A Chinese medicinal formulation ameliorates dextran sulfate sodium-induced experimental colitis by suppressing the activity of nuclear factor-kappaB signaling.

Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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A Chinese medicinal formulation ameliorates dextran sulfate sodium-induced experimental colitis by suppressing the activity of nuclear factor-kappaB signaling Siu Wai Tsang a, Siu Po Ip b, Justin Che-Yuen Wu c, Siew-Chien Ng c, Ken Kin-Lam Yung d, Zhao-Xiang Bian a,n a

School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China d Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China b c

art ic l e i nf o

a b s t r a c t

Article history: Received 10 April 2014 Received in revised form 1 October 2014 Accepted 22 December 2014

Ethnopharmacological relevance: Inflammatory bowel disease (IBD) is generally associated with a set of debilitating symptoms including abdominal pain, tenesmus, diarrhea and bloody stool. The standard approaches for treating IBD, which are the application of pharmaceuticals, are often unsatisfactory. IBD patients may suffer from repeated relapses and even exacerbation after taking these medications. Thus, patients are increasingly seeking relief through the use of complementary and alternative medicines. Aim of study: To provide scientific ground for the mode of actions of a Chinese medicinal formulation— modified ZenWu Decoction (MZWD) in ulcerative colitis. Materials and methods: C57BL6 mice were fed with 3 cycles of 2% dextran sulfate sodium (DSS) in drinking water for the induction of chronic colitis and then given MZWD at 17.47 g/kg/day. Effects of MZWD were evaluated by histopathological and biochemical assays. Results: When MZWD was given, inflammatory responses namely immune-cell infiltration, elevated serum levels of pro-inflammatory cytokines and mucosal lesions were notably suppressed. Further, MZWD treatment attenuated the activation of nuclear factor-kappaB (NF-κB), the vital regulator of inflammatory cascades, while lessening the degradation of I-kappaB-alpha and reducing the activity of protease-activated receptor 2 in DSS-induced colonic tissues. Consequently, diarrhea, bloody stool and colon shortening were reduced whilst mucosal integrity was improved in MZWD-treated colitis mice. Conclusions: Our findings suggest that MZWD is a potential remedy for treating IBD, and the mechanism of its efficacy is an anti-inflammatory effect associated with the suppression of the NF-κB pathway. & 2015 Published by Elsevier Ireland Ltd.

Keywords: Inflammatory bowel disease (IBD) Ulcerative colitis (UC) Diarrhea Bloody stool Dextran sulfate sodium (DSS) Nuclear factor-kappaB (NF-κB)

1. Introduction Inflammatory bowel disease (IBD), commonly refers to ulcerative colitis (UC) and Crohn's disease (CD), is ordinarily characterized by a set of complicated chronic inflammatory conditions of the intestines. In general, UC is solely restricted to the large

Abbreviations: DSS, dextran sulfate sodium; IL-1β, interleukin-1beta; IFN-γ, interferon-gamma; IκB-α, I-kappaB-alpha; MZWD, modified ZenWu Decoction; NF-κB, nuclear factor-kappaB; PAR2, protease-activated receptor 2; TNF-α, tumor necrosis factor-alpha n Correspondence to: School of Chinese Medicine, Hong Kong Baptist University, 3/F, SCM Building, 7 Baptist University Road, Kowloon Tong, Kowloon, Hong Kong SAR, China. Tel.: þ 852 3411 2905; fax: þ852 3411 2929. E-mail address: [email protected] (Z.-X. Bian).

intestine whereas CD may affect the whole gastrointestinal tract, literally from mouth to anus. IBD causes debilitating symptoms including abdominal pain, tenesmus, diarrhea and bloody stools; therefore, the quality of life of IBD sufferers is noticeably disturbed to varying degrees (Papadakis and Targan, 2000). According to recent reports, the incidence of IBD has been significantly increasing over the past two decades (Lakatos, 2006). However, the etiology of IBD is yet a mystery. Environmental, hereditary, infectious and autoimmune factors are considered as the plausible and intercalating causes (Xavier and Podolsky, 2007). Anti-inflammatory drugs, immune system suppressors and antibiotics are the conventional medications for the treatment of IBD (Stone et al., 2003). Among the mainstream measures, biologic anti-tumor necrosis factor-alpha (TNF-α) remedies such as Infliximab and non-selective anti-inflammatory drug aminosalicylates

http://dx.doi.org/10.1016/j.jep.2014.12.035 0378-8741/& 2015 Published by Elsevier Ireland Ltd.

Please cite this article as: Tsang, S.W., et al., A Chinese medicinal formulation ameliorates dextran sulfate sodium-induced experimental colitis by suppressing the.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2014.12.035i

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S.W. Tsang et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

are regarded as the mainstay of drug treatment (Magro, 2010); however, the anti-TNF-α protocol is not satisfactory as nearly 50% of the UC patients who received it suffered from repeated relapses or even exacerbations (Lok et al., 2007). Concerns about the safety of injecting anti-TNF-α antibodies have been expressed; these antibodies are associated with an increased risk of malignancy in the long term (Hudesman et al., 2013), and shorter-term risks of psoriasis (Guerra et al., 2012) and sarcoidosis (Fok et al., 2012). The immunomodulator Cyclosporine has been shown to be beneficial in severe flare-ups, though side effects including hypertension, nephrotoxicity and electrolyte imbalance are often associated with it (Mocciaro et al., 2012). If medications fail, colectomy, i.e. removing part of or the entire colon, becomes the last resort. As suggested by a number of medical reports, IBD patients are at a higher risk of developing colon cancer (Dobbins, 1984). Due to the unsatisfactory therapeutics of Western medicine, patients, physicians and researchers are turning to complementary and alternative medicine approaches such as Traditional Chinese Medicine (TCM) for a relief and a more effective way for treating IBD (Podolsky, 2002). According to the Chinese medical theory, the stagnation of Qi and blood followed by combined dampness or cold-dampness in the stomach and intestine leads to the symptoms now defined as UC. The 5-herb formula ZenWu Decoction (ZWD) comprising Poria [sclerotium of Poria cocos (Schw.) Wolf, family: Polyporaceae], Paeoniae Radix Alba (root of Paeonia lactiflora Pall., family: Ranunculaceae), Macrocephalae Rhizoma (rhizome of Atractylodes macrocephala Koidz., family: Asteraceae), Zingiberis Rhizoma (rhizome of Zingiber officinale Rosc., family: Zingiberaceae) and Aconiti Lateralis Radix Praeparata (processed root of Aconitum carmichaelii Debx., family: Ranunculaceae) has been used as a folk medicine for the alleviation of chronic diarrhea for centuries. To this traditional formula, we replaced the relatively toxic component Aconiti Lateralis Radix Praeparata with Codonopsis Radix [root of Codonopsis pilosula (Franch.) Nannf., family: Campanulaceae] and Coptidis Rhizoma (rhizome of Coptis chinensis Franch., family: Ranunculaceae), and created modified ZenWu Decoction (MZWD). Unfortunately, no scientific evidence on the mechanism of the efficacy of both the original ZWD and MZWD has been provided. Therefore, we conducted this study to confirm the efficacy of MZWD in treating experimental IBD and to explore its underlying mechanism. At the molecular level, inflammatory responses such as UC principally involve the activation of the pivotal inflammatory regulator nuclear factor-kappaB (NF-κB). In mammals, NF-κB plays important roles in a number of cellular and organismal processes (Hayden and Ghosh, 2008). The nuclear translocation of NF-κB dimmers implicates the transactivation of its target genes encoding inflammatory cytokines and mediators in response to tissue injury, repair and inflammation (Bonizzi and Karin, 2004). In IBD conditions, innate immune responses in the intestine are initiated by macrophages and dendritic cells for fighting against the luminal antigens and commensal bacteria conquering the mucosa (Platt and Mowat, 2008). Upon the activation of the immune cells, proinflammatory cytokines namely TNF-α, interleukin-1beta (IL-1β) and interferon-gamma (IFN-γ) are rapidly and massively produced. Transcription of the TNF-α gene in the activated immune cells results in consequent secretion of TNF-α in a positive feedback manner (Braegger et al., 1992). It is believed that TNF-α prolongs inflammatory conditions by activating NF-κB-dependent pathways by which ulceration and mucosal destruction is accelerated. Protease-activated receptor 2 (PAR2) is a pro-inflammatory mediator that expressed predominantly in epithelial cells of the internal milieu such as in the gastrointestinal tract (Bohm et al., 1996). It also largely contributes to NF-κB-mediated inflammatory processes and primes the activation of intracellular signaling

networks since PAR2 is the common target of various proteolytic enzymes including beta-tryptase (β-tryptase), thrombin and matrix metalloproteases (Cenac et al., 2002). In firing proinflammatory responses, the nuclear translocation of NF-κB is undoubtedly the most pivotal initiative process. Increased activities of NF-κB are often observed in experimental colitis as well as in UC patients (Andresen et al., 2005; Dong et al., 2010). Therefore, agents that suppress the activation of this transcription factor have the potential for therapeutic intervention. Among the several experimental animal models used for the investigation of IBD, oral administration of dextran sulfate sodium (DSS) has been widely acknowledged for accurately mimicking the UC situation in humans (Jurjus et al., 2004; Kim et al., 2012). In the current study, we examined the effectiveness of our modified 6-herb formula, MZWD, on DSS-induced colonic inflammation in mice by means of various histopathological and biochemical assays. Our results demonstrated that administration of MZWD significantly reduced the infiltration of immune cells and the secretion of pro-inflammatory cytokines. Most importantly, MZWD treatment suppressed the activation of NF-κB, the degradation of its cytosolic inhibitory complex and the activity of PAR2. As a result, the severity of colitis in the DSS-treated mice was reduced. Our findings not only elucidate the in vivo antiinflammatory effects of MZWD, but also partially reveal the underlying mechanism of the pathogenesis of IBD. We suggest that MZWD is potentially an effective therapeutic approach for the treatment of IBD.

2. Materials and methods 2.1. Preparation of modified ZenWu Decoction (MZWD) Top-grade herbal materials were obtained from GMP-certified pharmaceutical companies or certified suppliers. The herbal materials were authenticated by Ms Yu-ying Zong, School of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China. The site of origin and the name and address of the vendor for each acquired herbal material were documented. Individual herbs were assigned with unique voucher numbers for tracking, management and documentation purposes. Voucher samples (MZWD-01 to MZWD-06) have been deposited at the herbarium of the School of Chinese Medicine, The Chinese University of Hong Kong. Herbs were certified free of heavy metals, toxic elements and pesticide residues and within microbial limits. MZWD extracts were prepared by grinding and mixing of the 6 component dried raw herbs (Poria, Paeoniae Radix Alba, Macrocephalae Rhizoma, Zingiberis Rhizoma, Codonopsis Radix and Coptidis Rhizoma) in proportions as shown in Table 1. Herbs were decocted with boiling water at 100 g/L for 60 min twice. Decoctions were then filtered and pooled. For the purpose of oral feeding to mice in a smaller volume, the pooled decoction was further concentrated and freeze-dried. For quality control purposes, extracts were sonicated in methanol and subjected to high Table 1 Composition of herbs in the formulation MZWD on a dry weight basis. Chinese herbs

Alias

Composition (%)

Codonopsis Radix Macrocephalae Rhizoma Poria Zingiberis Rhizoma Paeoniae Radix Alba Coptidis Rhizoma

Dangshen Bai Zhu Fu Ling Gan Jiang Bai Shao Huanglian

17.6 17.6 35.4 11.8 11.8 5.9


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performance liquid chromatography (HPLC) analysis using a C18 column for detection at wavelengths 245 nm, 280 nm and 330 nm. 2.2. Animals C57BL6J mice 7–8 weeks old weighing 21–24 grams were purchased from The Chinese University of Hong Kong (Shatin, Hong Kong SAR, China). They were maintained in standard cages in a humidity-controlled room with an ambient temperature of 23 72 1C and 12-h light/dark cycle. The handling of mice and all experimental procedures performed had been obtained from the Committee on the Use of Human and Animal Subjects in Teaching and Research (HASC) of Hong Kong Baptist University (HASC registration# RC-IRMS/11-12/01) and were in accordance with the Animals Ordinance, Department of Health, Hong Kong SAR, China. 2.3. Induction of colitis Mice were randomly divided into 3 groups (n ¼ 9/group). Experimental colitis was induced by giving mice drinking water ad libitum containing 2% DSS in a period of 44 days. Throughout the experiment, the volumes of water or DSS-containing water consumed by the 3 different groups of mice were approximately equal. Group 1: saline treatment control (designated CON); Group

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2: 2% DSS treatment (designated DSS); Group 3: 2% DSS treatment plus oral gavage (o.g.) of MZWD (designated DSS þMZWD). Schematic timeline and grouping of our experimental design are shown in Fig. 1(a). For the induction of chronic colitis, mice in DSS group were subjected to 3 cycles of 2% DSS treatment during the 44-day experimental period and given o.g. of saline while mice in the CON group were fed with drinking water without DSS and orally administered with saline. The DSS þMZWD group received 3 cycles of DSS treatments along with oral administration of MZWD at the dose of 17.47 g raw herbs/kg/day, which is equivalent to the typical clinical dosage (1.42 g raw herbs/kg/day) for humans. At the time of sacrifice, blood was collected for the measurement of inflammatory cytokines, followed by the rapid removal of colonic tissues. 2.4. Macroscopic evaluation of severity of colitis The severity of colitis in mice was macroscopically assessed based on the loss of body weight, the scoring of stool consistency and the presence of fecal occult blood as reported previously (Islam et al., 2008). Scoring of stool consistency was determined as follows: 0: no diarrhea; 1: mild; 2: moderate; 3: severe and bloody diarrhea. Fecal occult blood in animal samples was detected utilizing the Hemoccult SENSA kits (Beckman Coulter, USA) according to the manufacturer's instruction. Scoring of fecal occult

Fig. 1. (a) Schematic chart showing the timeline of experimental design and grouping of animals. Mice were randomly divided into 3 groups (n¼ 9). Group 1 was kept as control and received drinking water without DSS throughout the entire experimental period. Group 2 served as the chronic colitis model and was induced by 3 cycles of 2% DSS treatment with 2 recovery periods in between. To examine the therapeutic effect of MZWD, group 3 was given MZWD o.g. starting from day 13 till the end of the experiment in addition to the same treatment as group 2 received. (b) Chromatographic fingerprints of MZWD at 3 wavelengths (245 nm, 280 nm and 330 nm). Peak 1 was identified as paeoniforin, peak 2 as palmatine and peak 3 as berberine.


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blood was set as follows: 0: negative; 1: very mild; 2: mild; 3: moderate; 4: severe. Disease activity index (DAI) was calculated by combining the scores of stool consistency and fecal occult blood. Colonic shortening was determined by measuring the length of the entire colon and the length between colo-cecal junction and anal verge. 2.5. Histological examination of colon After mice were sacrificed, distal colons were harvested, rinsed and fixed in 4% paraformaldehyde at 4 1C overnight. Colonic tissues were then processed with sequential clearing and dehydrating steps, and embedded in paraffin blocks. Samples were sectioned into 6 μm slices and subjected to standard Hematoxylin and Eosin (H&E) staining for the evaluation of colonic architecture, loss of crypts, extent of injury and mucosal damage and lymphocyte infiltration. Mucosal height was measured as the perpendicular distance between the membrane surface and the muscularis mucosae. Crypt length was measured as the distance between the basement membrane and the lowest point of the crypt. Number of crypts was counted in an area spanning 200 μm across the muscularis mucosae. Four regions were picked in each sample and 3 samples were selected from each experimental group for calculations. 2.6. Biochemical assessment of colonic injury Serum was obtained after centrifugation of the whole blood at 2000 g for 5 min at 4 1C. Colonic tissues (10% w/v) were homogenized in ice-cold potassium phosphate buffer containing protease inhibitor followed by 2 cycles of sonication. The colonic and serum levels of pro-inflammatory cytokines namely TNF-α, IL-1β, IL-10 and IFN-γ were measured using enzyme-linked immunosorbent assays (ELISAs, eBioscience) according to manufacturer's instructions. Myeloperoxidase (MPO) activity, an important indicator of lipid peroxidation, in colonic homogenate was measured according to our previous reported protocol (Qin et al., 2012). In brief, supernatant of colonic homogenate extracted with 0.3% hexadecyltrimenthyl ammonium bromide was reacted with 0.5 mM ο-dianisidine dihydrochloride and 0.05% hydrogen peroxide. MPO activity was calculated as the change in optical density at 460 nm over 2 min and expressed as mU/mg protein.

2.9. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay The in situ detection of cell apoptosis in colonic tissues was determined using the TUNEL apoptosis detection kit according to the manufacturer's instruction (Upstate Millipore). Three slides per sample block were evaluated. Micrographs were randomly selected and the numbers of normal and apoptotic cells were counted. The apoptosis index was expressed as a percentage of TUNEL-positive cells out of the total number of nucleated cells. 2.10. Statistical analysis Data were expressed as means 7standard derivation (SD) and analyzed by one-way ANOVA followed by Tukey's multiple comparison tests in order to detect inter-group differences. Significant difference from the saline control group was determined at 95% confident intervals (considered significant when a n p value ofo0.05).

3. Theory Our medicinal formulation MZWD ameliorates the severity of experimental ulcerative colitis in mice via attenuating the activities of NF-κB and PAR-2. Diarrhea, bloody stool, colon shortening and production of pro-inflammatory cytokines were reduced whilst mucosal integrity was improved in MZWD-treated colitis mice. Therefore, we suggested that MZWD is a potential remedy for treating inflammatory bowel disease in particular to ulcerative colitis.

4. Results 4.1. HPLC profiling and safety assessment of MZWD Three components with relatively high contents were found in our HPLC profiling of MZWD at wavelengths 245 nm, 280 nm and 330 nm. By comparison with reference standards, peak 1 was identified as paeoniforin, peak 2 as palmatine and peak 3 as berberine (Fig. 1(b)). We also conducted an acute toxicity test of MZWD on mice at a maximal oral dose of 338.28 g/kg (241 folds to clinical dose). No toxicological signs or any other negative effects were observed in the MZWD-treated animals during a test period of 2 weeks.

2.7. Immunohistochemistry (IHC) analysis Paraffin slides were de-paraffinized, exposed to anti-NF-κB p65 (Cell Signaling) or anti-I-kappaB-alpha (IκB-α, Santa-Cruz) or antiPAR2 (Santa-Cruz) antibodies for 16 h at 4 1C and probed with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody for 1 h at room temperature. Peroxidase activity was visualized by the addition of substrate-chromogen solution from the EnVision þ System-HRP (DAB) kit (DakoCytomation). Images were captured using Nikon microscope equipped with SPOT advanced software. 2.8. Western blotting analysis Colonic tissues were homogenized in ice-cold RIPA buffer containing protease inhibitor. Protein samples at 10 μg were separated by SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (Bio-rad). After blocking with 5% non-fat milk, membranes were incubated with a primary antibody followed by an appropriate secondary antibody conjugated with HRP. The positive immunoreactions were detected with x-ray film (Fuji) by chemiluminescence using an ECL kit (GE Healthcare).

4.2. MZWD decreased DAI scores in DSS-induced colitis in mice In C57BL6 mice, three cycles of 2% DSS administration were associated with significant clinical changes, including loss of body weight (Fig. 2(a)), appearance of diarrhea (Table 2a), and appearance of fecal occult blood (Table 2b). Treatment with MZWD at 17.47 g/kg/day o.g. during the 44-day experimental period suppressed the mentioned pathological conditions and resulted in a reduction of colitis in DSS-induced mice (Fig. 2(a), Tables 2a and 2b). Also, we evaluated the severity of colitis based on the DAI scores. The higher the DAI score, the more severe the colitis. The DAI scores of the DSS þMZWD group (4.0 70.82) were significantly lower than those of the DSS group (6.0 70.82). In contrast, the DAI score of the control group was nil (Fig. 2(b)). Macroscopically, ulcerative lesions were observed in colons of the DSS group, but not in the control or DSS þMZWD group (Fig. 2(c)). In addition, the severity of colitis was also implicated by the weight of colonic tissues and the colon lengths. The colons of the DSS group were significantly heavier as severe edema was found. Such edematous condition was reduced in the DSS þMZWD group (Fig. 2(d)). Colons were shortened by more than 10% under colitis


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Fig. 2. Evaluation of the severity of DSS-induced colitis in mice. (a) Change of body weight of each group was calculated as the percent difference between the original body weight (day1) and the body weight on any particular day during the experimental period. Values represent the averages of 6 animals. n Significantly different from CON (po 0.05); nn significantly different from CON (p o 0.001). (b) DAI scores of different groups of animals. n Significantly different from CON (p o 0.001); # significantly different from DSS (p o 0.05). (c) Images showing ulcerative lesions in colons of different groups of animals. (d) Weights of colons were measured at the time of sacrifice. n Significantly different among the 3 groups (p o 0.05). (e) Histology (H&E staining) of mucosal integrity in tissues of the CON, DSS and DSS þMZWD groups.

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Table 2a Evaluation of stool consistency after DSS induction and MZWD treatment. Stool Consistency

Day 6

Day 25

Day 44

CON DSS DSS þ MZWD

07 0 1.25 7 0.5n 1.25 7 0.5n

07 0 2.5 7 0.5774n 2.25 7 0.5n

07 0 2.75 7 0.5n 1.75 7 0.5n

Table 2d Therapeutic effects of MZWD on crypt length and number.

Crypt length No. of intact crypts

#

po 0.001 when comparing to control. p o0.05 when compare to DSS group.

#

Table 2b Evaluation of bloody stool after DSS induction and MZWD treatment. Day 6

Day 25

Day 44

CON DSS DSS þ MZWD

07 0 1.5 7 0.5774n 1.5 7 0.5774n

07 0 2.75 7 0.5n 2.25 7 0.5n

07 0 3.25 7 0.5n 2.25 7 0.5n

#

Scoring of hemoccult test: 0 ¼negative; 1¼ faint blue color (mild); 4¼ intense blue with wider area (most severe), the intensity of blue color correlates to the concentration of hemoglobin in feces. The scores of each group were the average of 6 animals. n

#

po 0.001 when comparing to control. p o0.05 when compare to DSS group.

Table 2c Therapeutic effects of MZWD on colon length. CON Entire colon

Mean (cm) 10.2 S.D. 0.5762 Colo–cecal junction to anal verge Mean (cm) 8.317 S.D. 0.4401

DSS þ MZWD

DSS n

8.825 0.4301 6.700nn 0.4243

DSS þ MZWD

167.7 μm 8.5

138.4 μmn 2.0n

149.6 μm# 5.5n #

Crypt length was measured as the distance between basement membrane and the lowest point of crypt. Number of crypt was counted in an area spanning 200 μm across the muscularis mucosae. Four regions were picked in each sample and 3 samples were selected from each group for the calculation of averages. n

Hemoccult

DSS

#

Scoring of stool consistency: 0¼no diarrhea; 1 ¼mild; 2¼ severe; 3¼ severe and bloody diarrhea. The scores of each group were the average of 6 animals. n

CON

p o 0.05 when comparing to control. po 0.05 when compare to DSS group.

mice, colonic levels of both TNF-α (3845 pg/mL71168) and IL-1β (748.2 pg/mL739.3) were elevated by nearly 40% when comparing to the control animals (TNF-α: 2147 pg/mL766.1; IL-1β: 384.87117.9); however, colonic levels of IL-6 or IL-10 were not significantly affected. With the application of MZWD, the elevated production of colonic TNF-α (2701 pg/mL7638.4) and IL-1β (630.4 pg/mL715.75) was suppressed by 25% (Fig. 3(a)–(e)). In the systemic circulation, serum level of TNF-α was elevated by approximately 40% after DSS induction. After MZWD was administered, the TNF-α elevation was restored notably to around the basal level (Fig. 3(f)), and the DSS-up-regulated serum level of IL-1β was also repressed (Fig. 3(g)). Further, colonic inflammation was quantitatively assayed by assessment of MPO activity. In the DSS group, colonic MPO activities were demonstrated with a roughly 5-fold increase when comparing to the non-DSStreated control group and such increase was found significantly attenuated in the DSSþ MZWD group (Fig. 3(h)).

4.5. MZWD inhibited activation of NF-κB in DSS-induced colitis

#

9.613 0.6770 7.225# 0.3615

Measurements of length of entire colon and colo-cecal portion. Values represent the mean 7 SD of each group (n¼ 6). n

po 0.05 when comparing to control. p o0.01 when comparing to control. # p o0.05 when compare to DSS group. nn

condition but were notably relieved by the treatment of MZWD (Table 2c). H&E stained images further revealed that DSS induction remarkably disrupted the colonic architecture, caused destruction of crypts and increased the thickness of the muscle layer, whereas MZWD treatment effectively corrected the architectural distortion (Fig. 2(e)). 4.3. MZWD restored crypt length and number in DSS-induced colitis in mice From the H&E stained colon sections, we found that the average crypt length of DSS group (138.4 μm) was nearly 20% shorter than that of control group (167.7 μm). The average crypt length of DSS þMZWD group was 149.6 μm. In a length of 200 μm, there were approximately 8.5 intact crypts in the control colons whereas only 2.0 intact crypts in colons of the DSS group. The number of intact crypts in colonic tissues of the MWZD treatment group was 5.5. Results are summarized in Table 2d. 4.4. Evaluation of anti-inflammatory effects of MZWD in DSS-induced colitis Production of pro-inflammatory cytokines was correlated to tissue injury and development of inflammatory events. In DSS-colitis

As NF-κB activity plays a critical role in inflammation, we examined the effect of MZWD on this pivotal transcription factor in the inflamed tissues. Immunohistochemical staining showed that expression of NF-κB p65 subunit in both epithelial cells and inflammatory cells of colonic tissues from DSS group was remarkably up-regulated when comparing to that of the CON group (Fig. 4). The activation of NF-κB is indicated by an up-regulation of its nuclear expression. When MZWD treatment was given, the expression of NF-κB p65 in colonic sections was notably suppressed, but still higher than that of control mice (Fig. 4(a)). In contrast, the expression of the inhibitory subunit of NF-κB, IκB-α was clearly lessened in colonic tissues of the DSS group. The stabilization of the IκB-α subunit in the DSSþMZWD group was comparable to that of the CON group (Fig. 4(b)). Furthermore, expression of PAR2, the key target of various proteolytic enzymes, was obviously elevated under colitis condition as shown in the immunohistochemical staining images. Such elevation of PAR2 was suppressed by the prolonged administration of MZWD (Fig. 4(c)). Similar suppressive effects of MZWD treatment on the expression of IκB-α and PAR2 proteins were shown in Western blotting images (Fig. 4(d)).

4.6. MZWD reduced apoptosis in colonic tissues of DSS-induced mice We analyzed the anti-apoptotic activity of MZWD in situ by TUNEL staining. As demonstrated in Fig. 5(a), the FITC signals representing fragmentation of DNA or apoptosis were substantially increased in colitis tissues of the DSS group but not in the CON group. Notably, the apoptotic signals were reduced in the colitis tissues of the DSS þMZWD group. The corresponding apoptosis indexes of the three experimental groups were provided in Fig. 5(b).


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Fig. 3. Biochemical assessment of the severity of DSS-induced colitis in mice. By means of ELISAs, levels of pro-inflammatory cytokines TNF-α (a), IL-1β (b) and IL-6 (c) and anti-inflammatory cytokines IL-10 (d) and IFN-γ (e) in colonic tissues and serum levels of pro-inflammatory cytokines TNF-α (f), IL-1β (g) were measured. n Significantly different among the 3 experimental groups (p o 0.05); nn significantly different among the 3 experimental groups (p o 0.001). (h) Colonic MPO activity was measured using a colorimetric approach and expressed as mOD/min/mg protein. n Significantly different among the 3 experimental groups (p o 0.01); nn significantly different among the 3 experimental groups (po 0.001).

5. Discussion To investigate the therapeutic effects of MZWD, we chose a mouse model of chemically induced colitis that mimics the UC

situation in humans. The model mice exhibited UC symptoms such as diarrhea, bloody stool, mucosal ulceration and shortened colons. We administered MZWD orally in DSS colitis mice and demonstrated that MZWD treatment increased body weight,


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Fig. 4. Histopathological assessment of the severity of colitis. Immunohistological images were probed with anti-NF-κB antibody (a), anti-IκB-α antibody (b) anti-PAR2 antibody (c), and visualized with chromogen-HRP. (d) By means of Western blotting analysis, the expression levels of IκB-α and PAR2 in colonic homogenates were examined.

improved stool consistency while reducing the incidence of bloody stools and colon shortening. In animal models of colitis, the production of pro-inflammatory cytokines namely TNF-α and IL1β is elicited after the activation of NF-κB in colonic tissue (Monteleone et al., 2002). Consistent with previous reports, the levels of pro-inflammatory cytokines—particularly TNF-α—in our DSS-induced mice were found positively correlated to the severity of colitis in the current study. Hence, the mainstay therapy for IBD patients is typically an anti-TNF-α protocol, for instance, injection of Infliximab (Baert et al., 1999; Baert et al., 2004). Our 6-herb formula, MZWD, significantly provided suppressive effects on both intrinsic and systemic TNF-α and IL-1β elevations under experimental colitis condition. A large number of experimental and clinical data suggests that chronic intestinal inflammation may be the result of a sustained overproduction of pro-inflammatory cytokines (Pahl, 1999; Papadakis and Targan, 2000). As the

progression of pro-inflammatory cascade was suspended, the severity of DSS colitis was alleviated in the DSS þMZWD mice. NF-κB activation is believed to be a critical step in the propagation of pro-inflammatory responses. Previous findings have shown that nuclear NF-κB protein expression was remarkably elevated in colonic tissues of rats with colitis (Dong et al., 2003; Andresen et al., 2005). In most cells, NF-κB/Rel transcription factors are predominantly associated with the inhibitory subunit IκB-α sequestered in the cytoplasm, and hence this cytosolic IκB/ NF-κB complex actually illustrates the inactivation of NF-κB (Baldwin, 1996). The dissociation of this IκB/NF-κB inactive complex exposes the nuclear translocation sequence of the Rel homology domain on NF-κB and triggers the transactivation of NF-κB target genes. In the present study, we demonstrated that NF-κB activation was increased along with an elevation of cytoplasmic IκB-α degradation in mice with DSS-induced colitis. In the


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Fig. 5. (a) In situ detection of apoptosis by means of TUNEL staining in DSS-induced colitis in mice. FITC signals represent fragmented DNA and cellular apoptosis. (b) Apoptosis indexes of the 3 experimental groups were calculated as a percentage of the TUNEL-positive cells to the total number of nucleated cells. n Significantly different among the 3 groups (p o 0.05).

DSS þMZWD mice, NF-κB p65 signals were significantly weakened whereas signals of the inhibitory subunit IκB-α were enhanced. The stabilization of IκB-α subunit in the cytoplasm implicated the NF-κB/TNF-α signaling was halted. Our results were in accordance with those of Zhang and colleagues that paeoniforin, a main constituent of our MZWD, ameliorated DSS-induced in mice through NF-κB activity in vivo and in vitro (Zhang et al., 2014). Moreover, from the HPLC spectra, we found that MZWD also comes with high contents of berberine and palmatine. In fact, berberine has been widely used as a traditional medicine for treating diarrhea (Rabbani et al., 1987) and gastrointestinal disorders (Zhou and Mineshita, 2000) whereas palmatine has been used for treatment of inflammation and liver-related diseases (Chang et al., 1999). The anti-inflammatory and therapeutic effects

of MZWD on the development of DSS-induced colitis are suggested to be associated with the blockade of NF-κB activation. PAR2 is a widely expressed G-protein-coupled receptor in human tissues, especially in those of the gastrointestinal tract (Nystedt et al., 1995; Bohm et al., 1996). It is activated by trypsin and various trypsin-like serine proteases including β-tryptase during tissue responses to injury, repair, inflammation and pain (Vergnolle et al., 2004; Vergnolle et al., 2001). According to a number of reports, expression of PAR2 is up-regulated by inflammatory mediators such as TNF-α and IL-1β upon damage to epithelial cells of the gut lining (Lohman et al., 2012). A significant up-regulated PAR2 expression was observed in mice with DSS colitis in the current in vivo experiment; however, such upregulation was notably diminished in mice treated with MZWD.


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This piece of result implicates a critical role of PAR2 in the pathogenesis of chronic colitis. In fact, PAR2 has been shown to contribute to inflammatory conditions as PAR2 antagonist GB88 attenuated chronic kidney diseases and pulmonary infection (Sharma et al., 2007; Moraes et al., 2008). From our results, we suggest that MZWD treatment restrains PAR2 activation in addition to inhibiting NF-κB/TNF-α signaling; hence, results in an amelioration of DSS-induced colitis. On the other hand, increased levels of fragmented DNA under colitis condition are indeed an indicator of cell death. Previous studies have demonstrated that the inhibition of NF-κB activation leads to a decrease in apoptosis (Pahl, 1999; Bonizzi and Karin, 2004). In this study, a positive correlation between NF-κB activity and the apoptotic index was clearly observed. With an inhibition of NF-κB activity, the epithelial damage in mucosa was thereby reduced in the DSS þMZWD mice. In summary, we have devised an herbal formula MZWD, based on TCM medical theory and ancient practice, for the treatment of IBD, a chronic and increasingly prevalent but recalcitrant condition. We have evaluated the effect of this formula in terms of clinical symptoms, physiological conditions and biochemical assays. We conclude that MZWD treatment attenuates the intrinsic and systemic inflammatory responses such as the upregulation of pro-inflammatory cytokines and related mediators via the inhibition of NF-κB activity. With protective effects provided, we strongly suggest that MZWD is a promising therapeutic means for treating IBD in general and UC in particular. More broadly, this study successfully integrates Eastern and Western sciences. Centuries of clinical experience provided the herbal formula while Western laboratory tests documented its specific mode of action. Combining the two not only validates the efficacy of a TCM formula, but also advances our understanding of human physiology, and may provide much needed relief to thousands of people. Acknowledgments This work was supported by HKBU RC-IRMS/11-12/01 from the Hong Kong Baptist University, Hong Kong SAR, China. References Andresen, L., Jorgensen, V.L., Perner, A., Hansen, A., Eugen-Olsen, J., Rask-Madsen, J., 2005. Activation of nuclear factor kappaB in colonic mucosa from patients with collagenous and ulcerative colitis. Gut 54 (4), 503–509. Baert, F., Vermeire, S., Noman, M., Van Assche, G., D'Haens, G., Rutgeerts, P., 2004. Management of ulcerative colitis and Crohn's disease. Acta Clinica Belgica 59 (5), 304–314. Baert, F.J., D'Haens, G.R., Peeters, M., Hiele, M.I., Schaible, T.F., Shealy, D., Geboes, K., Rutgeerts, P.J., 1999. Tumor necrosis factor alpha antibody (infliximab) therapy profoundly down-regulates the inflammation in Crohn's ileocolitis. Gastroenterology 116 (1), 22–28. Baldwin Jr., A.S., 1996. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annual Review of Immunology 14, 649–683. Bohm, S.K., Kong, W., Bromme, D., Smeekens, S.P., Anderson, D.C., Connolly, A., Kahn, M., Nelken, N.A., Coughlin, S.R., Payan, D.G., Bunnett, N.W., 1996. Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2. Biochemical Journal 314 (Pt 3), 1009–1016. Bonizzi, G., Karin, M., 2004. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends in Immunology 25 (6), 280–288. Braegger, C.P., Nicholls, S., Murch, S.H., Stephens, S., MacDonald, T.T., 1992. Tumour necrosis factor alpha in stool as a marker of intestinal inflammation. Lancet 339 (8785), 89–91. Cenac, N., Coelho, A.M., Nguyen, C., Compton, S., Andrade-Gordon, P., MacNaughton, W.K., Wallace, J.L., Hollenberg, M.D., Bunnett, N.W., Garcia-Villar, R., Bueno, L., Vergnolle, N., 2002. Induction of intestinal inflammation in mouse by activation of proteinaseactivated receptor-2. American Journal of Pathology 161 (5), 1903–1915. Chang, Y.L., Usami, S., Hsieh, M.T., Jiang, M.J., 1999. Effects of palmatine on isometric force and intracellular calcium levels of arterial smooth muscle. Life Sciences 64 (8), 597–606. Dobbins 3rd, W.O., 1984. Dysplasia and malignancy in inflammatory bowel disease. Annual Review of Medicine 35, 33–48.

67 Dong, J., Jimi, E., Zeiss, C., Hayden, M.S., Ghosh, S., 2010. Constitutively active NF-kappaB triggers systemic TNFalpha-dependent inflammation and localized 68 TNFalpha-independent inflammatory disease. Genes and Development 24 (16), 69 1709–1717. 70 Dong, W.G., Liu, S.P., Yu, B.P., Wu, D.F., Luo, H.S., Yu, J.P., 2003. Ameliorative effects of sodium ferulate on experimental colitis and their mechanisms in rats. World 71 Journal of Gastroenterology 9 (11), 2533–2538. 72 Fok, K.C., Ng, W.W., Henderson, C.J., Connor, S.J., 2012. Cutaneous sarcoidosis in a 73 patient with ulcerative colitis on infliximab. Journal of Crohn's and Colitis 6 (6), 708–712. 74 Guerra, I., Algaba, A., Perez-Calle, J.L., Chaparro, M., Marin-Jimenez, I., Garcia75 Castellanos, R., Gonzalez-Lama, Y., Lopez-Sanroman, A., Mancenido, N., 76 Martinez-Montiel, P., Quintanilla, E., Taxonera, C., Villafruela, M., RomeroMate, A., Lopez-Serrano, P., Gisbert, J.P., Bermejo, F., 2012. Induction of psoriasis 77 with anti-TNF agents in patients with inflammatory bowel disease: a report of 78 21 cases. Journal of Crohn's and Colitis 6 (5), 518–523. 79 Hayden, M.S., Ghosh, S., 2008. Shared principles in NF-kappaB signaling. Cell 132 80 (3), 344–362. Hudesman, D., Lichtiger, S., Sands, B., 2013. Risk of extraintestinal solid cancer with 81 anti-TNF therapy in adults with inflammatory bowel disease: review of the 82 literature. Inflammatory Bowel Disease 19 (3), 644–649. 83 Islam, M.S., Murata, T., Fujisawa, M., Nagasaka, R., Ushio, H., Bari, A.M., Hori, M., Ozaki, H., 2008. Anti-inflammatory effects of phytosteryl ferulates in colitis 84 induced by dextran sulphate sodium in mice. British Journal of Pharmacology 85 154 (4), 812–824. 86 Jurjus, A.R., Khoury, N.N., Reimund, J.M., 2004. Animal models of inflammatory bowel disease. Journal of Pharmacological and Toxicological Methods 50 (2), 87 81–92. 88 Kim, J.J., Shajib, M.S., Manocha, M.M., Khan, W.I., 2012. Investigating intestinal 89 inflammation in DSS-induced model of IBD. Journal of Visualized Experiments 60. 90 Lakatos, P.L., 2006. Recent trends in the epidemiology of inflammatory bowel 91 diseases: up or down? World Journal of Gastroenterology 12 (38), 6102–6108. 92 Lohman, R.J., Cotterell, A.J., Suen, J., Liu, L., Do, A.T., Vesey, D.A., Fairlie, D.P., 2012. 93 Antagonism of protease-activated receptor 2 protects against experimental colitis. Journal of Pharmacology and Experimental Therapeutics 340 (2), 94 256–265. 95 Lok, K.H., Hung, H.G., Ng, C.H., Li, K.K., Li, K.F., Szeto, M.L., 2007. The epidemiology 96 and clinical characteristics of Crohn's disease in the Hong Kong Chinese population: experiences from a regional hospital. Hong Kong Medical Journal 97 13 (6), 436–441. 98 Magro, F., 2010. Management of inflammatory bowel disease with infliximab and 99 other anti-tumor necrosis factor therapies. Foreword—BioDrugs 24 (Suppl 1), 1–2. 100 Mocciaro, F., Renna, S., Orlando, A., Rizzuto, G., Sinagra, E., Orlando, E., Cottone, M., 101 2012. Cyclosporine or infliximab as rescue therapy in severe refractory 102 ulcerative colitis: early and long-term data from a retrospective observational study. Journal of Crohn's and Colitis 6 (6), 681–686. 103 Monteleone, I., Vavassori, P., Biancone, L., Monteleone, G., Pallone, F., 2002. 104 Immunoregulation in the gut: success and failures in human disease. Gut. 50 Q3 105 (Suppl 3)III60–III64. 106 Moraes, T.J., Martin, R., Plumb, J.D., Vachon, E., Cameron, C.M., Danesh, A., Kelvin, D.J., Ruf, W., Downey, G.P., 2008. Role of PAR2 in murine pulmonary pseudomonal 107 infection. The American Journal of Physiology—Lung Cellular and Molecular 108 Physiology 294 (2), L368–L377. 109 Nystedt, S., Emilsson, K., Larsson, A.K., Strombeck, B., Sundelin, J., 1995. Molecular cloning and functional expression of the gene encoding the human proteinase110 activated receptor 2. European Journal of Biochemistry 232 (1), 84–89. 111 Pahl, H.L., 1999. Activators and target genes of Rel/NF-kappaB transcription factors. 112 Oncogene 18 (49), 6853–6866. Papadakis, K.A., Targan, S.R., 2000. The role of chemokines and chemokine 113 receptors in mucosal inflammation. Inflammatroy Bowel Diseases 6 (4), 114 303–313. 115 Platt, A.M., Mowat, A.M., 2008. Mucosal macrophages and the regulation of immune responses in the intestine. Immunology Letters 119 (1–2), 22–31. 116 Podolsky, D.K., 2002. Inflammatory bowel disease. The New England Journal of 117 Medicine 347 (6), 417–429. 118 Qin, H.Y., Xiao, H.T., Wu, J.C., Berman, B.M., Sung, J.J., Bian, Z.X., 2012. Key factors in 119 developing the trinitrobenzene sulfonic acid-induced post-inflammatory irritable bowel syndrome model in rats. World Journal of Gastroenterology 18 (20), 120 2481–2492. 121 Rabbani, G.H., Butler, T., Knight, J., Sanyal, S.C., Alam, K., 1987. Randomized 122 controlled trial of berberine sulfate therapy for diarrhea due to enterotoxigenic Escherichia coli and Vibrio cholerae. The Journal of Infectious Diseases 155 (5), 123 979–984. 124 Sharma, R., Prasad, V., McCarthy, E.T., Savin, V.J., Dileepan, K.N., Stechschulte, D.J., 125 Lianos, E., Wiegmann, T., Sharma, M., 2007. Chymase increases glomerular albumin permeability via protease-activated receptor-2. Molecular and Cellular 126 Biochemistry 297 (1–2), 161–169. 127 Stone, M.A., Mayberry, J.F., Baker, R., 2003. Prevalence and management of 128 inflammatory bowel disease: a cross-sectional study from central England. European Journal of Gastroenterology and Hepatology 15 (12), 1275–1280. 129 Vergnolle, N., Cellars, L., Mencarelli, A., Rizzo, G., Swaminathan, S., Beck, P., Steinhoff, 130 M., Andrade-Gordon, P., Bunnett, N.W., Hollenberg, M.D., Wallace, J.L., Cirino, G., 131 Fiorucci, S., 2004. A role for proteinase-activated receptor-1 in inflammatory bowel 132 diseases. Journal of Clinical Investigation 114 (10), 1444–1456.



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Vergnolle, N., Wallace, J.L., Bunnett, N.W., Hollenberg, M.D., 2001. Proteaseactivated receptors in inflammation, neuronal signaling and pain. Trends in Pharmacological Sciences 22 (3), 146–152. Xavier, R.J., Podolsky, D.K., 2007. Unravelling the pathogenesis of inflammatory bowel disease. Nature 448 (7152), 427–434. Zhang, J., Dou, W., Zhang, E., Sun, A., Ding, L., Wei, X., Chou, G., Mani, S., Wang, Z., 2014. Paeoniflorin abrogates DSS-induced colitis via a TLR4-dependent

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pathway. The American Journal of Physiology—Gastrointestinal and Liver Physiology 306 (1), G27–G36. Zhou, H., Mineshita, S., 2000. The effect of berberine chloride on experimental colitis in rats in vivo and in vitro. Journal of Pharmacology and Experimental Therapeutics. 294 (3), 822–829.


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Asiatic acid ameliorates dextran sulfate sodium-induced murine experimental colitis via suppressing mitochondria-mediated NLRP3 inflammasome activation.

Kefir treatment ameliorates dextran sulfate sodium-induced colitis in rats.

Uridine Ameliorates Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice.

Heat-killed VSL#3 ameliorates dextran sulfate sodium (DSS)-induced acute experimental colitis in rats.

Faecalibacterium prausnitzii supernatant ameliorates dextran sulfate sodium induced colitis by regulating Th17 cell differentiation.

AP-1 signaling.

Treatment of dextran sodium sulfate-induced experimental colitis by adoptive transfer of peritoneal cells.

TGF-beta signaling-dependent alleviation of dextran sulfate sodium-induced colitis by mesenchymal stem cell transplantation.

Ascorbic acid ameliorates oxidative stress and inflammation in dextran sulfate sodium-induced ulcerative colitis in mice.

Allyl isothiocyanate ameliorates angiogenesis and inflammation in dextran sulfate sodium-induced acute colitis.

Human Amnion-Derived Mesenchymal Stem Cell Transplantation Ameliorates Dextran Sulfate Sodium-Induced Severe Colitis in Rats.

Alloferon Alleviates Dextran Sulfate Sodium-induced Colitis.

Bifidobacterium longum alleviates dextran sulfate sodium-induced colitis by suppressing IL-17A response: involvement of intestinal epithelial costimulatory molecules.

Boehmeria nivea attenuates the development of dextran sulfate sodium-induced experimental colitis.

Treg Profile.

dextran sulfate sodium mouse model.

Treg Profile.

Dietary Propolis Ameliorates Dextran Sulfate Sodium-Induced Colitis and Modulates the Gut Microbiota in Rats Fed a Western Diet.

An experimental model of xanthoma by intradermal dextran sulfate injection.

Therapeutic potential of tonsil-derived mesenchymal stem cells in dextran sulfate sodium-induced experimental murine colitis.

Propionate Ameliorates Dextran Sodium Sulfate-Induced Colitis by Improving Intestinal Barrier Function and Reducing Inflammation and Oxidative Stress.

Fermented Pueraria Lobata extract ameliorates dextran sulfate sodium-induced colitis by reducing pro-inflammatory cytokines and recovering intestinal barrier function.

Carbon monoxide attenuates dextran sulfate sodium-induced colitis via inhibition of GSK-3β signaling.

Intramuscular injection of adenoviral hepatocyte growth factor at a distal site ameliorates dextran sodium sulfate-induced colitis in mice.