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Gut and Liver is an international journal of gastroenterology, focusing on the gastrointestinal tract, liver, biliary tree, pancreas, motility, and neurogastroenterology. Gut atnd Liver delivers up-to-date, authoritative papers on both clinical and research-based topics in gastroenterology. The Journal publishes original articles, case reports, brief communications, letters to the editor and invited review articles in the field of gastroenterology. The Journal is operated by internationally renowned editorial boards and designed to provide a global opportunity to promote academic developments in the field of gastroenterology and hepatology. +MORE
Yong Chan Lee |
Professor of Medicine Director, Gastrointestinal Research Laboratory Veterans Affairs Medical Center, Univ. California San Francisco San Francisco, USA |
Jong Pil Im | Seoul National University College of Medicine, Seoul, Korea |
Robert S. Bresalier | University of Texas M. D. Anderson Cancer Center, Houston, USA |
Steven H. Itzkowitz | Mount Sinai Medical Center, NY, USA |
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Kyu-Hyun Han1, Jong-Min Park2, Migyeong Jeong2, Young-Min Han2, Eun-Jin Go2, Juyeon Park3, Hocheol Kim4, Jae Gab Han5, Oran Kwon6, Ki Baik Hahm1,2
Correspondence to: Ki Baik Hahma and Oran Kwonb. aDigestive Disease Center, CHA University Bundang Medical Center, 59 Yatap-ro, Bundang-gu, Seongnam 13496, Korea, Tel: +82-31-780-5005, Fax: +82-31-881-7185, E-mail: hahmkb@cha.ac.kr. bDepartment of Nutritional Science and Food Management, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Korea, Tel: +82-2-3277-6860, Fax: +82-2-3277-2862, E-mail: orank@ewha.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gut Liver 2017;11(5):655-666. https://doi.org/10.5009/gnl16496
Published online June 27, 2017, Published date September 15, 2017
Copyright © Gut and Liver.
In inflammatory bowel disease (IBD), repeated bouts of remission and relapse occur in patients and can impose a risk of colitis-associated cancer. We hypothesized that plant extracts of Murine intestinal epithelial IEC-6 cells were pretreated with AM or TH before a lipopolysaccharide (LPS)-induced challenge. Acute colitis was induced with 7 days of dextran sulfate sodium (DSS) in male C57BL/6 mice, and extracts of AM and TH were administered for 2 weeks before DSS administration. Because the AM or TH extracts were far superior in preventing DSS-induced colitis than sulfasalazine, AM or TH extracts can be considered natural agents that can prevent IBD relapse.Background/Aims
Methods
Results
Conclusions
Keywords:
Recently concerns regarding phytochemicals, naturally organized substance from root, stem and leaves of plants, had been raised remarkably even in the era of modern medicine like biologics, the reason why preference for natural products even in the developmental stage of molecular targeted therapy can be explained by their orchestrated actions such as anti-inflammation and immune modulation including reducing cytokine secretion and regulating redox-sensitive transcription factors, antioxidative and regenerative action as well as antimutagenesis. In summary, beside of well-known safety, even though biologics or immune modulators covers specific merit under top-down approach achieving mucosal healing and complete healing, phytochemicals from natural products can also impose well-concerted actions of “anti-inflammation” and “regeneration,” being implicated in preventing relapse as well as removing oncogenic inflammatory activities.
Though current drug therapies including 5-aminosalicylates (5-ASA) and some immune modulators have been prescribed commonly, they have shown unexpected diverse side effects as well as some limitation in efficacy. Therefore, continuous efforts had been paid to find naturally occurring phytochemicals such as polyphenols, terpenoids, and flavonoids as alternative candidates for inflammatory bowel disease (IBD) therapy. Furthermore, unmet clinical need in the treatment of IBD is that one of clinical features of IBD is frequent relapse even during maintenance therapy and these repeated bouts of relapse threatened patient the fear of cancer complication. Though not so high, the risk of colitis-associated cancer (CAC) still demands the development of safer and effective anti-inflammatory agents. We hypothesized that possibly “natural agent” or “phytochemicals” can be answer to this unmet medical need.
In the current study, we have focused on to two phytochemicals,
Until now, several anti-inflammatory and antioxidant effects of AM or TH were explored in diverse disease models, but never evaluated for IBD. Considering the clinical features of IBD, vulnerable to relapse relevant to sustained inflammatory surge in spite of maintenance therapy, in this study, we aimed at documenting the anti-inflammatory and antioxidant activities of AM and/or TH on experimentally induced colitis, pretreatment in order to put the clinical implication of preventing relapse.
All chemical reagents were obtained from Sigma (St. Louis, MO, USA). AM and TH phytochemicals were supplied from NeuMed Inc. (Seoul, Korea), dissolved in dimethyl sulfoxide (DMSO) for experiment. Dextran sulfate sodium salt (DSS; molecular weight at 36,000 to 50,000 Da) was purchased from MP Biomedicals (Morgan Irvine, CA, USA). Primary antibodies for Western blotting were purchased as follows: α-tubulin, β-actin, and NQO-1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); COX-2 antibody was from Thermo Scientific (Fremont, CA, USA), other antibodies from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated anti-rat/rabbit/mouse/goat IgG was purchased Santa Cruz Biotechnology. All other materials were obtained in the highest available grade.
The dried aerial part of TH and rhizomes of AM were purchased from Gyeongdong Herbal Market, Jegi-dong Seoul, Korea. The samples were identified by professor Hocheol Kim and voucher specimens (#HP565 and #HP019) were deposited at the Department of Herbal Pharmacology, College of Oriental Medicine, Kyung Hee University, Seoul, Korea.
Dried aerial part of 50 g TH and root of 50 g AM were extracted separately with water by using a reflux apparatus twice for 3 hours at 100°C. The extracts were filtered and concentrated under reduced pressure, and samples were lyophilized to yield a yellow brown powder. The yield (%) of individual extracts was 24.4% and 68.5%, respectively. Then, two kinds of powder were mixed for preparing HT057 in the proportion of the powder. The quantitative authentication of HP565, HP019 and HT057 performed by a high performance liquid chromatography (HPLC) analysis system equipped with a Waters 1525 pump, a 2707 Autosampler and a 2998 PDA detector (See
Six-week-old female C57BL/6 mice (Orient Bio Inc., Seongnam, Korea) were fed sterilized commercial pellet diets (Biogenomics Co., Seoul, Korea) and sterilized water
During the period of DSS administration, all mice were subjected to following examination on daily basis; bleeding, diarrhea, poor oral intake. The disease activity index (DAI) was calculated by scoring changes described by Cooper
Total RNA was isolated with TRIzol (Life Technologies, Carlsbad, CA, USA). The tissues in TRIzol were incubated for 10 minutes at 4°C. Furthermore, 100 μL of chloroform was added and gently mixed. After incubation for 10 minutes on ice, samples were centrifuged at 12,000 rpm for 15 minutes. Supernatants were mixed with 500 μL of isopropanol and incubated at 4°C for 15 minutes. After centrifuging at 12,000 rpm for 10 minutes, pellets were washed with 70% (v/v) ethanol. After allowing the ethanol to completely evaporate, pellets were dissolved in 100 μL of diethylenepyrocarbonate-treated water (Invitrogen Life Technologies, Carlsbad, CA, USA). Complementary DNA was prepared using reverse transcriptase originating from Murine Moloney leukemia virus (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Polymerase chain reaction was performed for 35 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds. Oligonucleotide primers were Table 1.
The tissues were homogenized with ice-cold cell lysis buffer (Cell Signaling Technology) containing 1 mM phenylmethylsulfonyl fluoride. After 1 hour of incubation, samples were centrifuged at 12,000 rpm for 15 minutes. Supernatants were then collected. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, which were incubated with appropriate antibodies and visualized using an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK).
Following harvesting of the colon, and homogenized in 10 mM sodium phosphate buffer, pH 7.4 (1 mL). After centrifugation (9,000 ×
After paraffin blocks were dewaxed and rehydrated with graded alcohol, these tissue sections were heated in pressure jars filled with 10 mM/L citrate buffer using microwave for 10 minutes. After that, slides were cooled in water for 15 minutes and then washed in phosphate buffered saline. The slides were incubated overnight with the primary antibody. The primary antibodies were specific rabbit polyclonal antibodies against COX-2 (Thermo), F4/80, and CD3 (Santa Cruz Biotechnology). Each antibody was diluted 1:100. After incubation, the subsequent reaction was formed using an Envision kit (DakoCytomation, Glostrup, Denmark). Finally, the slides were incubated with 3,3′-diaminobenzidine (DakoCytomation) and counter-stained with hematoxylin (Sigma). Number of antibody positive cells was determined in five fields of colon mucosa and submucosa area selected at random in each mouse and examined at ×40 and ×100 magnification. Values are given as mean±standard error of the mean (SEM).
Rat intestinal epithelial cells (IEC-6) was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA) and maintained according to the ATCC’s instructions. IEC-6 cells were maintained in DMEM medium containing 10% fetal bovine serum (Hyclone; Thermo, Logan, UT, USA), and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. MTT was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The filtrate (50 g) was mixed with water 1 L and then lyophilized. The cells were plated into 96-well plates at 104 cells/mL and allowed to adhere for 24 hours. AM and TH extract was applied in the test wells at various concentrations for 24 hours.
Statistical analysis was carried out using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Data (mean±SEM) from cell lines and animals were analyzed using Kruskal-Wallis tests and group comparisons were performed with Mann-Whitney U tests.
Using animal model of 4% DSS-induced colitis as shown in protocol (Fig. 1A), 100 mg/kg AM (group 3), 100 mg/kg TH (group 4), 50 mg/kg AM+50 mg/kg TH (group 5), and 30 mg/kg sulfasalazine (group 6) were pretreated 2 weeks before DSS administration to evaluate the preventive effects of AM or TH. Four percent DSS administration provoked significant levels of colitis as manifested with reduced body weight (Fig. 1B) and clinical symptoms of hematochezia, diarrhea, animal activities, abdominal pain, and poor oral intake, and so forth (Fig. 1C). Mean body weights were significantly decreased only in DSS-induced colitis group 2 (p<0.05), but no significant decreases in body weight were noted on other group. DAI score included the severity of rectal bleeding, diarrhea and poor oral intake, which was measured every day after DSS administration (score from 0 to 5).18 DSS-administrated group had significantly increased DAI score compared with normal group 1 (p<0.001) (Fig. 1C). However, oral administration of AM and/or TH significantly improved the severity of rectal bleeding, diarrhea and poor oral intake (Fig. 1C). As seen in Fig. 1D, DSS administration significantly shortened colon length (p<0.001), but AM and/or TH pretreatment group showed significantly longer colon length than DSS control group (p<0.05). Significant finding was noted from that sulfasalazine treated group (group 6) that sulfasalazine was only effective in the change of body weight, whereas sulfasalazine neither decrease DAI nor prevented DSS-induced colon length shortening. According to pathological score, total pathological scores were made according to group. On the pathological findings as seen in Fig. 1E and Fig. 2A, 4% DSS administered in drinking water have induced significant levels of colon inflammations, extensive colon ulcerations as well as submucosal edema (p<0.001). However, these pathological scores were significantly decreased in group 3, group 4, and group 5 (p<0.05). On separate analysis of colon ulceration according to group, only group 3, group 4, and group 5 showed significantly decreased scores, whereas group 6 did not (p<0.01), signifying that AM and/or TH played significant preventive effects of colitis. In this setting, sulfasalazine showed inferior outcome compared to AM and/or TH in preventing DSS-induced colitis. Interestingly, sulfasalazine was found to be inferior to AM and/or TH on these pathological analyses (Fig. 1E). When pathological changes were scored according to inflammation, ulceration, and regeneration, respectively, group 3 and group 5 showed significant decreases in inflammation score (p<0.05), group 3, group 4, and group 5 showed significant decreases in colon ulceration (p<0.05) (Fig. 2A). AM and/or TH pretreatment imposed significant anti-inflammatory and antiulceration activities in colitis model, better than sulfasalazine in some pathogenesis of experimental colitis (Fig. 2A).
On further detailed evaluation of colon inflammation and colon ulcer after DSS administration, AM and/or TH afforded significant decreases in either inflammation score or ulceration score (p<0.05) (Fig. 2A). Hence, COX-2 and iNOS are frequently overexpressed in IBD as well as colon cancer16,20–22 and inflammatory cytokines including IL-1β and IL-6 closely reflected disease activities of IBD,23,24 we have measured
Since the sources of all of these mediators implicated in DSS-induced colitis are infiltrated monocytes such as lymphocytes and macrophages, we did do the immunohistochemical staining with F4/80 for macrophage and CD3 for T lymphocytes in DSS induced colitis (Fig. 4A). As observed, F4/80 and CD3 were all significantly increased in DSS administered control group (p<0.001), but the levels of F4/80 and CD3 were all significantly decreased with pretreatment of AM, TH, and sulfasalazine (p<0.05). Signal transducer and activator of transcription 3 (STAT3), activated via IL-6-gp130-JAK signaling, has a role of regulation of inflammation pathway such as iNOS expression and cyclin D1 expression when it is phosphorylated and bind to the promoter region of target gene.25–27 Nuclear factor (NF)-κB has been considered as transcription factor to regulate expression of COX-2 and iNOS.28 Activation of NF-κB was followed when the inhibitory subunit IκBα was dissociated, which requires phosphorylation of IκBα. The phosphorylation of IκBα-relased free NF-κB to translocated to the nucleus and regulate of transcription of target genes subsequently. MAPKs (mitogen-activated protein kinases) such as ERK (extracellular signal regulated kinase) were associated with activation of NF-κB.29,30 In this condition, NF-κB p65 and STAT3 expressions were all significantly increased after DSS administration (p<0.001), but pretreatment of TH and AT+TH significantly decreased NF-κB and STAT3 (p<0.05) (Fig. 4B). Sulfasalazine was inferior to AM and TH in repressing DSS-induced NF-κB as well as STAT3 inactivation. On serial measurement of ERK, JNK, and p38 (data not shown), we have found ERK played main signal transduction in DSS-induced colitis (Fig. 4C) and AM extract significantly inactivated ERK. Based on these elucidations, we have stimulated IEC-6 intestinal cells with lipopolysaccharide (LPS) and checked the changes of
On detailed evaluation based on scoring system for regeneration, the scores for colon regeneration were significantly increased in AT, TH, but not sulfasalazine pretreated group (p<0.01) (Fig. 5A). As seen in the
In the current study, we have found that AM and/or TH extracts were superior to current anti-inflammatory drug, sulfasalazine, in the preventing DSS-induced colitis, featured with potent anti-inflammatory, antioxidative, and regenerative mechanisms. Translating our findings into clinical implication, these edible and safe plant extracts, AM and/or TH, can be applied to prevent the relapse of IBD as supplementary agent during maintenance therapy. Though compared in animal models, we believe sulfasalazine, though currently acknowledged gold standard as maintenance therapeutics, was proven to be insufficient in preventing relapse due to lack in regenerating action such as HO-1.
Before our study, it was reported that AM had anti-inflammatory activities in several studies2,4 and even tumor suppressive effect in various cancer treatment such as lung cancer, advanced colon cancer and brain glioma.5–7,31 Though not touched in the current study, significant STAT3 inactivation as well as NF-κB repression of AM can also be clue to the prevention of CAC, if we followed up colitis model for longer time. Since carcinogenic effect might be caused by chronic inflammation because inflammation caused mutation or instability of genome,28 the significant anti-inflammatory actions of AM and/or TH can exert both the preventive effect of IBD relapse and possibly prevention of CAC. Though there was no previous study explaining how AM anti-inflammatory effect, in this study, we found that COX-2, iNOS, TNF-α, NF-κB, pIκBα, p-STAT3 and ERK, mediators all reported to be intervened in colitis as well as CAC, were significantly regulated by AM and/or TH in both
Multiple data showed anti-inflammatory activities of TH.12,32,33 Kim
Recently, Jeong
Currently, in clinic, starting 5-ASA as basic anti-inflammatory drugs including sulfasalazine and mesalazine, some antibiotics and probiotics, systemic or locally acting steroids, and immune modulators have been used to induce remission and to reduce the symptoms. After remission induction, some of which were continued for maintaining remission. However, since long-term uses of these agents has been found to lead to severe toxicities, the concerns to alternate management including certain dietary agents, healthy spices, omega-3 polyunsaturated fatty acids, and some anti-inflammatory phytochemicals, AM and/or TH in the current study, are increasing to offer further beneficial or additional preventive or ameliorating effects in the treatment of IBD.44,45 Surely concerted mechanisms as well as beneficiary actions of dietary planta extracts from natural products have merits in the treatment of IBD further supported with safety in addition to symptomatic improvement.46
In summary, we found that planta extracts like AM and/or TH, based on potent anti-inflammatory and antioxidative effect as well as regeneration spurting action via HO-1 induction, significantly rescued from DSS-induced colitis. Thinking safety and efficacy, we concluded that these natural extracts can be potential candidate to treat IBD targeting to prevent relapse of IBD as supplementary intervention. However, further detailed clinical trials should be followed to put our phytoextracts as beneficiary relapse preventing strategy.
This research was supported by National Center of Efficacy Evaluation for the Development of Health Products Targeting Digestive Disorders (NCEED).
Primers Used in the Current Experiments
Primer | Size, bp | Anneal temperature, °C | ||
---|---|---|---|---|
Sense primer, 5′→3′ | Antisense primer, 5′→3′ | |||
COX-2 | GAA ATG GCT GCA GAG TTG AA | TCA TCT AGT CTG GAG TGG GA | 356 | 58 |
IL-6 | CTT CCA GCC AGT TGC CTT CT | GAG AGC ATT GGA AGT TGG GG | 496 | 58 |
TNF-α | CCC TCA CAC TCA GAT CAT CTT CTC AA | TCT AAG GTA CTT GGG CAG GTT GAC CTC | 428 | 58 |
HO-1 | GAC AGC ATG TCC CAG GAT TT | GGT TCT GCT TGT TTC GCT CT | 198 | 60 |
GAPDH | GGT GCT GAG TAT GTC GTG GA | TTC AGC TCT GGG ATG ACC TT | 404 | 58 |
COX-2 | CAT CCT GCC AGC TCC ACC GC | GGG AGG AAG GGC CCT GGT GT | 474 | 58 |
IL-6 | CCG GAG AGG AGA CTT CAC AG | TGG TCT TGG TCC TTA GCC AC | 479 | 58 |
IL-1β | CAG GCT CCG AGA TGA ACA ACA AAA | TGG GGA ACT CTG CAG ACT CAA ACT | 332 | 58 |
GAPDH | AAT GTA TCC GTT GTG GAT CT | TCC ACC ACC CTG TTG CTG TA | 300 | 58 |
Gut and Liver 2017; 11(5): 655-666
Published online September 15, 2017 https://doi.org/10.5009/gnl16496
Copyright © Gut and Liver.
Kyu-Hyun Han1, Jong-Min Park2, Migyeong Jeong2, Young-Min Han2, Eun-Jin Go2, Juyeon Park3, Hocheol Kim4, Jae Gab Han5, Oran Kwon6, Ki Baik Hahm1,2
1Digestive Disease Center, CHA University Bundang Medical Center, CHA Cancer Institute, CHA University, Seongnam, Korea, 2CHA Cancer Prevention Research Center, CHA Cancer Institute, CHA University, Seongnam, Korea, 3Korea Institute of Science and Technology for Eastern Medicine (KISTEM), NeuMed Inc., Korea, 4Department of Herbal Pharmacology, College of Korean Medicine, Kyung Hee University, Seoul, Korea, 5Department of Health Food Research & Development, Daesang Corp., Icheon, Korea, 6Department of Nutritional Science and Food Management, Ewha Womans University, Seoul, Korea
Correspondence to: Ki Baik Hahma and Oran Kwonb. aDigestive Disease Center, CHA University Bundang Medical Center, 59 Yatap-ro, Bundang-gu, Seongnam 13496, Korea, Tel: +82-31-780-5005, Fax: +82-31-881-7185, E-mail: hahmkb@cha.ac.kr. bDepartment of Nutritional Science and Food Management, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Korea, Tel: +82-2-3277-6860, Fax: +82-2-3277-2862, E-mail: orank@ewha.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
In inflammatory bowel disease (IBD), repeated bouts of remission and relapse occur in patients and can impose a risk of colitis-associated cancer. We hypothesized that plant extracts of Murine intestinal epithelial IEC-6 cells were pretreated with AM or TH before a lipopolysaccharide (LPS)-induced challenge. Acute colitis was induced with 7 days of dextran sulfate sodium (DSS) in male C57BL/6 mice, and extracts of AM and TH were administered for 2 weeks before DSS administration. Because the AM or TH extracts were far superior in preventing DSS-induced colitis than sulfasalazine, AM or TH extracts can be considered natural agents that can prevent IBD relapse.Background/Aims
Methods
Results
Conclusions
Keywords:
Recently concerns regarding phytochemicals, naturally organized substance from root, stem and leaves of plants, had been raised remarkably even in the era of modern medicine like biologics, the reason why preference for natural products even in the developmental stage of molecular targeted therapy can be explained by their orchestrated actions such as anti-inflammation and immune modulation including reducing cytokine secretion and regulating redox-sensitive transcription factors, antioxidative and regenerative action as well as antimutagenesis. In summary, beside of well-known safety, even though biologics or immune modulators covers specific merit under top-down approach achieving mucosal healing and complete healing, phytochemicals from natural products can also impose well-concerted actions of “anti-inflammation” and “regeneration,” being implicated in preventing relapse as well as removing oncogenic inflammatory activities.
Though current drug therapies including 5-aminosalicylates (5-ASA) and some immune modulators have been prescribed commonly, they have shown unexpected diverse side effects as well as some limitation in efficacy. Therefore, continuous efforts had been paid to find naturally occurring phytochemicals such as polyphenols, terpenoids, and flavonoids as alternative candidates for inflammatory bowel disease (IBD) therapy. Furthermore, unmet clinical need in the treatment of IBD is that one of clinical features of IBD is frequent relapse even during maintenance therapy and these repeated bouts of relapse threatened patient the fear of cancer complication. Though not so high, the risk of colitis-associated cancer (CAC) still demands the development of safer and effective anti-inflammatory agents. We hypothesized that possibly “natural agent” or “phytochemicals” can be answer to this unmet medical need.
In the current study, we have focused on to two phytochemicals,
Until now, several anti-inflammatory and antioxidant effects of AM or TH were explored in diverse disease models, but never evaluated for IBD. Considering the clinical features of IBD, vulnerable to relapse relevant to sustained inflammatory surge in spite of maintenance therapy, in this study, we aimed at documenting the anti-inflammatory and antioxidant activities of AM and/or TH on experimentally induced colitis, pretreatment in order to put the clinical implication of preventing relapse.
All chemical reagents were obtained from Sigma (St. Louis, MO, USA). AM and TH phytochemicals were supplied from NeuMed Inc. (Seoul, Korea), dissolved in dimethyl sulfoxide (DMSO) for experiment. Dextran sulfate sodium salt (DSS; molecular weight at 36,000 to 50,000 Da) was purchased from MP Biomedicals (Morgan Irvine, CA, USA). Primary antibodies for Western blotting were purchased as follows: α-tubulin, β-actin, and NQO-1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); COX-2 antibody was from Thermo Scientific (Fremont, CA, USA), other antibodies from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated anti-rat/rabbit/mouse/goat IgG was purchased Santa Cruz Biotechnology. All other materials were obtained in the highest available grade.
The dried aerial part of TH and rhizomes of AM were purchased from Gyeongdong Herbal Market, Jegi-dong Seoul, Korea. The samples were identified by professor Hocheol Kim and voucher specimens (#HP565 and #HP019) were deposited at the Department of Herbal Pharmacology, College of Oriental Medicine, Kyung Hee University, Seoul, Korea.
Dried aerial part of 50 g TH and root of 50 g AM were extracted separately with water by using a reflux apparatus twice for 3 hours at 100°C. The extracts were filtered and concentrated under reduced pressure, and samples were lyophilized to yield a yellow brown powder. The yield (%) of individual extracts was 24.4% and 68.5%, respectively. Then, two kinds of powder were mixed for preparing HT057 in the proportion of the powder. The quantitative authentication of HP565, HP019 and HT057 performed by a high performance liquid chromatography (HPLC) analysis system equipped with a Waters 1525 pump, a 2707 Autosampler and a 2998 PDA detector (See
Six-week-old female C57BL/6 mice (Orient Bio Inc., Seongnam, Korea) were fed sterilized commercial pellet diets (Biogenomics Co., Seoul, Korea) and sterilized water
During the period of DSS administration, all mice were subjected to following examination on daily basis; bleeding, diarrhea, poor oral intake. The disease activity index (DAI) was calculated by scoring changes described by Cooper
Total RNA was isolated with TRIzol (Life Technologies, Carlsbad, CA, USA). The tissues in TRIzol were incubated for 10 minutes at 4°C. Furthermore, 100 μL of chloroform was added and gently mixed. After incubation for 10 minutes on ice, samples were centrifuged at 12,000 rpm for 15 minutes. Supernatants were mixed with 500 μL of isopropanol and incubated at 4°C for 15 minutes. After centrifuging at 12,000 rpm for 10 minutes, pellets were washed with 70% (v/v) ethanol. After allowing the ethanol to completely evaporate, pellets were dissolved in 100 μL of diethylenepyrocarbonate-treated water (Invitrogen Life Technologies, Carlsbad, CA, USA). Complementary DNA was prepared using reverse transcriptase originating from Murine Moloney leukemia virus (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Polymerase chain reaction was performed for 35 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds. Oligonucleotide primers were Table 1.
The tissues were homogenized with ice-cold cell lysis buffer (Cell Signaling Technology) containing 1 mM phenylmethylsulfonyl fluoride. After 1 hour of incubation, samples were centrifuged at 12,000 rpm for 15 minutes. Supernatants were then collected. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, which were incubated with appropriate antibodies and visualized using an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK).
Following harvesting of the colon, and homogenized in 10 mM sodium phosphate buffer, pH 7.4 (1 mL). After centrifugation (9,000 ×
After paraffin blocks were dewaxed and rehydrated with graded alcohol, these tissue sections were heated in pressure jars filled with 10 mM/L citrate buffer using microwave for 10 minutes. After that, slides were cooled in water for 15 minutes and then washed in phosphate buffered saline. The slides were incubated overnight with the primary antibody. The primary antibodies were specific rabbit polyclonal antibodies against COX-2 (Thermo), F4/80, and CD3 (Santa Cruz Biotechnology). Each antibody was diluted 1:100. After incubation, the subsequent reaction was formed using an Envision kit (DakoCytomation, Glostrup, Denmark). Finally, the slides were incubated with 3,3′-diaminobenzidine (DakoCytomation) and counter-stained with hematoxylin (Sigma). Number of antibody positive cells was determined in five fields of colon mucosa and submucosa area selected at random in each mouse and examined at ×40 and ×100 magnification. Values are given as mean±standard error of the mean (SEM).
Rat intestinal epithelial cells (IEC-6) was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA) and maintained according to the ATCC’s instructions. IEC-6 cells were maintained in DMEM medium containing 10% fetal bovine serum (Hyclone; Thermo, Logan, UT, USA), and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. MTT was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The filtrate (50 g) was mixed with water 1 L and then lyophilized. The cells were plated into 96-well plates at 104 cells/mL and allowed to adhere for 24 hours. AM and TH extract was applied in the test wells at various concentrations for 24 hours.
Statistical analysis was carried out using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Data (mean±SEM) from cell lines and animals were analyzed using Kruskal-Wallis tests and group comparisons were performed with Mann-Whitney U tests.
Using animal model of 4% DSS-induced colitis as shown in protocol (Fig. 1A), 100 mg/kg AM (group 3), 100 mg/kg TH (group 4), 50 mg/kg AM+50 mg/kg TH (group 5), and 30 mg/kg sulfasalazine (group 6) were pretreated 2 weeks before DSS administration to evaluate the preventive effects of AM or TH. Four percent DSS administration provoked significant levels of colitis as manifested with reduced body weight (Fig. 1B) and clinical symptoms of hematochezia, diarrhea, animal activities, abdominal pain, and poor oral intake, and so forth (Fig. 1C). Mean body weights were significantly decreased only in DSS-induced colitis group 2 (p<0.05), but no significant decreases in body weight were noted on other group. DAI score included the severity of rectal bleeding, diarrhea and poor oral intake, which was measured every day after DSS administration (score from 0 to 5).18 DSS-administrated group had significantly increased DAI score compared with normal group 1 (p<0.001) (Fig. 1C). However, oral administration of AM and/or TH significantly improved the severity of rectal bleeding, diarrhea and poor oral intake (Fig. 1C). As seen in Fig. 1D, DSS administration significantly shortened colon length (p<0.001), but AM and/or TH pretreatment group showed significantly longer colon length than DSS control group (p<0.05). Significant finding was noted from that sulfasalazine treated group (group 6) that sulfasalazine was only effective in the change of body weight, whereas sulfasalazine neither decrease DAI nor prevented DSS-induced colon length shortening. According to pathological score, total pathological scores were made according to group. On the pathological findings as seen in Fig. 1E and Fig. 2A, 4% DSS administered in drinking water have induced significant levels of colon inflammations, extensive colon ulcerations as well as submucosal edema (p<0.001). However, these pathological scores were significantly decreased in group 3, group 4, and group 5 (p<0.05). On separate analysis of colon ulceration according to group, only group 3, group 4, and group 5 showed significantly decreased scores, whereas group 6 did not (p<0.01), signifying that AM and/or TH played significant preventive effects of colitis. In this setting, sulfasalazine showed inferior outcome compared to AM and/or TH in preventing DSS-induced colitis. Interestingly, sulfasalazine was found to be inferior to AM and/or TH on these pathological analyses (Fig. 1E). When pathological changes were scored according to inflammation, ulceration, and regeneration, respectively, group 3 and group 5 showed significant decreases in inflammation score (p<0.05), group 3, group 4, and group 5 showed significant decreases in colon ulceration (p<0.05) (Fig. 2A). AM and/or TH pretreatment imposed significant anti-inflammatory and antiulceration activities in colitis model, better than sulfasalazine in some pathogenesis of experimental colitis (Fig. 2A).
On further detailed evaluation of colon inflammation and colon ulcer after DSS administration, AM and/or TH afforded significant decreases in either inflammation score or ulceration score (p<0.05) (Fig. 2A). Hence, COX-2 and iNOS are frequently overexpressed in IBD as well as colon cancer16,20–22 and inflammatory cytokines including IL-1β and IL-6 closely reflected disease activities of IBD,23,24 we have measured
Since the sources of all of these mediators implicated in DSS-induced colitis are infiltrated monocytes such as lymphocytes and macrophages, we did do the immunohistochemical staining with F4/80 for macrophage and CD3 for T lymphocytes in DSS induced colitis (Fig. 4A). As observed, F4/80 and CD3 were all significantly increased in DSS administered control group (p<0.001), but the levels of F4/80 and CD3 were all significantly decreased with pretreatment of AM, TH, and sulfasalazine (p<0.05). Signal transducer and activator of transcription 3 (STAT3), activated via IL-6-gp130-JAK signaling, has a role of regulation of inflammation pathway such as iNOS expression and cyclin D1 expression when it is phosphorylated and bind to the promoter region of target gene.25–27 Nuclear factor (NF)-κB has been considered as transcription factor to regulate expression of COX-2 and iNOS.28 Activation of NF-κB was followed when the inhibitory subunit IκBα was dissociated, which requires phosphorylation of IκBα. The phosphorylation of IκBα-relased free NF-κB to translocated to the nucleus and regulate of transcription of target genes subsequently. MAPKs (mitogen-activated protein kinases) such as ERK (extracellular signal regulated kinase) were associated with activation of NF-κB.29,30 In this condition, NF-κB p65 and STAT3 expressions were all significantly increased after DSS administration (p<0.001), but pretreatment of TH and AT+TH significantly decreased NF-κB and STAT3 (p<0.05) (Fig. 4B). Sulfasalazine was inferior to AM and TH in repressing DSS-induced NF-κB as well as STAT3 inactivation. On serial measurement of ERK, JNK, and p38 (data not shown), we have found ERK played main signal transduction in DSS-induced colitis (Fig. 4C) and AM extract significantly inactivated ERK. Based on these elucidations, we have stimulated IEC-6 intestinal cells with lipopolysaccharide (LPS) and checked the changes of
On detailed evaluation based on scoring system for regeneration, the scores for colon regeneration were significantly increased in AT, TH, but not sulfasalazine pretreated group (p<0.01) (Fig. 5A). As seen in the
In the current study, we have found that AM and/or TH extracts were superior to current anti-inflammatory drug, sulfasalazine, in the preventing DSS-induced colitis, featured with potent anti-inflammatory, antioxidative, and regenerative mechanisms. Translating our findings into clinical implication, these edible and safe plant extracts, AM and/or TH, can be applied to prevent the relapse of IBD as supplementary agent during maintenance therapy. Though compared in animal models, we believe sulfasalazine, though currently acknowledged gold standard as maintenance therapeutics, was proven to be insufficient in preventing relapse due to lack in regenerating action such as HO-1.
Before our study, it was reported that AM had anti-inflammatory activities in several studies2,4 and even tumor suppressive effect in various cancer treatment such as lung cancer, advanced colon cancer and brain glioma.5–7,31 Though not touched in the current study, significant STAT3 inactivation as well as NF-κB repression of AM can also be clue to the prevention of CAC, if we followed up colitis model for longer time. Since carcinogenic effect might be caused by chronic inflammation because inflammation caused mutation or instability of genome,28 the significant anti-inflammatory actions of AM and/or TH can exert both the preventive effect of IBD relapse and possibly prevention of CAC. Though there was no previous study explaining how AM anti-inflammatory effect, in this study, we found that COX-2, iNOS, TNF-α, NF-κB, pIκBα, p-STAT3 and ERK, mediators all reported to be intervened in colitis as well as CAC, were significantly regulated by AM and/or TH in both
Multiple data showed anti-inflammatory activities of TH.12,32,33 Kim
Recently, Jeong
Currently, in clinic, starting 5-ASA as basic anti-inflammatory drugs including sulfasalazine and mesalazine, some antibiotics and probiotics, systemic or locally acting steroids, and immune modulators have been used to induce remission and to reduce the symptoms. After remission induction, some of which were continued for maintaining remission. However, since long-term uses of these agents has been found to lead to severe toxicities, the concerns to alternate management including certain dietary agents, healthy spices, omega-3 polyunsaturated fatty acids, and some anti-inflammatory phytochemicals, AM and/or TH in the current study, are increasing to offer further beneficial or additional preventive or ameliorating effects in the treatment of IBD.44,45 Surely concerted mechanisms as well as beneficiary actions of dietary planta extracts from natural products have merits in the treatment of IBD further supported with safety in addition to symptomatic improvement.46
In summary, we found that planta extracts like AM and/or TH, based on potent anti-inflammatory and antioxidative effect as well as regeneration spurting action via HO-1 induction, significantly rescued from DSS-induced colitis. Thinking safety and efficacy, we concluded that these natural extracts can be potential candidate to treat IBD targeting to prevent relapse of IBD as supplementary intervention. However, further detailed clinical trials should be followed to put our phytoextracts as beneficiary relapse preventing strategy.
This research was supported by National Center of Efficacy Evaluation for the Development of Health Products Targeting Digestive Disorders (NCEED).
Table 1 Primers Used in the Current Experiments
Primer | Size, bp | Anneal temperature, °C | ||
---|---|---|---|---|
Sense primer, 5′→3′ | Antisense primer, 5′→3′ | |||
COX-2 | GAA ATG GCT GCA GAG TTG AA | TCA TCT AGT CTG GAG TGG GA | 356 | 58 |
IL-6 | CTT CCA GCC AGT TGC CTT CT | GAG AGC ATT GGA AGT TGG GG | 496 | 58 |
TNF-α | CCC TCA CAC TCA GAT CAT CTT CTC AA | TCT AAG GTA CTT GGG CAG GTT GAC CTC | 428 | 58 |
HO-1 | GAC AGC ATG TCC CAG GAT TT | GGT TCT GCT TGT TTC GCT CT | 198 | 60 |
GAPDH | GGT GCT GAG TAT GTC GTG GA | TTC AGC TCT GGG ATG ACC TT | 404 | 58 |
COX-2 | CAT CCT GCC AGC TCC ACC GC | GGG AGG AAG GGC CCT GGT GT | 474 | 58 |
IL-6 | CCG GAG AGG AGA CTT CAC AG | TGG TCT TGG TCC TTA GCC AC | 479 | 58 |
IL-1β | CAG GCT CCG AGA TGA ACA ACA AAA | TGG GGA ACT CTG CAG ACT CAA ACT | 332 | 58 |
GAPDH | AAT GTA TCC GTT GTG GAT CT | TCC ACC ACC CTG TTG CTG TA | 300 | 58 |
COX-2, cyclooxygenase-2; IL, interleukin; TNF, tumor necrosis factor; HO-1, heme oxygenase-1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.