved by The Animal Ethics Committees of Hong Kong Baptist University, in accordance with “Institutional Guidelines and Animal Ordinance” (Department of Health, Hong Kong Special Administrative Region). Induction of Colitis and Treatment. Experimental colitis was induced by giving mice a DSS drinking solution ad libitum for 5 consecutive days, as previously described.25 Briefly, DSS was dissolved in distilled water at a concentration of 2.0% (w/v) and administered to the mice. The first day and the last day of DSS treatment were designated as day 0 and day 5, respectively. Mice were marked and 2123
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Figure 5. Effect of coriligin (1) on protein expression of apoptotic markers in the colon of animals with DSS-induced colitis. (A) Representative images of Western blot. (B) Protein expression of cleaved caspase-3. (C) Protein expression of cleaved caspase-9. (D) Protein expression of Bcl-2. Colitis was induced in all groups except the control group. Compound 1 and infliximab were administered to mice from day 6 to day 12. On day 13, the mice were sacrificed, and protein expression of cleaved caspase-3, cleaved caspase-9, and Bcl-2 were determined in colon homogenates using Western blot. Data are expressed as means ± SEM, n = 5 (#p < 0.05, ##p < 0.01, and ###p < 0.001, compared with the control group; *p < 0.05 and **p < 0.01, compared with DSS model group). checked daily for body weight. On day 6, body weight, stool consistency, and gross bleeding of each DSS-treated mouse were assessed, and animals showing body weight loss, diarrhea, and bleeding were selected for the investigation. All colitic mice were then randomly divided into five groups with different interventions once daily for seven days, with eight mice in each group. The DSS model group was administered with saline as negative control, while infliximab was given as positive control (ip, 5 mg/kg) and corilagin (1) treatment groups were given different doses of 1 (ip, 7.5, 15, or 30 mg/kg, respectively). In parallel, a vehicle control group was also set up to receive drinking water without DSS throughout the entire experimental period. Evaluation of Disease Activity Index (DAI). The DAI was determined by scoring changes in body weight, stool consistency, and bleeding, in accordance with a previously reported method.12,26 In brief, body weight, stool consistency, and bleeding in the stool were monitored daily for a determination of DAI. Each score was given as follows: body weight loss (0, none; 1, 1−5%; 2, 5−10%; 3, 11−15%; 4, >15%), diarrhea (0, normal; 2, loose stools; 4, watery diarrhea), and bleeding (0, normal; 2, slight bleeding; 4, gross bleeding). The DAI score ranged from 0 to 12 (total score). At the end of the experiment, mice were killed, the colon was dissected from each mouse, and the length was measured from the ileo-cecal junction to the anal verge. Assessment of Histological Score. Mouse colons were opened longitudinally, gently washed with ice-cold PBS, fixed in 4% paraformaldehyde overnight, and embedded in paraffin. Five-micrometer sections were stained with hematoxylin/eosin according to a standard procedure to evaluate colonic damage. The histological scoring system was used as described previously.27 Briefly, each colon was scored considering (1) the severity of inflammation (0, none; 1, mild; 2, moderate; 3, severe); (2) the extent of inflammation (0, none; 1, mucosa; 2, mucosa and submucosa; 3, transmural); and (3) crypt damage (0, none; 1, 1/3 damaged; 2, 2/3 damaged; 3, crypt loss but surface epithelium present; 4, both crypt and surface epithelium lost).
Scores were then added, resulting in a total histological score that ranged from 0 to 10. Myeloperoxidase Assay. Myeloperoxidase activity was measured as described in a previous study.28 In brief, the colon tissues were homogenized in 0.5% hexadecyltrimethylammonium bromide (1 mL per 100 mg of colon tissue). The homogenates were centrifuged at 19000g at 4 °C for 15 min. Aliquots of 80 μL of supernatant were mixed with 120 μL of potassium phosphate buffer (50 mmol, pH 6.0) with 0.0005% o-dianisidine dihydrochloride and 0.1% hydrogen peroxide. Changes in optical density were measured at 460 nm at room temperature (25 °C). MPO activity was calculated from the rate of optical density changes, and one unit of MPO activity was defined as the amount of enzyme present that produced a change in optical density of 1.0 U/min at 25 °C in the final reaction volume. The results were normalized to the wet weight of colon tissue and quantified as units/g tissue. Measurement of Cytokines. Colon levels of cytokines were assayed using commercially available ELISA kits. Briefly, colon samples were homogenized in phosphate buffer containing 0.05% Tween-20, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 IU aprotinin A. The homogenates were centrifuged at 16000g at 4 °C for 15 min, and the supernatants were collected for the determination of levels of the cytokines, TNF-α, IL-1β, IL-6, and IL-10, according to the manufacturer’s protocols. The amount of protein in each sample was measured by the Bradford method,29 using bovine serum albumin as a standard. The levels of each cytokine were evaluated in each sample and expressed in pg/mg. Western Blot Analysis. The colon tissues were homogenized in RIPA buffer [1% Triton, 0.1% SDS, 0.5% deoxycholate, 1 mM EDTA, 20 mM Tris (pH 7.4), 150 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 0.1 mM phenylmethylsulfonyl fluoride], and 0.1 g of each colon tissue was homogenized in 1 mL of ice-cold RIPA buffer. After homogenization and centrifugation (15000g at 4 °C for 15 min), the supernatant was collected in aliquots and stored at −80 °C for 2124
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(13) Moodley, K.; Mackraj, I.; Naidoo, Y. J. Ethnopharmacol. 2013, 146, 225−231. (14) Boussetta, T.; Raad, H.; Letteron, P.; Gougerot-Pocidalo, M. A.; Marie, J. C.; Driss, F.; El-Benna, J. PLoS One 2009, 4, e6458. (15) Yang, X. L.; Guo, T. K.; Wang, Y. H.; Huang, Y. H.; Liu, X.; Wang, X. X.; Li, W.; Zhao, X.; Wang, L. P.; Yan, S.; Wu, D.; Wu, Y. J. Int. Immunopharmacol. 2012, 12, 408−414. (16) Muzes, G.; Molnar, B.; Tulassay, Z.; Sipos, F. World J. Gastroenterol. 2012, 18, 5848−5861. (17) Neuman, M. G. Rom. J. Gastroenterol. 2004, 13, 309−316. (18) Papadakis, K. A.; Targan, S. R. Annu. Rev. Med. 2000, 51, 289− 298. (19) Lawrence, T. Cold Spring Harb. Perspect. Biol. 2009, 1, a001651. (20) Liu, X.; Wang, J. M. PLoS One 2011, 6, e24740. (21) Yamamoto, Y.; Gaynor, R. B. Trends Biochem. Sci. 2004, 29, 72− 79. (22) Lee, J. Y.; Kim, J. S.; Kim, J. M.; Kim, N.; Jung, H. C.; Song, I. S. Int. Immunopharmacol. 2007, 7, 241−248. (23) Le’Negrate, G.; Selva, E.; Auberger, P.; Rossi, B.; Hofman, P. J. Cell. Biol. 2000, 150, 1479−1488. (24) Araki, Y.; Mukaisyo, K.; Sugihara, H.; Fujiyama, Y.; Hattori, T. Oncol. Rep. 2010, 24, 869−874. (25) Wirtz, S.; Neufert, C.; Weigmann, B.; Neurath, M. F. Nat. Protoc. 2007, 2, 541−546. (26) Han, E. S.; Oh, J. Y.; Park, H. J. J. Ethnopharmacol. 2011, 134, 703−710. (27) Andujar, I.; Recio, M. C.; Giner, R. M.; Cienfuegos-Jovellanos, E.; Laghi, S.; Muguerza, B.; Rios, J. L. J. Agric. Food Chem. 2011, 59, 6474−6483. (28) Qin, H. Y.; Xiao, H. T.; Wu, J. C.; Berman, B. M.; Sung, J. J.; Bian, Z. X. World J. Gastroenterol. 2012, 18, 2481−2492. (29) Bradford, M. M. Anal. Biochem. 1976, 72, 248−254.
Western blot analysis. Protein concentrations were quantified by the Bradford method.29 For Western blot analysis, samples were diluted to a concentration of 2 mg/mL in SDS loading buffer and boiled for 5 min. Fifty micrograms of protein from each sample was separated by 10−12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 0.5% bovine serum albumin in TBST buffer (50 mM Tris, 150 mM NaCl, and 0.05% Tween-20; pH 8.0) for 1.5 h and then incubated overnight at 4 °C with suitably diluted primary antibodies. The expression of β-actin was used to quantify the identical loading amount of protein. After extensive washing with TBST (3 × 10 min), the membranes were incubated in horseradish peroxidase-linked antibiotin and the appropriate secondary antibody in TBST with 0.5% bovine serum albumin for 1 h at room temperature and then rewashed (TBST, 3 × 10 min). The blots were detected using the enhanced chemiluminescence (ECL) reaction. Quantification of protein bands was achieved by densitometric analysis using Image-Pro Plus software (Media Cybernetics, Inc., Silver Spring, MD, USA). Statistical Analysis. The data were evaluated as mean values ± standard deviations. Statistical significances were evaluated using oneway ANOVA, followed by Duncan’s multiple range tests. GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA) was used for all calculations, and p < 0.05 was considered as statistically significant.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86-852-34112905. Fax: +86-852-34112929. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Faculty Research Grants from Hong Kong Baptist University (Project Nos. FRG 2/10-11/027 and IRMC/11-12/02).
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REFERENCES
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