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Toki-shakuyaku-san, a Japanese kampo medicine, reduces colon inflammation in a mouse model of acute colitis
International Immunopharmacology 29 (2015) 869–875
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Toki-shakuyaku-san, a Japanese kampo medicine, reduces colon inflammation in a mouse model of acute colitis Remya Sreedhar a, Somasundaram Arumugam a, Rajarajan A. Thandavarayan b, Vijayasree V. Giridharan c, Vengadeshprabhu Karuppagounder a, Vigneshwaran Pitchaimani a, Rejina Afrin a, Meilei Harima a, Takashi Nakamura a, Kazuyuki Ueno d, Masahiko Nakamura e, Kenji Suzuki f, Kenichi Watanabe a,⁎ a
Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata City 956-8603, Japan Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, TX 77030, USA c J.K.K Nataraja College of Pharmacy, Natarajapuram, Komarapalayam, 638183, Namakkal District, Tamil Nadu, India d Department of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata City 956-8603, Japan e Department of Cardiology, Yamanashi Prefectural Central Hospital, Kofu, Yamanashi, Japan f Department of Gastroenterology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City 951-8510, Japan b
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Article history: Received 29 May 2015 Received in revised form 20 August 2015 Accepted 24 August 2015 Available online 4 September 2015 Keywords: Apoptosis Endoplasmic reticulum stress Inflammation Inflammatory bowel diseases Kampo medicine Toki-shakuyaku-san
a b s t r a c t Toki-shakuyaku-san (TOKI) is a Japanese kampo medicine, which consists of a mixture of herbal medicines and considered to be a promising remedial agent due to its immunomodulatory and anti-inflammatory effects. We examined the beneficial effects of TOKI in inflammatory bowel disease associated with the inflammation of the intestinal barrier. A study was designed, using C57BL/6 female mice and were administered with 3% DSS in drinking water for 8 days with or without 1 g/kg/day TOKI orally for the last 3 days and a normal group supplied with plain drinking water for 8 days. TOKI treatment attenuated the clinical symptoms of acute murine colitis and also alleviated the inflammatory mechanism by reducing the inflammatory mediators, such as IL-1β, IL-2, TGF-β, RAGE and TLR2. It has also decreased the levels of CHOP, caspase12, cleaved caspase3 and cleaved caspase7 and thereby down-regulated the endoplasmic reticulum stress and apoptotic signaling induced by DSS. Moreover, the expression levels of cyclin D1 and c-kit have also confirmed the beneficial role of TOKI in colitis. All these data suggested that TOKI can be a promising agent for the treatment of colitis since it alleviates the disease progression and severity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis (UC) are characterized by chronic and relapsed inflammation in the intestine, but the etiology and pathogenesis have not been completely understood [1–3]. UC is an intractable IBD that causes inflammation and sores on the inner lining of the digestive tract mainly large intestine [3]. Key features of UC include diffuse mucosal inflammation that reverts proximally from the rectum to a varying degree in conjunction with severe inflammation and the coincident production of a complex mixture of inflammatory mediators, extensive superficial mucosal ulceration and is characterized by rectal bleeding, diarrhea, and abdominal pain [1]. Researchers presumed a number of factors associated with the pathogenesis of this disease probably environmental and genetic factors, which interact with the intestinal mucosal barrier and trigger an event that leads ultimately to a chronic activation of immune and nonimmune cells in the gut [4,5]. Oral administration of ⁎ Corresponding author. E-mail address: [email protected] (K. Watanabe).
http://dx.doi.org/10.1016/j.intimp.2015.08.029 1567-5769/© 2015 Elsevier B.V. All rights reserved.
dextran sulfate sodium (DSS) instigates UC in hamster and mice [5,6]. DSS consumption provokes immense macrophage and granulocyte infiltration, distortion of the anatomy of glands, and ulceration in the colon, which replicates the pathological features in the acute phase of colitis patients and therefore used as a conventional animal model of UC. DSS can damage enterocytes directly and disturb the integrity of the gut barrier and eventually promotes the invasion of intraluminal commensal bacteria [6–8]. Therapy for IBD mainly focuses on the regulation of inflammatory cells and their secretion of various inflammatory mediators like proinflammatory cytokines, chemokines, etc. [9]. Recently, endoplasmic reticulum (ER) stress has been recognized for the induction of inflammatory cytokines [10,11]. Inflammatory cytokines released after the induction of ER stress functions as an alarming or danger signal to communicate with other cells or to recruit immune cells [12]. The incorporation of natural products into the therapeutic regimens is an attractive approach for improving disease treatment due to their generally low toxicity profiles and high patient compliance [13]. Japanese herbal medicines of traditional Chinese origin, also called kampo medicines, are highly standardized for their quality and widely
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used for the treatment of various diseases [14]. Recently several kampo medicines have been investigated using animal studies and clinical trials to evaluate their potential beneficial effects [15]. Almost 150 kampo formulations have been approved as prescription drugs by the Ministry of Health, Labour and Welfare, Japan, and the treatment with kampo medicine is a symptom- and patient-based, therefore, a form of tailor-made drug therapy [16]. Toki-shakuyaku-san (TOKI; “Dang gui shao yao san” in Chinese) is a traditional kampo medicine, which is widely used in Japan, Korea and China to improve blood circulation and to treat various gynecological disorders such as irregular menstruation, dysmenorrhea, endometriosis, menopausal syndromes and sterility [17–22]. The TOKI kampo formulation is made from six different herbs such as Hoelin (Fu ling), Cnidium rhizome (Chuan xiong), Angelica sinensis (Dang gui), Peony root (Shao yao), Atractylodes rhizome (Bai zhu), and Alisma rhizome (Ze xie) [23]. In this study, we used TOKI in an animal model of UC to find out the cardinal effects of this drug in IBD and to find out the role of this drug in the anti-inflammatory cascade. 2. Materials and methods 2.1. Drugs and chemicals DSS (MW 36,000–50,000) was purchased from Wako, Japan. TOKI was obtained from Tsumura & Co., Tokyo, Japan (Prepared under GMP; Standard Commodity Classification No. of Japan—875200). All other chemicals used in this study were purchased from Sigma, Tokyo, Japan until mentioned otherwise. 2.2. Animals C57BL/6J female mice were maintained and housed in the animal facility of the Faculty of Pharmacy, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan. The mice were fed with normal chow diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and water ad libitum. Animals were kept under temperature and humidity controlled conditions and a light-dark cycle of 12:12 h. All the experiments were approved by the regulations of the Committee on Bioethics for Animal Experiments of our institute (approval number—H2705) and were performed under the relevant regulations and guidelines during the light phase of the cycle [24]. 2.3. Experimental design A protocol was designated for 8 days and the mice were allocated into three groups of matching age, sex and number (n = 6–8). In the first group, designated as the normal control, the mice received normal diet and plain drinking water (DW) ad libitum. The second group, designated as the DSS control, received 3% DSS in DW throughout the protocol. The third group, designated as the TOKI group, received 3% DSS in DW and once daily treatment with TOKI suspension in water (1 g/kg/day) orally during the last three days. 2.4. Change in body weight and disease activity index (DAI) Body weight was measured on a daily basis from the prior day of colitis induction and throughout the study protocol. The stool consistency and the presence of bloody stools, as well as rectal bleeding, were observed and scored as described previously [25]. Weight loss was scored as 0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, over 20. Stool consistency was scored as 0, well-formed pellets; 2, loose stools; and 4, diarrhea. Fecal blood was scored as 0, negative hemoccult test; 2, positive hemoccult test; and 4, gross bleeding. The clinical disease activity index (DAI) was the sum of the above parameters.
2.5. Colon length analysis and histological scoring The mice were sacrificed at the end of the protocol and their colons were excised from cecum to one cm above to anus. The colon length was measured, which indirectly stipulate the inflammatory index of the colon. For the histological analysis, the distal colon samples were fixed immediately in 10% formaldehyde solution, embedded in paraffin, cut into 5 μm thick transversal sections, mounted on glass slides, deparaffinized and stained with hematoxylin and eosin stain (H&E). The slides were monitored under light microscope to check the colonic damage [26]. 2.6. Western immunoblotting analysis Western immunoblotting analysis was carried out as per the earlier method [27]. The tissue samples obtained from the colons were homogenized in lysis buffer. Protein concentration of the homogenates was then measured by the bicinchoninic acid method. For Western blots, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified with the following antibodies to quantify their levels: rabbit anti-growth arrest damage-inducible protein (GADD)-153 (also called as C/EBP homologous protein (CHOP)), rabbit anti-caspase12, rabbit anti cleaved caspase3, rabbit anti-cleaved caspase7, rabbit anti-interleukin (IL)-1β, goat anti-receptor for advanced glycation end products (RAGE), goat anti-toll like receptor (TLR)-2, mouse anti-cyclin D1, and rabbit anti-c-kit and rabbit antiglyceraldehyde 3 phosphate dehydrogenase (GAPDH) antibodies (Santa Cruz Biotechnology, TX, USA or Cell Signaling Technology, Tokyo, Japan). We have used 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA), and the proteins separated were electrophoretically transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk (Sigma, St. Louis, MO, USA) in TBS-T (20 mM/L Tris, pH 7.6, 137 mm/L NaCl and 0.05% Tween) and incubated with primary antibody diluted in the blocking solution (1:1000) at 4 °C overnight. The bound antibody was visualized with respective horseradish peroxidase coupled secondary antibody (Santa Cruz Biotechnology, TX, USA) and chemiluminescence developing agents (Amersham Biosciences, Buckinghamshire, UK). The level of GAPDH was estimated in every sample. Films were scanned and band densities were measured by densitometric analysis using the Scion Image program (Epson GT-X700, Tokyo, Japan). Finally, Western blot data were normalized with GAPDH. 2.7. Evaluation of colonic gene expression by realtime reverse transcriptionpolymerase chain reaction (RT-PCR) RT-PCR was carried out by the already prescribed method from our lab [27]. Colon tissues were preserved in RNAlater (Ambion Inc., Austin, TX, USA) immediately after sacrifice. RNA extraction was performed by using MagNA Pure Compact RNA Isolation Kit (Roche Diagnostics K. K., Tokyo, Japan) according to the manufacturer's protocol. Synthesis of cDNA was performed by reverse transcription using total RNA (2 μg) as a template (Super Sript II; Invitrogen Corp. Carlsbad, CA, USA). Analysis of gene expression was carried out by RT-PCR (Smart cycler; Cepheid, Sunnyvale, CA, USA) using cDNA synthesized from colon tissues. RT-PCR was performed by monitoring with the following TaqMan probes; IL-1β (Mm00434228_ml), IL-2 (Mm00434256_ml), transforming growth factor (TGF)-β (Mm03024053_m1) and GAPDH (Mm99999915_gl) (Applied Biosystems, Foster City, CA, USA), according to the following protocol: 600 s at 95 °C, followed by thermal cycles of 15 s at 95 °C, and 60 s at 60 °C for extension. Relative standard curves representing several 10 fold dilutions (1:10:100:1000:10,000:100,000) of cDNA from colon tissue samples. Results were used for linear regression analysis of other samples. Results were normalized to GAPDH mRNA as an internal control and thus shown as relative mRNA levels.
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2.9. Statistical analysis
3. Results
All the values are expressed as mean ± SEM. Statistical analysis of differences between the groups was performed by one way analysis of variance (ANOVA), followed by Tukey's multiple comparison methods. A value of p b 0.05 was considered statistically significant.
3.1. TOKI ameliorated pathological symptoms in a murine model of DSS colitis Along the experimental period, the control group mice showed clinical pathogenesis of colitis and the TOKI group mice showed milder
Fig. 1. Effect of TOKI treatment on disease severity of DSS-induced acute colitis. A, graph shows the images of colons; B, graph shows disease activity index (DAI) scores; C and D, bar graphs show the percentage body weight change and colon lengths respectively; E, H&E stained colon images. N, Normal mice; DSS, mice supplied 3% DSS in drinking water for 8 days; TOKI, DSS supplied mice treated with TOKI (1 g/kg/day/oral suspension in water) on last 3 days. Data were analyzed by oneway ANOVA followed by Tukey's multiple comparison test where ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 vs N; ###p b 0.001 vs DSS.
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symptoms of colitis when compared to the DSS control group mice (Fig. 1A). In mice with DSS-induced acute colitis, we observed hemorrhage in the colonic lumen, body weight loss and marked diarrhea with bloody stools, which resulted in a sharp increase in DAI, compared with normal control mice (Fig. 1B). TOKI treatment did not completely abolish the abrupt increase in DAI, but reduced its severity. DSS administration was associated with significant body weight reduction and TOKI treatment could not prevent this body weight reduction (Fig. 1C). DSS administration associated with the inflammation of the colon which results in the reduction in colon length. TOKI treatment showed some protection against the inflammation and hence the length of the colon did not reduce significantly (Fig. 1D) compared to control group mice.
apoptosis marker proteins such as cleaved caspase3 and cleaved caspase7 were decreased markedly on treatment with TOKI when compared with the DSS control group (Fig. 2C and D). 3.4. The role of TOKI in the inflammatory mechanism of DSS induced colitis TOKI treatment protected the colon from the infiltration of inflammatory cytokines. Western blotting data showed that there is a marked reduction in the protein expression of inflammatory cytokine IL-1β in the TOKI group when compared with the DSS control group (Fig. 3A). RTPCR analysis has confirmed the effect of TOKI on IL-1β, IL-2 and TGF-β mRNA expressions (Fig. 3B–D). Besides this, RAGE and TLR2 levels were also decreased in the mice treated with TOKI when compared with DSS control group (Fig. 3E and F).
3.2. Effect of TOKI treatment on the colonic architecture 3.5. Effect of TOKI on c-kit and cyclin D1 protein levels H&E stained sections from the colons of the three different groups of mice were observed under light microscope. It showed marked damage in the colonic architecture of the DSS control group mice and the TOKI treatment group showed some protection against the damage induced by DSS administration (Fig. 1E). 3.3. TOKI attenuated DSS induced ER stress and apoptosis in colon The protein expression of CHOP is upregulated in the colon of DSS control group mice and is considered to be an important marker of the ER stress mediated apoptosis. Notably, treatment with TOKI showed a trend towards reduced expression of CHOP in the colon of the TOKI group mice (Fig. 2A). Caspase12 plays a major role in the ER stress mediated apoptosis. Treatment with TOKI significantly decreased the colonic protein level of caspase 12 in TOKI group mice when compared with the DSS control group mice (Fig. 2B). In addition, the downstream
Cyclin D1 is a cell cycle regulatory protein which is upregulated both in immune and epithelial cells during IBD. TOKI treatment showed a significant reduction in the protein level of the colonic cyclin D1 when compared with the DSS control group (Fig. 4A). In addition, c-kit, a tyrosine kinase receptor, level was decreased in DSS control group whereas TOKI treatment showed significantly increased expression (Fig. 4B). 4. Discussion IBD is characterized by the disruption of intestinal barrier, which leads to an excessive production of inflammatory cytokines [1]. The present study used an acute model of colitis with features resembling human UC, such as ulceration, epithelial damage and mucosal inflammatory cellular infiltration. Recent studies have suggested the use of
Fig. 2. Effect of TOKI treatment on ER stress and apoptosis. A–D, the graph shows Western blotting data of CHOP, caspase12, cleaved caspase3 and cleaved caspase7 respectively normalized to their GAPDH content. N, Normal mice; DSS, mice supplied 3% DSS in drinking water for 8 days; TOKI, DSS supplied mice treated with TOKI (1 g/kg/day/oral suspension in water) on last 3 days. Data were analyzed by oneway ANOVA followed by Tukey's multiple comparison test where ⁎⁎p b 0.05, ⁎⁎⁎p b 0.001, vs N; #p b 0.05, ##p b 0.01 vs DSS.
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Fig. 3. Effect of TOKI treatment on inflammatory mechanism of colitis. A & B, Western blotting and mRNA analysis of IL-1β respectively; C & D, mRNA analysis of IL-2 and TGF-β respectively; E & F, Western blotting data of RAGE and TLR2 respectively. GAPDH was used as a control protein. N, Normal mice; DSS, mice supplied 3% DSS in drinking water for 8 days; TOKI, DSS supplied mice treated with TOKI (1 g/kg/day/oral suspension in water) on last 3 days. Data were analyzed by oneway ANOVA followed by Tukey's multiple comparison test where ⁎p b 0.05 vs N; #p b 0.05, ##p b 0.01 vs DSS.
alternative and complementary approach to managing the inflammatory system such as the use of natural products and alternative medicines, which improve the disease condition by preventing inflammatory
cascade [5]. Present study results demonstrated that the Japanese kampo medicine, TOKI, significantly abrogated colitis in the DSSinduced murine model of UC, and the beneficial effects of TOKI
Fig. 4. Protective effect of TOKI on cellular loss in DSS colitis. A & B, graphs show Western blotting analysis of cyclin D1 and c-kit using GAPDH as control. N, Normal mice; DSS, mice supplied 3% DSS in drinking water for 8 days; TOKI, DSS supplied mice treated with TOKI (1 g/kg/day/oral suspension in water) on last 3 days. Data were analyzed by one way ANOVA followed by Tukey's multiple comparison test where ⁎p b 0.05, ⁎⁎⁎p b 0. 001, vs N; #p b 0.05, ###p b 0.001 vs DSS.
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treatment could be linked in part to the inhibition of inflammatory cascade and ER stress mediated apoptosis. DAI has been considered to be an important measure for the clinical pathogenesis of colitis and this may help to assess the condition of patient [28]. It was found that the DSS control group mice suffered from severe colitis as evidenced by the increased DAI score and decreased colon length. Protection from the colon length shortening and stool consistency changes was offered by the treatment with TOKI in mice with DSS induced colitis (Fig. 1). In addition, TOKI offered significant protection to the colonic architecture in colitis mice treated with it (Fig. 1E). Enhanced ER stress has been implicated in various pathological situations including inflammation and there is a definite association between ER stress response and the development of IBD [29]. The transcription factor CHOP is a good marker for ER stress, because it is expressed specifically under the conditions of ER dysfunction [30]. Overexpression of CHOP has been reported to lead to cell cycle arrest and/or apoptosis [31]. The expression of CHOP is upregulated by the induction of colitis with DSS in mice while treatment with TOKI showed decrease in its expression suggesting its protective effect against ER stress (Fig. 2A). Caspase12, a protein associated with the ER membrane, is upregulated in severe ER stress conditions, which leads to apoptosis [32]. In addition, the short prodomain-containing caspases such as caspase3 and caspase7 (downstream apoptosis signals) are activated through the direct proteolytic cleavage by caspase12 [32]. In our present study DSS control group mice showed elevated protein levels of caspase12, cleaved caspase3 and cleaved caspase7 in their colons, while TOKI treatment significantly protected from these changes (Fig. 2B–D). Immune cells, which are infiltrated into the intestinal lumen of patients with IBD, produce various cytokines and that trigger inflammatory responses [5]. In addition, TLRs play a significant role in intestinal homeostasis. Recent studies suggest an important association of TLR2 in the inflammatory cytokine infiltration [33]. Our present study showed an increased level of TLR2 protein expression in DSS induced colitis mice and significant reduction in TOKI treated group (Fig. 3F). TGF-β is well known to play an important role in intestinal fibrosis by inducing collagen production in fibroblast and intestinal smooth muscle cells [34]. mRNA analysis of TGF-β showed a reduced level in TOKI treated mice when compared with the DSS control mice (Fig. 3D). IL-1β, a proinflammatory cytokine, is able to develop several biological effects associated with infection, inflammation and autoimmunity. It can act by inducing upregulated expression of IL-2 by promoting T-cell survival [35]. Both protein expression and mRNA levels of IL-1β were significantly increased in the DSS induced colitis mice, whereas TOKI treatment showed reduction in their levels (Fig. 3A and B). Data from mRNA analysis for IL-2 levels also showed a significant reduction when treated with TOKI (Fig. 3C). RAGE has been implicated in the pathogenesis of numerous inflammatory conditions and is upregulated in the UC patients [36–39]. Our data also suggested the increased level of RAGE in DSS induced colitis in mice (Fig. 3E). Meanwhile, treatment with TOKI reduced its protein expression and offered protection against RAGE mediated inflammation in colitis. Cyclin D1 is a member of cyclin family and cell cycle regulatory protein that is upregulated in IBD in both epithelial and immune cells. It is overexpressed in many human cancers and inflammatory diseases. Because of its importance, it serves as a potential target for the treatment of inflammatory diseases such as IBD [40–43]. Our data from the present study also supported the role of cyclin D1 in UC. In DSS induced colitis mice, the protein expression level of cyclin D1 was significantly elevated and treatment with the kampo medicine TOKI significantly reduced its level (Fig. 4A). There are several studies suggesting the importance of tyrosine kinase receptor c-kit during damage repair and regeneration [44–47]. In our present study c-kit protein expression in the DSS induced colitis mice was significantly lesser than normal mice. But TOKI treated mice showed an elevated level of c-kit protein, which has demonstrated the protective effect of TOKI against DSS induced murine colitis (Fig. 4B).
Taken together TOKI treatment offered significant protection against the clinical features of DSS induced colitis. Reduction in the protein level of CHOP and caspase proteins suggested the ameliorative effect of TOKI treatment on ER stress and ER stress mediated apoptosis. The mRNA, and Western blotting analysis of various inflammatory cytokines such as IL-1β, IL-2, and TGF-β showed marked reduction in their level upon treatment with TOKI. In addition to that, the protein levels of RAGE and TLR2 also decreased markedly with TOKI treatment. All these data suggested the strong anti-inflammatory effect of TOKI in DSS induced colitis. Protein expression of cyclin D1 and c-kit also supported the protective role of TOKI against DSS induced colitis. Therefore, we can strongly suggest the use of this kampo medicine as a better therapeutic option for IBD. Further clinical trials would confirm the beneficial effects in patients with IBD. Conflicts of interest None. Acknowledgment We acknowledge both the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Promotion and Mutual Aid Corporation for Private Schools, Japan for their grant support (23602012 and 26460239 respectively). References [1] R.J. Xavier, D.K. Podolsky, Unravelling the pathogenesis of inflammatory bowel disease, Nature 448 (2007) 427–434. [2] S. Yamada, T. Koyama, H. Noguchi, Y. Ueda, R. Kitsuyama, H. Shimizu, et al., Marine hydroquinone zonarol prevents inflammation and apoptosis in dextran sulfate sodium-induced mice ulcerative colitis, PLoS One 9 (2014), e113509. [3] R. Aldini, R. Budriesi, G. Roda, M. Micucci, P. Ioan, A. D'Errico-Grigioni, et al., Curcuma longa extract exerts a myorelaxant effect on the ileum and colon in a mouse experimental colitis model, independent of the anti-inflammatory effect, PLoS One 7 (2012), e44650. [4] G. Paul, F. Bataille, F. Obermeier, J. Bock, F. Klebl, U. Strauch, et al., Analysis of intestinal haem-oxygenase-1 (HO-1) in clinical and experimental colitis, Clin. Exp. Immunol. 140 (2005) 547–555. [5] S. Arumugam, R.A. Thandavarayan, V. Pitchaimani, V. Karuppagounder, M. Harima, Y. Nishizawa, et al., Prevention of DSS induced acute colitis by Petit Vert, a newly developed function improved vegetable, in mice, PharmaNutrition 2 (2014) 129–134. [6] R.S. Blumberg, L.J. Saubermann, W. Strober, Animal models of mucosal inflammation and their relation to human inflammatory bowel disease, Curr. Opin. Immunol. 11 (1999) 648–656. [7] F.I. Kostadinova, T. Baba, Y. Ishida, T. Kondo, B.K. Popivanova, N. Mukaida, Crucial involvement of the CX3CR1–CX3CL1 axis in dextran sulfate sodium-mediated acute colitis in mice, J. Leukoc. Biol. 88 (2010) 133–143. [8] B. Egger, M. Bajaj-Elliott, T.T. MacDonald, R. Inglin, V.E. Eysselein, M.W. Buchler, Characterisation of acute murine dextran sodium sulphate colitis: cytokine profile and dose dependency, Digestion 62 (2000) 240–248. [9] T. Hanawa, K. Suzuki, Y. Kawauchi, M. Takamura, H. Yoneyama, G.D. Han, et al., Attenuation of mouse acute colitis by naked hepatocyte growth factor gene transfer into the liver, J. Gene Med. 8 (2006) 623–635. [10] S. Bogaert, M. De Vos, K. Olievier, H. Peeters, D. Elewaut, B. Lambrecht, et al., Involvement of endoplasmic reticulum stress in inflammatory bowel disease: a different implication for colonic and ileal disease? PLoS One 6 (2011), e25589. [11] K. Hino, A. Saito, R. Asada, S. Kanemoto, K. Imaizumi, increased susceptibility to dextran sulfate sodium-induced colitis in the endoplasmic reticulum stress transducer OASIS deficient mice, PLoS One 9 (2014), e88048. [12] B. Guo, Z. Li, Endoplasmic reticulum stress in hepatic steatosis and inflammatory bowel diseases, Front. Genet. 5 (2014) 242. [13] D.C. Montrose, N.A. Horelik, J.P. Madigan, G.D. Stoner, L.S. Wang, R.S. Bruno, et al., Anti-inflammatory effects of freeze-dried black raspberry powder in ulcerative colitis, Carcinogenesis 32 (2011) 343–350. [14] N. Kageyama-Yahara, P. Wang, X. Wang, T. Yamamoto, M. Kadowaki, The Inhibitory effect of ergosterol, a bioactive constituent of a traditional Japanese herbal medicine saireito on the activity of mucosal-type mast cells, Biol. Pharm. Bull. 33 (2010) 142–145. [15] S. Ohnishi, H. Takeda, Herbal medicines for the treatment of cancer chemotherapyinduced side effects, Front. Pharmacol. 6 (2015) 14. [16] T. Minagawa, O. Ishizuka, Status of urological kampo medicine: a narrative review and future vision, Int. J. Urol. 22 (2015) 254–263. [17] T. Akase, E. Hihara, T. Shimada, K. Kojima, T. Akase, S. Tashiro, et al., Efficacy of Tokishakuyakusan on the anemia in the iron-deficient pregnant rats, Biol. Pharm. Bull. 30 (2007) 1523–1528.
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Toki-shakuyaku-san, a Japanese kampo medicine, reduces colon inflammation in a mouse model of acute colitis Remya Sreedhar a, Somasundaram Arumugam a, Rajarajan A. Thandavarayan b, Vijayasree V. Giridharan c, Vengadeshprabhu Karuppagounder a, Vigneshwaran Pitchaimani a, Rejina Afrin a, Meilei Harima a, Takashi Nakamura a, Kazuyuki Ueno d, Masahiko Nakamura e, Kenji Suzuki f, Kenichi Watanabe a,⁎ a
Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata City 956-8603, Japan Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, TX 77030, USA c J.K.K Nataraja College of Pharmacy, Natarajapuram, Komarapalayam, 638183, Namakkal District, Tamil Nadu, India d Department of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata City 956-8603, Japan e Department of Cardiology, Yamanashi Prefectural Central Hospital, Kofu, Yamanashi, Japan f Department of Gastroenterology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City 951-8510, Japan b
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Article history: Received 29 May 2015 Received in revised form 20 August 2015 Accepted 24 August 2015 Available online 4 September 2015 Keywords: Apoptosis Endoplasmic reticulum stress Inflammation Inflammatory bowel diseases Kampo medicine Toki-shakuyaku-san
a b s t r a c t Toki-shakuyaku-san (TOKI) is a Japanese kampo medicine, which consists of a mixture of herbal medicines and considered to be a promising remedial agent due to its immunomodulatory and anti-inflammatory effects. We examined the beneficial effects of TOKI in inflammatory bowel disease associated with the inflammation of the intestinal barrier. A study was designed, using C57BL/6 female mice and were administered with 3% DSS in drinking water for 8 days with or without 1 g/kg/day TOKI orally for the last 3 days and a normal group supplied with plain drinking water for 8 days. TOKI treatment attenuated the clinical symptoms of acute murine colitis and also alleviated the inflammatory mechanism by reducing the inflammatory mediators, such as IL-1β, IL-2, TGF-β, RAGE and TLR2. It has also decreased the levels of CHOP, caspase12, cleaved caspase3 and cleaved caspase7 and thereby down-regulated the endoplasmic reticulum stress and apoptotic signaling induced by DSS. Moreover, the expression levels of cyclin D1 and c-kit have also confirmed the beneficial role of TOKI in colitis. All these data suggested that TOKI can be a promising agent for the treatment of colitis since it alleviates the disease progression and severity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis (UC) are characterized by chronic and relapsed inflammation in the intestine, but the etiology and pathogenesis have not been completely understood [1–3]. UC is an intractable IBD that causes inflammation and sores on the inner lining of the digestive tract mainly large intestine [3]. Key features of UC include diffuse mucosal inflammation that reverts proximally from the rectum to a varying degree in conjunction with severe inflammation and the coincident production of a complex mixture of inflammatory mediators, extensive superficial mucosal ulceration and is characterized by rectal bleeding, diarrhea, and abdominal pain [1]. Researchers presumed a number of factors associated with the pathogenesis of this disease probably environmental and genetic factors, which interact with the intestinal mucosal barrier and trigger an event that leads ultimately to a chronic activation of immune and nonimmune cells in the gut [4,5]. Oral administration of ⁎ Corresponding author. E-mail address: [email protected] (K. Watanabe).
http://dx.doi.org/10.1016/j.intimp.2015.08.029 1567-5769/© 2015 Elsevier B.V. All rights reserved.
dextran sulfate sodium (DSS) instigates UC in hamster and mice [5,6]. DSS consumption provokes immense macrophage and granulocyte infiltration, distortion of the anatomy of glands, and ulceration in the colon, which replicates the pathological features in the acute phase of colitis patients and therefore used as a conventional animal model of UC. DSS can damage enterocytes directly and disturb the integrity of the gut barrier and eventually promotes the invasion of intraluminal commensal bacteria [6–8]. Therapy for IBD mainly focuses on the regulation of inflammatory cells and their secretion of various inflammatory mediators like proinflammatory cytokines, chemokines, etc. [9]. Recently, endoplasmic reticulum (ER) stress has been recognized for the induction of inflammatory cytokines [10,11]. Inflammatory cytokines released after the induction of ER stress functions as an alarming or danger signal to communicate with other cells or to recruit immune cells [12]. The incorporation of natural products into the therapeutic regimens is an attractive approach for improving disease treatment due to their generally low toxicity profiles and high patient compliance [13]. Japanese herbal medicines of traditional Chinese origin, also called kampo medicines, are highly standardized for their quality and widely
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used for the treatment of various diseases [14]. Recently several kampo medicines have been investigated using animal studies and clinical trials to evaluate their potential beneficial effects [15]. Almost 150 kampo formulations have been approved as prescription drugs by the Ministry of Health, Labour and Welfare, Japan, and the treatment with kampo medicine is a symptom- and patient-based, therefore, a form of tailor-made drug therapy [16]. Toki-shakuyaku-san (TOKI; “Dang gui shao yao san” in Chinese) is a traditional kampo medicine, which is widely used in Japan, Korea and China to improve blood circulation and to treat various gynecological disorders such as irregular menstruation, dysmenorrhea, endometriosis, menopausal syndromes and sterility [17–22]. The TOKI kampo formulation is made from six different herbs such as Hoelin (Fu ling), Cnidium rhizome (Chuan xiong), Angelica sinensis (Dang gui), Peony root (Shao yao), Atractylodes rhizome (Bai zhu), and Alisma rhizome (Ze xie) [23]. In this study, we used TOKI in an animal model of UC to find out the cardinal effects of this drug in IBD and to find out the role of this drug in the anti-inflammatory cascade. 2. Materials and methods 2.1. Drugs and chemicals DSS (MW 36,000–50,000) was purchased from Wako, Japan. TOKI was obtained from Tsumura & Co., Tokyo, Japan (Prepared under GMP; Standard Commodity Classification No. of Japan—875200). All other chemicals used in this study were purchased from Sigma, Tokyo, Japan until mentioned otherwise. 2.2. Animals C57BL/6J female mice were maintained and housed in the animal facility of the Faculty of Pharmacy, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan. The mice were fed with normal chow diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and water ad libitum. Animals were kept under temperature and humidity controlled conditions and a light-dark cycle of 12:12 h. All the experiments were approved by the regulations of the Committee on Bioethics for Animal Experiments of our institute (approval number—H2705) and were performed under the relevant regulations and guidelines during the light phase of the cycle [24]. 2.3. Experimental design A protocol was designated for 8 days and the mice were allocated into three groups of matching age, sex and number (n = 6–8). In the first group, designated as the normal control, the mice received normal diet and plain drinking water (DW) ad libitum. The second group, designated as the DSS control, received 3% DSS in DW throughout the protocol. The third group, designated as the TOKI group, received 3% DSS in DW and once daily treatment with TOKI suspension in water (1 g/kg/day) orally during the last three days. 2.4. Change in body weight and disease activity index (DAI) Body weight was measured on a daily basis from the prior day of colitis induction and throughout the study protocol. The stool consistency and the presence of bloody stools, as well as rectal bleeding, were observed and scored as described previously [25]. Weight loss was scored as 0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, over 20. Stool consistency was scored as 0, well-formed pellets; 2, loose stools; and 4, diarrhea. Fecal blood was scored as 0, negative hemoccult test; 2, positive hemoccult test; and 4, gross bleeding. The clinical disease activity index (DAI) was the sum of the above parameters.
2.5. Colon length analysis and histological scoring The mice were sacrificed at the end of the protocol and their colons were excised from cecum to one cm above to anus. The colon length was measured, which indirectly stipulate the inflammatory index of the colon. For the histological analysis, the distal colon samples were fixed immediately in 10% formaldehyde solution, embedded in paraffin, cut into 5 μm thick transversal sections, mounted on glass slides, deparaffinized and stained with hematoxylin and eosin stain (H&E). The slides were monitored under light microscope to check the colonic damage [26]. 2.6. Western immunoblotting analysis Western immunoblotting analysis was carried out as per the earlier method [27]. The tissue samples obtained from the colons were homogenized in lysis buffer. Protein concentration of the homogenates was then measured by the bicinchoninic acid method. For Western blots, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified with the following antibodies to quantify their levels: rabbit anti-growth arrest damage-inducible protein (GADD)-153 (also called as C/EBP homologous protein (CHOP)), rabbit anti-caspase12, rabbit anti cleaved caspase3, rabbit anti-cleaved caspase7, rabbit anti-interleukin (IL)-1β, goat anti-receptor for advanced glycation end products (RAGE), goat anti-toll like receptor (TLR)-2, mouse anti-cyclin D1, and rabbit anti-c-kit and rabbit antiglyceraldehyde 3 phosphate dehydrogenase (GAPDH) antibodies (Santa Cruz Biotechnology, TX, USA or Cell Signaling Technology, Tokyo, Japan). We have used 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA), and the proteins separated were electrophoretically transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk (Sigma, St. Louis, MO, USA) in TBS-T (20 mM/L Tris, pH 7.6, 137 mm/L NaCl and 0.05% Tween) and incubated with primary antibody diluted in the blocking solution (1:1000) at 4 °C overnight. The bound antibody was visualized with respective horseradish peroxidase coupled secondary antibody (Santa Cruz Biotechnology, TX, USA) and chemiluminescence developing agents (Amersham Biosciences, Buckinghamshire, UK). The level of GAPDH was estimated in every sample. Films were scanned and band densities were measured by densitometric analysis using the Scion Image program (Epson GT-X700, Tokyo, Japan). Finally, Western blot data were normalized with GAPDH. 2.7. Evaluation of colonic gene expression by realtime reverse transcriptionpolymerase chain reaction (RT-PCR) RT-PCR was carried out by the already prescribed method from our lab [27]. Colon tissues were preserved in RNAlater (Ambion Inc., Austin, TX, USA) immediately after sacrifice. RNA extraction was performed by using MagNA Pure Compact RNA Isolation Kit (Roche Diagnostics K. K., Tokyo, Japan) according to the manufacturer's protocol. Synthesis of cDNA was performed by reverse transcription using total RNA (2 μg) as a template (Super Sript II; Invitrogen Corp. Carlsbad, CA, USA). Analysis of gene expression was carried out by RT-PCR (Smart cycler; Cepheid, Sunnyvale, CA, USA) using cDNA synthesized from colon tissues. RT-PCR was performed by monitoring with the following TaqMan probes; IL-1β (Mm00434228_ml), IL-2 (Mm00434256_ml), transforming growth factor (TGF)-β (Mm03024053_m1) and GAPDH (Mm99999915_gl) (Applied Biosystems, Foster City, CA, USA), according to the following protocol: 600 s at 95 °C, followed by thermal cycles of 15 s at 95 °C, and 60 s at 60 °C for extension. Relative standard curves representing several 10 fold dilutions (1:10:100:1000:10,000:100,000) of cDNA from colon tissue samples. Results were used for linear regression analysis of other samples. Results were normalized to GAPDH mRNA as an internal control and thus shown as relative mRNA levels.
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2.9. Statistical analysis
3. Results
All the values are expressed as mean ± SEM. Statistical analysis of differences between the groups was performed by one way analysis of variance (ANOVA), followed by Tukey's multiple comparison methods. A value of p b 0.05 was considered statistically significant.
3.1. TOKI ameliorated pathological symptoms in a murine model of DSS colitis Along the experimental period, the control group mice showed clinical pathogenesis of colitis and the TOKI group mice showed milder
Fig. 1. Effect of TOKI treatment on disease severity of DSS-induced acute colitis. A, graph shows the images of colons; B, graph shows disease activity index (DAI) scores; C and D, bar graphs show the percentage body weight change and colon lengths respectively; E, H&E stained colon images. N, Normal mice; DSS, mice supplied 3% DSS in drinking water for 8 days; TOKI, DSS supplied mice treated with TOKI (1 g/kg/day/oral suspension in water) on last 3 days. Data were analyzed by oneway ANOVA followed by Tukey's multiple comparison test where ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 vs N; ###p b 0.001 vs DSS.
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symptoms of colitis when compared to the DSS control group mice (Fig. 1A). In mice with DSS-induced acute colitis, we observed hemorrhage in the colonic lumen, body weight loss and marked diarrhea with bloody stools, which resulted in a sharp increase in DAI, compared with normal control mice (Fig. 1B). TOKI treatment did not completely abolish the abrupt increase in DAI, but reduced its severity. DSS administration was associated with significant body weight reduction and TOKI treatment could not prevent this body weight reduction (Fig. 1C). DSS administration associated with the inflammation of the colon which results in the reduction in colon length. TOKI treatment showed some protection against the inflammation and hence the length of the colon did not reduce significantly (Fig. 1D) compared to control group mice.
apoptosis marker proteins such as cleaved caspase3 and cleaved caspase7 were decreased markedly on treatment with TOKI when compared with the DSS control group (Fig. 2C and D). 3.4. The role of TOKI in the inflammatory mechanism of DSS induced colitis TOKI treatment protected the colon from the infiltration of inflammatory cytokines. Western blotting data showed that there is a marked reduction in the protein expression of inflammatory cytokine IL-1β in the TOKI group when compared with the DSS control group (Fig. 3A). RTPCR analysis has confirmed the effect of TOKI on IL-1β, IL-2 and TGF-β mRNA expressions (Fig. 3B–D). Besides this, RAGE and TLR2 levels were also decreased in the mice treated with TOKI when compared with DSS control group (Fig. 3E and F).
3.2. Effect of TOKI treatment on the colonic architecture 3.5. Effect of TOKI on c-kit and cyclin D1 protein levels H&E stained sections from the colons of the three different groups of mice were observed under light microscope. It showed marked damage in the colonic architecture of the DSS control group mice and the TOKI treatment group showed some protection against the damage induced by DSS administration (Fig. 1E). 3.3. TOKI attenuated DSS induced ER stress and apoptosis in colon The protein expression of CHOP is upregulated in the colon of DSS control group mice and is considered to be an important marker of the ER stress mediated apoptosis. Notably, treatment with TOKI showed a trend towards reduced expression of CHOP in the colon of the TOKI group mice (Fig. 2A). Caspase12 plays a major role in the ER stress mediated apoptosis. Treatment with TOKI significantly decreased the colonic protein level of caspase 12 in TOKI group mice when compared with the DSS control group mice (Fig. 2B). In addition, the downstream
Cyclin D1 is a cell cycle regulatory protein which is upregulated both in immune and epithelial cells during IBD. TOKI treatment showed a significant reduction in the protein level of the colonic cyclin D1 when compared with the DSS control group (Fig. 4A). In addition, c-kit, a tyrosine kinase receptor, level was decreased in DSS control group whereas TOKI treatment showed significantly increased expression (Fig. 4B). 4. Discussion IBD is characterized by the disruption of intestinal barrier, which leads to an excessive production of inflammatory cytokines [1]. The present study used an acute model of colitis with features resembling human UC, such as ulceration, epithelial damage and mucosal inflammatory cellular infiltration. Recent studies have suggested the use of
Fig. 2. Effect of TOKI treatment on ER stress and apoptosis. A–D, the graph shows Western blotting data of CHOP, caspase12, cleaved caspase3 and cleaved caspase7 respectively normalized to their GAPDH content. N, Normal mice; DSS, mice supplied 3% DSS in drinking water for 8 days; TOKI, DSS supplied mice treated with TOKI (1 g/kg/day/oral suspension in water) on last 3 days. Data were analyzed by oneway ANOVA followed by Tukey's multiple comparison test where ⁎⁎p b 0.05, ⁎⁎⁎p b 0.001, vs N; #p b 0.05, ##p b 0.01 vs DSS.
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Fig. 3. Effect of TOKI treatment on inflammatory mechanism of colitis. A & B, Western blotting and mRNA analysis of IL-1β respectively; C & D, mRNA analysis of IL-2 and TGF-β respectively; E & F, Western blotting data of RAGE and TLR2 respectively. GAPDH was used as a control protein. N, Normal mice; DSS, mice supplied 3% DSS in drinking water for 8 days; TOKI, DSS supplied mice treated with TOKI (1 g/kg/day/oral suspension in water) on last 3 days. Data were analyzed by oneway ANOVA followed by Tukey's multiple comparison test where ⁎p b 0.05 vs N; #p b 0.05, ##p b 0.01 vs DSS.
alternative and complementary approach to managing the inflammatory system such as the use of natural products and alternative medicines, which improve the disease condition by preventing inflammatory
cascade [5]. Present study results demonstrated that the Japanese kampo medicine, TOKI, significantly abrogated colitis in the DSSinduced murine model of UC, and the beneficial effects of TOKI
Fig. 4. Protective effect of TOKI on cellular loss in DSS colitis. A & B, graphs show Western blotting analysis of cyclin D1 and c-kit using GAPDH as control. N, Normal mice; DSS, mice supplied 3% DSS in drinking water for 8 days; TOKI, DSS supplied mice treated with TOKI (1 g/kg/day/oral suspension in water) on last 3 days. Data were analyzed by one way ANOVA followed by Tukey's multiple comparison test where ⁎p b 0.05, ⁎⁎⁎p b 0. 001, vs N; #p b 0.05, ###p b 0.001 vs DSS.
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treatment could be linked in part to the inhibition of inflammatory cascade and ER stress mediated apoptosis. DAI has been considered to be an important measure for the clinical pathogenesis of colitis and this may help to assess the condition of patient [28]. It was found that the DSS control group mice suffered from severe colitis as evidenced by the increased DAI score and decreased colon length. Protection from the colon length shortening and stool consistency changes was offered by the treatment with TOKI in mice with DSS induced colitis (Fig. 1). In addition, TOKI offered significant protection to the colonic architecture in colitis mice treated with it (Fig. 1E). Enhanced ER stress has been implicated in various pathological situations including inflammation and there is a definite association between ER stress response and the development of IBD [29]. The transcription factor CHOP is a good marker for ER stress, because it is expressed specifically under the conditions of ER dysfunction [30]. Overexpression of CHOP has been reported to lead to cell cycle arrest and/or apoptosis [31]. The expression of CHOP is upregulated by the induction of colitis with DSS in mice while treatment with TOKI showed decrease in its expression suggesting its protective effect against ER stress (Fig. 2A). Caspase12, a protein associated with the ER membrane, is upregulated in severe ER stress conditions, which leads to apoptosis [32]. In addition, the short prodomain-containing caspases such as caspase3 and caspase7 (downstream apoptosis signals) are activated through the direct proteolytic cleavage by caspase12 [32]. In our present study DSS control group mice showed elevated protein levels of caspase12, cleaved caspase3 and cleaved caspase7 in their colons, while TOKI treatment significantly protected from these changes (Fig. 2B–D). Immune cells, which are infiltrated into the intestinal lumen of patients with IBD, produce various cytokines and that trigger inflammatory responses [5]. In addition, TLRs play a significant role in intestinal homeostasis. Recent studies suggest an important association of TLR2 in the inflammatory cytokine infiltration [33]. Our present study showed an increased level of TLR2 protein expression in DSS induced colitis mice and significant reduction in TOKI treated group (Fig. 3F). TGF-β is well known to play an important role in intestinal fibrosis by inducing collagen production in fibroblast and intestinal smooth muscle cells [34]. mRNA analysis of TGF-β showed a reduced level in TOKI treated mice when compared with the DSS control mice (Fig. 3D). IL-1β, a proinflammatory cytokine, is able to develop several biological effects associated with infection, inflammation and autoimmunity. It can act by inducing upregulated expression of IL-2 by promoting T-cell survival [35]. Both protein expression and mRNA levels of IL-1β were significantly increased in the DSS induced colitis mice, whereas TOKI treatment showed reduction in their levels (Fig. 3A and B). Data from mRNA analysis for IL-2 levels also showed a significant reduction when treated with TOKI (Fig. 3C). RAGE has been implicated in the pathogenesis of numerous inflammatory conditions and is upregulated in the UC patients [36–39]. Our data also suggested the increased level of RAGE in DSS induced colitis in mice (Fig. 3E). Meanwhile, treatment with TOKI reduced its protein expression and offered protection against RAGE mediated inflammation in colitis. Cyclin D1 is a member of cyclin family and cell cycle regulatory protein that is upregulated in IBD in both epithelial and immune cells. It is overexpressed in many human cancers and inflammatory diseases. Because of its importance, it serves as a potential target for the treatment of inflammatory diseases such as IBD [40–43]. Our data from the present study also supported the role of cyclin D1 in UC. In DSS induced colitis mice, the protein expression level of cyclin D1 was significantly elevated and treatment with the kampo medicine TOKI significantly reduced its level (Fig. 4A). There are several studies suggesting the importance of tyrosine kinase receptor c-kit during damage repair and regeneration [44–47]. In our present study c-kit protein expression in the DSS induced colitis mice was significantly lesser than normal mice. But TOKI treated mice showed an elevated level of c-kit protein, which has demonstrated the protective effect of TOKI against DSS induced murine colitis (Fig. 4B).
Taken together TOKI treatment offered significant protection against the clinical features of DSS induced colitis. Reduction in the protein level of CHOP and caspase proteins suggested the ameliorative effect of TOKI treatment on ER stress and ER stress mediated apoptosis. The mRNA, and Western blotting analysis of various inflammatory cytokines such as IL-1β, IL-2, and TGF-β showed marked reduction in their level upon treatment with TOKI. In addition to that, the protein levels of RAGE and TLR2 also decreased markedly with TOKI treatment. All these data suggested the strong anti-inflammatory effect of TOKI in DSS induced colitis. Protein expression of cyclin D1 and c-kit also supported the protective role of TOKI against DSS induced colitis. Therefore, we can strongly suggest the use of this kampo medicine as a better therapeutic option for IBD. Further clinical trials would confirm the beneficial effects in patients with IBD. Conflicts of interest None. Acknowledgment We acknowledge both the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Promotion and Mutual Aid Corporation for Private Schools, Japan for their grant support (23602012 and 26460239 respectively). References [1] R.J. Xavier, D.K. Podolsky, Unravelling the pathogenesis of inflammatory bowel disease, Nature 448 (2007) 427–434. [2] S. Yamada, T. Koyama, H. Noguchi, Y. Ueda, R. Kitsuyama, H. Shimizu, et al., Marine hydroquinone zonarol prevents inflammation and apoptosis in dextran sulfate sodium-induced mice ulcerative colitis, PLoS One 9 (2014), e113509. [3] R. Aldini, R. Budriesi, G. Roda, M. Micucci, P. Ioan, A. D'Errico-Grigioni, et al., Curcuma longa extract exerts a myorelaxant effect on the ileum and colon in a mouse experimental colitis model, independent of the anti-inflammatory effect, PLoS One 7 (2012), e44650. [4] G. Paul, F. Bataille, F. Obermeier, J. Bock, F. Klebl, U. Strauch, et al., Analysis of intestinal haem-oxygenase-1 (HO-1) in clinical and experimental colitis, Clin. Exp. Immunol. 140 (2005) 547–555. [5] S. Arumugam, R.A. Thandavarayan, V. Pitchaimani, V. Karuppagounder, M. Harima, Y. Nishizawa, et al., Prevention of DSS induced acute colitis by Petit Vert, a newly developed function improved vegetable, in mice, PharmaNutrition 2 (2014) 129–134. [6] R.S. Blumberg, L.J. Saubermann, W. Strober, Animal models of mucosal inflammation and their relation to human inflammatory bowel disease, Curr. Opin. Immunol. 11 (1999) 648–656. [7] F.I. Kostadinova, T. Baba, Y. Ishida, T. Kondo, B.K. Popivanova, N. Mukaida, Crucial involvement of the CX3CR1–CX3CL1 axis in dextran sulfate sodium-mediated acute colitis in mice, J. Leukoc. Biol. 88 (2010) 133–143. [8] B. Egger, M. Bajaj-Elliott, T.T. MacDonald, R. Inglin, V.E. Eysselein, M.W. Buchler, Characterisation of acute murine dextran sodium sulphate colitis: cytokine profile and dose dependency, Digestion 62 (2000) 240–248. [9] T. Hanawa, K. Suzuki, Y. Kawauchi, M. Takamura, H. Yoneyama, G.D. Han, et al., Attenuation of mouse acute colitis by naked hepatocyte growth factor gene transfer into the liver, J. Gene Med. 8 (2006) 623–635. [10] S. Bogaert, M. De Vos, K. Olievier, H. Peeters, D. Elewaut, B. Lambrecht, et al., Involvement of endoplasmic reticulum stress in inflammatory bowel disease: a different implication for colonic and ileal disease? PLoS One 6 (2011), e25589. [11] K. Hino, A. Saito, R. Asada, S. Kanemoto, K. Imaizumi, increased susceptibility to dextran sulfate sodium-induced colitis in the endoplasmic reticulum stress transducer OASIS deficient mice, PLoS One 9 (2014), e88048. [12] B. Guo, Z. Li, Endoplasmic reticulum stress in hepatic steatosis and inflammatory bowel diseases, Front. Genet. 5 (2014) 242. [13] D.C. Montrose, N.A. Horelik, J.P. Madigan, G.D. Stoner, L.S. Wang, R.S. Bruno, et al., Anti-inflammatory effects of freeze-dried black raspberry powder in ulcerative colitis, Carcinogenesis 32 (2011) 343–350. [14] N. Kageyama-Yahara, P. Wang, X. Wang, T. Yamamoto, M. Kadowaki, The Inhibitory effect of ergosterol, a bioactive constituent of a traditional Japanese herbal medicine saireito on the activity of mucosal-type mast cells, Biol. Pharm. Bull. 33 (2010) 142–145. [15] S. Ohnishi, H. Takeda, Herbal medicines for the treatment of cancer chemotherapyinduced side effects, Front. Pharmacol. 6 (2015) 14. [16] T. Minagawa, O. Ishizuka, Status of urological kampo medicine: a narrative review and future vision, Int. J. Urol. 22 (2015) 254–263. [17] T. Akase, E. Hihara, T. Shimada, K. Kojima, T. Akase, S. Tashiro, et al., Efficacy of Tokishakuyakusan on the anemia in the iron-deficient pregnant rats, Biol. Pharm. Bull. 30 (2007) 1523–1528.
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