Zanthoxylum bungeanum pericarp extract prevents dextran sulfate sodium-induced experimental colitis in mice via the regulation of TLR4 and TLR4-related signaling pathways

INTIMP-04367; No of Pages 9 International Immunopharmacology xxx (2016) xxx–xxx

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Zanthoxylum bungeanum pericarp extract prevents dextran sulfate sodium-induced experimental colitis in mice via the regulation of TLR4 and TLR4-related signaling pathways Zecai Zhang, Jiuxi Liu, Peng Shen, Yongguo Cao, Xiaojie Lu, Xuejiao Gao, Yunhe Fu, Bo Liu ⁎, Naisheng Zhang ⁎ College of Veterinary Medicine, Jilin University, Changchun 130062, People's Republic of China

a r t i c l e

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Article history: Received 21 June 2016 Received in revised form 27 October 2016 Accepted 27 October 2016 Available online xxxx Keywords: Zanthoxylum bungeanum J774.1 cells Colitis Inflammation Signaling pathway

a b s t r a c t Zanthoxylum bungeanum, which belongs to the Zanthoxylum genus of the Rutaceae family, is now wildly distributed in most parts of China and some Southeast Asian countries. The pericarp of Zanthoxylum bungeanum has been known to exhibit antibacterial, anti-inflammatory and other important therapeutic activities. The purpose of this study was to investigate the effects and mechanisms of Zanthoxylum bungeanum pericarp extract (ZBE) on DSS-induced experimental colitis in mice. The results demonstrated that the major flavonoid composition of ZBE includes rutin (32.36%), quercetin (13.61%) and isoquercitrin (24.89%). ZBE alleviated DSS-induced body weight loss, colon length shortening and colonic pathological damage. Furthermore, ZBE inhibited the expression of TNFα, IL-1β and IL-12 via the regulation of TLR4 and TLR4-related pathways in DSS-induced experimental colitis in mice and LPS-triggered inflammation in J774.1 cells. Our findings suggest that ZBE is effective in ameliorating experimental colitis, and further investigation is necessary on the use of ZBE as a new dietary strategy to lower the risk of ulcerative colitis (UC). © 2016 Elsevier B.V. All rights reserved.

1. Introduction Ulcerative colitis (UC), a chronic and relapsing inflammatory disease of the gastrointestinal tract, not only affects millions of patients worldwide, but also increases the risk of colon cancer [1,2]. To study this disease, a model of colitis in mice has been used by the oral administration of dextran sulfate sodium (DSS) [3]. This model could research into the pathogenesis of UC and is similar to human UC. Although the etiology and pathogenesis of UC are complicated and remain uncertain, the intestinal mucosa of UC patients is reported to be characterized by excessive immune responses and lead to the damage of intestinal epithelial barrier by the abnormal activity of some pro-inflammatory signals, such as toll-like receptor 4 (TLR4). Several pathways are correlated with colonic inflammation. However, TLR4 represents one of the important mechanisms. It has been reported that the targeted suppression of the TLR4 pathway has also become a treatment strategy for UC in recent years [4]. Nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) are two major inflammatory pathways downstream of TLR4. Several studies have also indicated that the inhibition of NF-κB and MAPK activation are effective treatments for the prevention of UC ⁎ Corresponding authors. E-mail addresses: [email protected] (B. Liu), [email protected] (N. Zhang).

[5]. Currently, most therapeutic drugs have been used to relieve the symptoms of patients with UC, including glucocorticosteroids, immunosuppressive agents and anti-TNF-a monoclonal antibody. However, most of these agents are not very effective and have severe side effects particularly for long-term therapy [6]. To address these problems, scientists have tried to develop novel agents, especially from natural compounds [7]. Zanthoxylum bungeanum, which belongs to the Zanthoxylum genus of the Rutaceae family, is now wildly distributed in most parts of China and some Southeast Asian countries. Z. bungeanum is rich in flavonoids and mainly contains rutin, quercetin, foeniculin, hyperin and isoquercitrin [8]. The pericarp of Z. bungeanum is now wildly used as a kind of spice to produce a fresh flavor and as traditional Chinese medicine due to its therapeutic properties [9]. Z. bungeanum has wide variety of biological and pharmacological activities, including anti-inflammation, antioxidant and antibacterial properties [10,11]. It is well known that abdominal pain and diarrhea are the most common symptoms in patients with colitis. Z. bungeanum has been reported to improve the function of the gastrointestinal tract and relieve diarrhea. Furthermore, Z. bungeanum is effective for the treatment of epigastric pain, stomachache, ascariosis and dysentery. However, direct evidence for the effect of ZBE on mice colitis has not yet been elucidated. Here, we examined the protective effects and mechanisms of ZBE on mice J774.1 cells and DSS-

http://dx.doi.org/10.1016/j.intimp.2016.10.021 1567-5769/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Z. Zhang, et al., Zanthoxylum bungeanum pericarp extract prevents dextran sulfate sodium-induced experimental colitis in mice via the regulation of TLR4 and TLR4-related.., Int Immunopharmacol (2016), http://dx.doi.org/10.1016/j.intimp.2016.10.021

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induced UC model mice to provide an experimental basis for a new dietary strategy for the prevention of UC. 2. Materials and methods

b.wt.); High dose ZBE group (H-ZBE, 2 g/kg b.wt.), and DSS group. Acute colitis was induced with 2.5% (wt/vol) DSS for 7 d. In the ZBE groups, the mice were treated with different doses of ZBE 14 days before and during DSS treatment via oral gavage once per day [14,15]. Mice in the control group and DSS group were given the same volume of water.

2.1. Reagents and chemicals LPS (Escherichia coli 055:B5) was purchased from Sigma Chemical CO (St. Louis, MO, USA). DSS (molecular weight 36–50 kDa) was obtained from MP Biomedicals, Morgan Irvine, CA. Mouse TNF-α, IL-1β and IL6 enzyme-linked immunosorbent assay (ELISA) kits were obtained from Biolegend (San Diego, CA, USA). Rabbit mAb, IκBα, p65, p38, ERK, and JNK, and mouse monoclonal antibodies p-IκBα, p-p65, pp38, p-ERK, and p-JNK were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Mouse mAb TLR4 were purchased from GeneTex. β-Actin and horseradish peroxidase conjugated goat anti-rabbit and goat anti-mouse antibodies were provided by Tianjin Sungene Biotech Co., Ltd. (Tianjin, China). The Nuclear and Cytoplasmic Protein Extraction Kit was provided by Beyotime Institute of Biotechnology. All other chemicals were of reagent grade. 2.2. Composition and preparation of ZBE The pericarp of Z. bungeanum (red huajiao) was purchased at Hanyuan, Sichuan Province, China. Z. bungeanum 100 g was soaked in the sixfold distilled water for 2 h before heated. The herbs were heated to 100 °C for 30 min in aqueous extract after adding 600 ml of water, and the decoction was filtered. Approximately 250 ml of water was added for a second extraction under the same conditions [12]. The supernatant obtained from the twice decoction was vacuum evaporated to a final density of 1 g/ml at 65 °C by a rotavapor. The supernatant of ZBE filtered through a 0.22 μm filter were used in vitro and in vivo experiment. Flavonoids were determined by HPLC. The test was performed on an Agilent 1100 series apparatus (Agilent Technologies, Palo Alto, CA). Chromatography was performed through an ODS-3 analytical HPLC column (5 μm, 150 × 4.6 mm, Phenomenex, Torrance, CA). Elution was carried out with acetonitrile/ultrapure water (v/v, 70:30), operating at a flow rate of 1 ml/min with UV detection at 294 nm. The column temperature was controlled at 35 °C. The total flavonoid content (TFC) was detected according to a SBC assay using sodium borohydride/chloranil [13].

2.5. Clinical scoring and histological analysis Body weight was measured on a daily basis. Stool consistency and blood in the stool were detected from the prior day of colitis induction and throughout the study protocol, and observed and scored as described previously [16]. The clinical disease activity index (DAI) was the sum of the clinical score [17]. On the 7 days of the colitis induced by DSS, mice were sacrificed, and the colon was excised from cecum to one cm above the anus. The length of the colon was detected, which indirectly stipulated the inflammatory index of the colon. For the histological analysis, the colon specimens were fixed in 10% formalin. The colon specimens were embedded in paraffin and then deparaffinized with xylene and rehydrated. Histological grading was conducted according to a previously described method [18]. 2.6. Cytokine analysis by ELISA The J774.1 cells were pretreated with or without ZBE (100, 200, and 400 μg/ml) for 1 h, and then treated with 1 μg/ml of LPS for 18 h. Adipose tissue was removed from excised colons and the colon segment was opened longitudinally and washed with PBS. Colon tissue (50 mg per well) was cultured in RPMI 1640 medium with 100 U/ml of penicillin and 100 mg/ml of streptomycin at 37 °C with 5% CO2 for 24 h. After 24 h, colon supernatants were collected and centrifuged at 12,000 ×g at 4 °C for 10 min. The TNF-α, IL-1β and IL-12 autocrine levels in the colon were quantified with ELISA kits according to the manufacturer's protocol. 2.7. Preparation of caecal bacterial lysates Caecal bacterial lysates (CBL) were prepared as described by Dieleman et al. [19]. Briefly, the caecal contents in each group were solubilized by vortexing the contents in RPMI-1640 medium, and then incubating them with 0.01 M MgCl2, 10 μg/ml DNase. Then, the contents were homogenized with 0.1 mm glass beads for 3 min. The homogenate

2.3. Cell culture and viability assay The J774.1 cells were purchased from China Cell Line Bank (Beijing, China) and cultured in RPMI-1640 medium with 10% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C with 5% CO2. The J774.1 cells were pretreated with or without ZBE (100, 200, and 400 μg/ml) for 1 h. After that, J774.1 cells were treated with LPS (1 μg/ml). After 18 h of LPS stimulation, MTT (20 μl of 5 mg/ml) was added to each well for 4 h. The supernatant was removed and dimethyl sulfoxide (150 μl per well) was added. The optical density was tested at 570 nm using a microplate reader. 2.4. Animals and mice model of DSS-induced colitis Male C57BL/6 mice (21–23 g) were purchased from the Center of Experimental Animals of Jilin University (Jilin, China). The mice were provided water and food ad libitum. The laboratory temperature maintained at 24 ± 1 °C. All experimental protocols were guided in accordance with the approval of the Institutional Animal Care and Use Committee of our university under the approved protocol number SCXXK (JI-2011-0004). The samples was randomly divided into five groups of eight animals per group: Control group; Low dose ZBE (L-ZBE, 0.5 g dry weight of crude extract/kg body weight); Middle dose ZBE group (M-ZBE, 1 g/kg

Fig. 1. High-performance liquid chromatography (HPLC) analysis of ZBE. Peaks: 1, rutin (32.36%); 2, quercetin (13.61%); 3, isoquercitrin (24.89%).

Please cite this article as: Z. Zhang, et al., Zanthoxylum bungeanum pericarp extract prevents dextran sulfate sodium-induced experimental colitis in mice via the regulation of TLR4 and TLR4-related.., Int Immunopharmacol (2016), http://dx.doi.org/10.1016/j.intimp.2016.10.021

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described by Ruyssers [20]. Approximately 4 × 105 MLN cells and 20 μg/ml CBL were cultured in RPMI-1640 medium with 10% fetal bovine serum and 50 mg/ml gentamicin at 37 °C with 5% CO2 for 72 h. The culture media was then collected for cytokine analysis and stored at −20 °C. 2.9. Western blotting analysis

Fig. 2. Effect of ZBE on the cell viability of J774.1 cells. Cells were treated with different concentrations of ZBE (0–400 μg/ml) in the absence or presence of LPS (1 μg/ml) for 24 h. Cell viability was determined using MTT assay. The values presented are the means ± SEM of three independent experiments.

was centrifuged at 10,000g for 10 min. The supernatant was filtered through a 0.45 μM filter. 2.8. Mesenteric lymph node cell cultures Mesenteric lymph nodes (MLNs) were harvested from mice of five experimental groups. Single cell suspensions were prepared as

J774.1 cells were treated with ZBE (100, 200, and 400 μg/ml) for 1 h. After LPS treatment for 1 h, total cellular proteins were extracted. Segments of the colon were homogenized, and total protein was extracted. Protein contents in all samples were quantified with the Pierce BCA protein assay kit (23,227, Thermo, USA). Samples with equal amounts of protein (40 μg) were fractionated on 10% SDS polyacrylamide gels and transferred onto PVDF membranes. Next, the blocked membrane with 5% nonfat milk was probed with primary antibodies. Then, an appropriate secondary antibody was applied for 1 h. The blots were tested using Western blotting detection program. The β-actin was used as an internal control of protein loading. 2.10. Statistical analysis Statistical analyses of the data were performed using SPSS software (ver. 17 for Windows; SPSS Inc., Chicago, IL, USA). Differences between the mean values of normally distributed data were assessed with oneway ANOVA (Dunnett's test) and the two-tailed Student's t-test. P values of 0.05 or less were considered statistically significant.

Fig. 3. ZBE treatment improved DSS-induced colitis in mice. (A) Body weight change of each group. (B) Disease activity index (DAI). (C) The lengths of colons from each group of mice were measured. Data were presented as the means ± SEM (n = 8 per group). (∗) p b 0.05 and (∗∗) p b 0.01 versus the DSS-treated group on the same day; (#) p b 0.05 compared with control group.

Please cite this article as: Z. Zhang, et al., Zanthoxylum bungeanum pericarp extract prevents dextran sulfate sodium-induced experimental colitis in mice via the regulation of TLR4 and TLR4-related.., Int Immunopharmacol (2016), http://dx.doi.org/10.1016/j.intimp.2016.10.021

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3. Results

3.2. Effects of ZBE on cell viability

3.1. Analysis of ZBE

The potential cytotoxicity of ZBE was evaluated by the MTT assay after incubating cells for 18 h in the absence or presence of LPS. The results are shown in Fig. 2. The result showed that the cell viabilities were not affected by the ZBE (100, 200, and 400 μg/ml). This result indicated that the viabilities of J774.1 cells were irrelevant to ZBE supplementation.

In the present study, quantitative phytochemical analysis of ZBE was performed to determine the flavonoid content. For repeatability and reproducibility, three samples were chosen for quantitative analysis. The results showed that the content of total flavonoids in ZBE was 40.46 ± 1.56 mg/ml. The total amount was estimated to be equal to 80.92 ± 3.12 mg per g of dried extract. In qualitative terms (as shown in Fig. 1), the major three peaks obtained from flavonoids were rutin (32.36%), quercetin (13.61%) and isoquercitrin (24.89%), which indicated that they may be the major components in the ZBE.

3.3. ZBE attenuated colitis induced by DSS To investigate the protective effect of ZBE on UC, we established a model of DSS-induced mice colitis. The results are shown in Fig. 3.

Fig. 4. ZBE treatment prevented DSS-induced colon damage in mice (H&E staining ×100). (A) The colons from each experimental group were processed for histological evaluation. Representative histological changes of colons obtained from mice in different groups. (B) Histopathological scores. Data are presented as means ± SEM (n = 7). (∗) p b 0.05, (∗∗) p b 0.01 versus DSS-treated alone group; (#) p b 0.05 compared with control group.

Please cite this article as: Z. Zhang, et al., Zanthoxylum bungeanum pericarp extract prevents dextran sulfate sodium-induced experimental colitis in mice via the regulation of TLR4 and TLR4-related.., Int Immunopharmacol (2016), http://dx.doi.org/10.1016/j.intimp.2016.10.021

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Compared with the control group, mice in the DSS group had a significant loss of body weight. However, ZBE (0.5, 1, and 2 g/kg b.wt.) significantly attenuated the body weight loss during the progression of experimental colitis in mice (Fig. 3A). ZBE significantly decreased the DAI, a clinical parameter reflecting the severity of weight loss, stool consistency and blood in stool (Fig. 3B), in a dose-dependent manner. In addition, DSS typically caused colonic shortening, while this change was significantly improved in the M-ZBE and H-ZBE groups (Fig. 3C). These results suggested that the DSS-induced colon damage was markedly restored by ZBE treatment in a dose-dependent manner. 3.4. ZBE decreased histopathological changes in DSS-induced colitis in mice H&E stained sections of colon segments were observed under a light microscope. The control group showed intact surface epithelium, cryptal glands, and submucosa, whereas overall damage to the surface epithelium, disruption of cryptal glands, and infiltration of inflammatory cells were observed in the DSS group. The ZBE (0.5, 1 g/kg b.wt.) treatment groups showed a relatively intact surface epithelium and cryptal glands. The ZBE (2 g/kg b.wt.) treatment group presented more intact surface epithelium and cryptal glands than those in the DSS and ZBE (0.5, 1 g/kg b.wt.) treatment groups (Fig. 4A). All treatment

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groups had significantly lower histological scores than that observed in the DSS group (Fig. 4B). 3.5. ZBE decreased pro-inflammatory cytokines in ex vivo and in vitro As an additional marker of local inflammation, the concentrations of cytokines were measured in ex vivo and in vitro. The results showed that the levels of TNF-α, IL-1β and IL-12 were remarkably enhanced after DSS challenge and LPS stimulation (Fig. 5). The administration of ZBE (0.5, 1, and 2 g/kg b.wt.) down-regulated the production of inflammatory cytokines in colon explants and J774.1 cells in a dose-dependent manner (Fig. 5A, B). Moreover, to investigate the effect of ZBE on hostdependent immune responses, we analyzed the cytokine responses of the MLN cells to CBL. The results showed that ZBE also significantly reduced the elevated expression of these cytokines in a dose-dependent manner (Fig. 5C). 3.6. ZBE reduced DSS-induced TLR4 expression in vivo and in vitro Many studies have shown that the TLR4 plays a key role in the pathogenesis of UC. Therefore, the targeted suppression of the TLR4 pathway has become a treatment strategy for UC in recent years [4]. In the

Fig. 5. Cytokine concentrations. (A) Cells were treated with 1 μg/ml LPS in absence or presence of ZBE (100, 200, and 400 μg/ml) for 18 h. Levels of TNF-α, IL-β, and IL-12 in culture supernatants were measured by ELISA (n = 3). (B) Effects of different doses of ZBE (0.5, 1, and 2 g/kg b.wt.) on TNF-α, IL-β, and IL-12 levels in colonic cultures, and (C) in MLN (n = 7). Data are presented as means ± SEM. (∗) p b 0.05, (∗∗) p b 0.01 versus DSS-treated alone group; (#) p b 0.05 compared with control group.

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present study, there was a significant increase in the expression of TLR4 in the DSS-induced group compared with the control group. However, treatment with ZBE (0.5, 1 g/kg b.wt.) could decrease TLR4 expression. The ZBE (2 g/kg b.wt.) treatment group presented more obvious inhibitory effect than those in the DSS and ZBE (0.5, 1 g/kg b.wt.) treatment groups (Fig. 6B). Furthermore, the result also demonstrated that ZBE could decrease the increase of LPS-stimulated TLR4 expression (Fig. 6A). These results suggested that ZBE might provide protection from colitis by the suppression of the TLR4 pathway. 3.7. The effect of ZBE on the NF-κB signaling pathway in vivo and in vitro NF-κB, an important signaling pathway, plays a pivotal role in regulating cytokine expression. To detect whether the suppression of inflammation by ZBE is mediated by the NF-κB pathway, NF-κB p65 and IκBα phosphorylation levels were determined. As shown in Fig. 7A, a significant increase in the phosphorylation of NF-κB p65 and IκBα was found in the LPS group compared with the control group. However, the phosphorylation of NF-κB p65 and IκBα were reduced significantly in the ZBE groups. Furthermore, we examined the effect of ZBE on the expression of NF-κB in colon tissue using Western blot. The results are shown in Fig. 7B. Treatment with DSS activated NF-κB signal pathway and obviously enhanced the level of NF-κB (p65 NF-κB) protein expression in the colon tissue. While treatment with ZBE clearly inhibited NF-κB p65 and IκBα phosphorylation levels in a dose dependent manner. 3.8. Effect of ZBM on the MAPK signaling pathway in vivo and in vitro MAPKs are also important pathways in regulating the development of inflammation. To further elucidate the detailed mechanisms, we investigated whether ZBE could affect MAPKs pathways. As expected, treatment with DSS obviously increased the phosphorylation of P38, ERK, and JNK in colon tissues compared with control group. The administration of ZBE alleviated the DSS-induced increase of the protein phosphorylation of three MAPKs in a dose dependent manner (Fig. 8B). Furthermore, we also investigated the effect of ZBE on the phosphorylation of P38, ERK, and JNK in vitro. The results are shown in Fig. 8A. The

ZBE (2 g/kg b.wt.) treatment group presented more dramatically reduced the DSS-induced increase of the protein phosphorylation of three MAPKs than those in the DSS and ZBE (0.5, 1 g/kg b.wt.) treatment groups. This result was consistent with the findings in vivo. 4. Discussion Ulcerative colitis (UC) is a chronic and relapsing inflammatory disease of gastrointestinal tract. UC is prevalent in developed countries and affects 8–12 per hundred thousand individuals [21,22]. Although many therapeutic drugs have been used for this disease, most of these agents are not very effective and have severe side effects particularly for long-term therapy. Therefore, it is necessary to develop novel therapeutics. Zanthoxylum bungeanum belongs to the Zanthoxylum genus of the Rutaceae family and is rich in flavonoids [8]. In the present study, ZBE was abundant in flavonoids (80.92 ± 3.12 mg/g). In qualitative terms, rutin, quercetin and isoquercitrin were identified as the three potential active compounds in flavonoids (Fig. 1). Rutin is the most common flavonoid glycoside in nature. Quercetin, a well-known flavonoid, is commonly added to food due to its biological role [23]. Additionally, rutin and quercetin have been reported to play anti-inflammatory, antioxidant and antimicrobial roles and protect against inflammatory bowel disease [24,25], which implies that rutin and quercetin could be the potential active compounds in ZBE that improved DSS-induced experimental colitis in our study. Isoquercitrin, a naturally occurring form of quercetin, has anti-oxidative and anti-inflammatory properties [26, 27]. These previous studies suggested that ZBE may be beneficially associated with intestinal disease; therefore ZBE was further investigated to determine the effects and mechanisms of ZBE on UC mice in the following assays. The DSS-induced colitis model could be used to explore the pathogenesis of UC and is similar to human UC. Its clinical features include weight loss, diarrhea, rectal bleeding and abdominal pain. To determine the preventive effect of ZBE on UC, we generated a model of DSS-induced mouse UC. In DSS stimulated mice, we observed significant benefits from the ZBE treatments in diarrhea, visible fecal blood and colon shortening (Fig. 3C). It is well known that weight loss and DAI are the main parameters used to estimate the level of inflammation in UC,

Fig. 6. Effect of ZBE on TLR4 expression in vitro and in vivo. (A) Cells were preincubated with ZBE (100, 200, and 400 μg/ml) for 1 h and then treated with 1 μg/ml LPS for 1 h. Protein samples were analyzed by western blot with specific antibodies. (B) Protein levels in the colon tissue were determined by Western blot. β-Actin was used as a control. Data are presented as means ± SEM (n = 3). (∗) p b 0.05, (∗∗) p b 0.01 versus DSS-treated alone group; (#) p b 0.05 compared with control group.

Please cite this article as: Z. Zhang, et al., Zanthoxylum bungeanum pericarp extract prevents dextran sulfate sodium-induced experimental colitis in mice via the regulation of TLR4 and TLR4-related.., Int Immunopharmacol (2016), http://dx.doi.org/10.1016/j.intimp.2016.10.021

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Fig. 7. Effect of ZBE on NF-κB activation in vitro and in vivo. (A) Cells were preincubated with ZBE (100, 200, and 400 μg/ml) for 1 h and then treated with 1 μg/ml LPS for 1 h. Protein samples were analyzed by western blot with specific antibodies. (B) Protein levels in the colon tissue were determined by Western blot. β-Actin was used as a control. Data are presented as means ± SEM (n = 3). (∗) p b 0.05, (∗∗) p b 0.01 versus DSS-treated alone group; (#) p b 0.05 compared with control group.

and our findings showed that body weight loss and DAI score were down-regulated compared with the control group (Fig. 3A, B). From histopathological observations, we found typical images of HE-stained colon tissue. Compared with the control group, the crypt structure and submucosa in the DSS group were irregular, whereas in ZBE-supplemented groups, the irregularity of the structure was significantly ameliorated (Fig. 4). Furthermore, ZBE (2 g/kg b.wt.) only have not generated histopathological change in the colons of mice, which showed the content of ZBE could be used. These findings indicated that ZBE had a protective effect on the UC induced by DSS. To our knowledge, this is the first report to indicate that ZBE is highly effective in ameliorating experimental colitis. Much evidence suggests that intense local immune response of UC is associated with release of pro-inflammatory cytokines [28,29]. In the current study, the levels of TNF-α, IL-1β and IL-12 were remarkably enhanced after DSS challenge and LPS stimulation, which were remarkably suppressed by ZBE in a dose-dependent manner (Fig. 5A, B). TNFα is the primary and earliest endogenous mediator in inflammatory diseases and could change the function of the intestinal barrier [30]. IL-1β could also regulate inflammation, and it is necessary in the early stages of the inflammation, which leads to the inflamed colon [31]. IL-12, a preinflammation factor, induces CD4+ T cells to Th1 cells, and promotes natural killer cells and T cells to secrete TNF-α cytokines to mediate inflammation [32]. The roles of these cytokines in host defenses and

inflammatory diseases have been well established [33]. Thus, our findings indicated that ZBE had an anti-inflammatory role in LPS-triggered J774.1 cells, inhibiting the levels of TNF-α, IL-1β and IL-12. Moreover, the immune responses of the host are closely associated with UC. Therefore, we investigated the secretion of cytokines in MLN cells after in vitro stimulation with CBL. As shown in Fig. 5C, ZBE decreased the expression of TNF-α, IL-1β and IL-12 in MLN cells treated with CBL. These results suggested that ZBE alleviated DSS-induced colitis. TLR4 is pattern recognition receptor that has been best characterized in the intestine. The activation of TLR4 plays a key role in the pathogenesis of UC. The targeted suppression of the TLR4 pathway has also become a treatment strategy for UC in recent years [4]. In the present study, the expression of TLR4 was remarkably increased after DSS challenge. However, treatment with ZBE (0.5, 1 g/kg b.wt.) could decrease TLR4 expression. The ZBE (2 g/kg b.wt.) treatment group presented more obvious inhibitory effect than those in the DSS and ZBE (0.5, 1 g/kg b.wt.) treatment groups (Fig. 6B). NF-κB and MAPK, two major inflammatory pathways downstream of TLR4, could regulate the development of inflammation. A previous study has also shown that NF-κB and MAPK could modulate the expression of many cytokines and regulate the inflammatory processes in inflammatory bowel diseases (IBD) [34,35]. The inhibition of NF-κB and MAPK activation is an effective treatment for the prevention of UC. As expected, our results showed that ZBE inhibited the NF-κB and MAPK pathways in a dose dependent manner, which indicated that ZBE could

Please cite this article as: Z. Zhang, et al., Zanthoxylum bungeanum pericarp extract prevents dextran sulfate sodium-induced experimental colitis in mice via the regulation of TLR4 and TLR4-related.., Int Immunopharmacol (2016), http://dx.doi.org/10.1016/j.intimp.2016.10.021

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Fig. 8. Effect of ZBE on MAPK activation in vitro and in vivo. (A) Cells were preincubated with ZBE (100, 200, and 400 μg/ml) for 1 h and then treated with 1 μg/ml LPS for 1 h. Protein samples were analyzed by western blot with specific antibodies. (B) Protein levels in the colon tissue were determined by Western blot. β-Actin was used as a control. Data are presented as means ± SEM (n = 3). (∗) p b 0.05, (∗∗) p b 0.01 versus DSS-treated alone group; (#) p b 0.05 compared with control group.

regulate NF-κB and MAPK pathway activation through TLR4. However, these results are preliminary, and more work is still needed to illustrate the exact target of ZBE in exerting protective effects on DSS-induced mice and determine the effect of ZBE on UC in humans. In conclusion, we investigated a novel protective strategy for UC mice. Treatment with ZBE significantly attenuated DSS-induced UC in mice. This protective mechanism may be due to the inhibition of the activation of TLR4 and TLR4-related signaling pathways and subsequent pro-inflammatory cytokines.

Acknowledgements This work was supported by a grant from the National Natural Science Foundation of China (nos. 31272622 and 31472248), Jilin Province Science Foundation for Youths (no. 20130522087JH), and the Key Project of Chinese National Programs for Research and Development (no. 2016YFD0501009).

References Declaration of interest The authors declare that they have no competing interest.

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Please cite this article as: Z. Zhang, et al., Zanthoxylum bungeanum pericarp extract prevents dextran sulfate sodium-induced experimental colitis in mice via the regulation of TLR4 and TLR4-related.., Int Immunopharmacol (2016), http://dx.doi.org/10.1016/j.intimp.2016.10.021