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Magnolol treatment attenuates dextran sulphate sodium-induced murine experimental colitis by regulating inflammation and mucosal damage
Life Sciences 196 (2018) 69–76
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Magnolol treatment attenuates dextran sulphate sodium-induced murine experimental colitis by regulating inflammation and mucosal damage
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Peng Shen1, Zecai Zhang1, Yue He, Cong Gu, Kunpeng Zhu, Shan Li, Yanxin Li, Xiaojie Lu, ⁎ Jiuxi Liu, Naisheng Zhang, Yongguo Cao College of Veterinary Medicine, Jilin University, Changchun 130062, People's Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords: Magnolol Colitis Dextran sulphate sodium Inflammation Mucosal damage
Magnolol, the main and active ingredient of the Magnolia officinalis, has been widely used in traditional prescription to the human disorders. Magnolol has been proved to have several pharmacological properties including anti-bacterial, anti-oxidant and anti-inflammatory activities. However, the effects of magnolol on ulcerative colitis (UC) have not been reported. The aim of this study was to investigate the protective effects and mechanisms of magnolol on dextran sulphate sodium (DSS)-induced colitis in mice. The results showed that magnolol significantly alleviated DSS-induced body weight loss, disease activities index (DAI), colon length shortening and colonic pathological damage. In addition, magnolol restrained the expression of TNF-α, IL-1β and IL-12 via the regulation of nuclear factor-κB (NF-κB) and Peroxisome proliferator-activated receptor-γ (PPAR-γ) pathways. Magnolol also enhanced the expression of ZO-1 and occludin in DSS-induced mice colonic tissues. These results showed that magnolol played protective effects on DSS-induced colitis and may be an alternative therapeutic reagent for colitis treatment.
1. Introduction Ulcerative colitis (UC), a form of inflammatory bowel disease (IBD), is a type of chronic and relapsing inflammatory of the gastrointestinal tract. Nowadays, millions of patients around the world are suffering with UC, and the risk of colon cancer is also significantly increasing [1,2]. Although multiple factors such as environmental changes, gene variations, gut microbiota, and immunological stimulus are thought to be associated with UC, its etiology and pathogenesis are complicated and remain uncertain [3]. To study this disease, a model of mice colitis has been used by the oral administration of dextran sulphate sodium (DSS), which could research into the pathogenesis of UC and is similar to human UC [4]. Emerging evidences show that the intestinal barrier plays a vital role in intestinal inflammation in experimental colitis [5]. In addition, it has been reported that the damage of intestinal epithelial barrier is regulated by the abnormal activity of some pro-inflammatory signals. Specifically, nuclear factor-κB (NF-κB), a transcription factor, promotes transcription of genes encoding pro-inflammatory cytokines. Its activation induces the production of important immune mediators, such as TNF-α, IL-1β and IL-12 [6]. The activation of NF-κB has been observed in UC patients and DSS-induced colitis mice [7]. Moreover, Peroxisome
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proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear hormone receptor family. Its activation could regulate NF-κB activation which decreases the levels of pro-inflammatory cytokines [8,9]. Currently, many therapeutic drugs have been used for UC, but most of these drugs exists many resistances problem, and the therapies for UC have major adverse effects. Therefore, the development of novel therapies for UC is urgently needed. Magnolol (Fig. 1), the main and active ingredient of the Magnolia officinalis, is a traditional Chinese medicine herb which has been widely used in traditional prescription to the human disorders. Magnolol has been proved to have several pharmacological effects including antibacterial, anti-oxidant and anti-inflammatory activities [10]. Previous studies in our laboratory have confirmed that magnolol inhibits the inflammatory response in mouse mastitis and acute lung injury [11,12]. Furthermore, magnolol also has been widely used for treatment of gastrointestinal disorders [13], indicating it may have the potential to be become an available anti-UC agent. Thus, we investigated the potential protective effects and mechanisms of magnolol on DSS induced UC model mice.
Corresponding author. E-mail address: [email protected] (Y. Cao). These authors contributed equally to this work and should be considered co-first authors.
https://doi.org/10.1016/j.lfs.2018.01.016 Received 9 October 2017; Received in revised form 12 January 2018; Accepted 15 January 2018 Available online 02 February 2018 0024-3205/ © 2018 Elsevier Inc. All rights reserved.
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measured on a daily basis. The disease activity index (DAI) was calculated with a previously established scoring system [15]. 2.4. Myeloperoxidase (MPO) assay MPO is an enzyme that is directly proportional to the number of neutrophils in the tissue. The MPO activity was measured according to the manufacturer's instructions. Briefly, colitis tissue, weighing approximately 100 mg, was fixed with phosphate buffered saline (PBS, weight/volume ratio 1:9) and homogenized. The supernatants were analyzed using the MPO kit (Jiancheng biotechnology, China) and detected by spectrophotometer at an absorbance value of 460 nm. Fig. 1. The chemical structure of magnolol.
2.5. Histologic analysis 2. Materials and methods
For the histological analysis, day 5 after colitis induction with 2.5% DSS, the colon was washed in PBS. Then, the colonic tissues were fixed in 10% formalin. The colon specimens were embedded in paraffin and then deparaffinized with xylene and rehydrated for hematoxylin & eosin (H&E). Histological grading was assessed according to a scoring scheme in the H&E-stained sections [16].
2.1. Chemicals Magnolol (> 98% HPLC) was purchased from (Chengdu, China). DSS (molecular weight 36–50 kDa) was obtained from MP Bio medicals, Morgan Irvine, CA. Mouse TNF-α, IL-1β and IL-12 enzyme-linked immunosorbent assay (ELISA) kits were obtained from Biolegend (San Diego, CA, USA). Rabbit monoclonal antibodies IκB, p65, p-IκB, and pp65 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The primary antibody that was raised against zonula occludens-1 (ZO-1) and occludin were obtained from Wanleibio Co., Ltd. (Liaoning, China). Rabbit monoclonal antibody PPAR-γ was purchased from GeneTex (Shanghai, China). β-actin and horseradish peroxidase conjugated goat anti-rabbit antibodies were provided by Tianjin Sungene Biotech Co., Ltd. (Tianjin, China).
2.6. Preparation of cecal bacterial lysates Cecal bacterial lysates (CBLs) were prepared as described by Dieleman et al. [17]. Briefly, the cecal contents in each group were solubilized by vortexing the contents in Roswell Park Memorial Institute (RPMI) 1640 medium and then incubating them with 10 μg/ml DNase and 0.01 M MgCl2. Then, the contents were homogenized for 3 min using 0.1 mm glass beads. The homogenate was centrifuged at 10000g for 10 min. The supernatant was filtered through a 0.45 μm syringe filter.
2.2. Animals and mice model of DSS-induced colitis
2.7. Mesenteric lymph node cell cultures
Male C57BL/6 mice, weighing approximately 21 to 23 g, were obtained from the Center of Experimental Animals of Bethune Medical College of Jilin University (Jilin, China). All mice were housed in a temperature maintained room (24 ± 1 °C). Before experimentation, all mice were adapted to their new environment for a minimum of 1 week, and supplied with standard diet and tap water ad libitum. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Mice were randomly divided into six groups of six mice each: Group I: Control group Group II: DSS group Group III: magnolol (25 mg/kg body weight) + DSS group Group IV: magnolol (50 mg/kg body weight) + DSS group Group V: magnolol (100 mg/kg body weight) + DSS group Group VI: magnolol (100 mg/kg body weight) group. For acute colitis model, the DSS group mice were exposed to 2.5% DSS, which was dissolved in drinking water, continuously for 5 days. In the magnolol + DSS groups, the mice were treated with different doses of magnolol 3 days before and during DSS treatment via oral gavage once per day [14]. Mice in the control group and DSS group were given the same volume of water. On the 5 days of the colitis induced by DSS, mice were sacrificed, samples were collected, and the colon was excised from cecum to 1 cm above the anus. The lengths of the colon were detected, which indirectly stipulated the inflammatory index of the colon.
Mesenteric lymph node (MLN) was harvested from mice of six experimental groups. Single cell suspensions were prepared as described by Ruyssers [18]. 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. 2.8. Cytokine analysis by ELISA The colonic tissues were weighed and homogenized with PBS [1:9 (w/v)] on ice and then centrifuged at 2000g for 40 min at 4 °C. The supernatant was collected and stored at −20 °C. The TNF-α, IL-1β and IL-12 levels were detected by ELISA kits according to the manufacturer's protocol. 2.9. RNA isolation and real-time PCR Total RNA from colonic tissues were extracted using TRIzol (Invitrogen, Carlsbad, CA) as per manufacturer's instructions. The RNA was reverse transcribed into cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Thermo). The quantification of relative mRNA concentrations, including TNF-α, IL-1β, IL-12 and β-actin mRNA, was performed with qRT-PCR using a 7500 Fast Real-Time PCR System (Applied Biosystems) and the SYBR Green Plus reagent kit (Roche), as described elsewhere [19]. The sequences of primers are listed in Table 1.
2.3. Measurements of UC and clinical colitis scoring 2.10. Western blot The baseline clinical disease activity index (DAI) was the sum evaluation of the clinical score [15]. In brief, body weight, feces condition and fecal blood test scores were added together. Body weight was
The colonic tissues were homogenated, then centrifuged for 10 min at 4 °C. The supernatant was collected and its protein concentration was 70
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control of protein loading.
Table 1 Sequence of primers used in current investigation in qRT-PCR. Name
Primer sequence
TNF-α
Sense: 5′-GCCTCCCTCTCATCAGTTCTA-3′ Anti-sense: 5′-GGCAGCCTTGTCCCTTG-3′ Sense: 5′-ACCTGTGTCTTTCCCGTGG-3′ Anti-sense: 5′-TCATCTCGGAGCCTGTAGTG-3′ Sense: 5′-GGTCACACTGGACCAAAGGGACTATG-3′ Anti-sense: 5′-ATTCTGCTGCCGTGCTTCCAAC-3′ Sense: 5′-CTACCGTCGTGACTTCGC-3′ Anti-sense: 5′-GGGTGACATCTCCCTGTT-3′
IL-1β IL-12 β-actin
2.11. Statistical analysis All data are shown as the mean ± standard deviation (SD). Data sets that involved more than two groups were assessed by one-way ANOVA followed by Tukey's multiple-comparison test. All experiments were repeated at least three times. P ≤ 0.05 were considered statistically significant. 3. Results 3.1. Magnolol attenuated colitis induced by DSS
measured using a BCA protein assay kit (23227, Thermo). The total protein was extracted as per the manufacturer's protocol. Protein extracts from colons were analyzed by western blot. Briefly, 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. Finally, protein expression was detected using the Bio-rad Imaging System (Bio-rad Biosciences, USA). The β-actin was used as an internal
It is well known that UC is characterized by namely diarrhea, bloody feces, body weight loss and shortening of the colon length. The results are shown in Fig. 2A. Mice in the DSS group had a significant loss of body weight compared with the control group. However, magnolol treatment significantly attenuated the body weight loss during the progression of murine experimental colitis. Magnolol treatment also markedly decreased the DAI scores induced by DSS (Fig. 2B), which is a clinical parameter reflects the severity of weight loss, stool consistency
Fig. 2. Magnolol treatment improved DSS-induced colitis in mice. (A) Bodyweight 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 ± SD (n = 6 per group). (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01 vs the DSS-treated group on the same day; (#) p < 0.05 vs the control group.
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Fig. 3. Magnolol treatment prevented DSS-induced colon damage in mice (H&E staining ×200). (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. (C) MPO activity in the colonic tissues was detected. Data are presented as means ± SD (n = 6). (∗) p ≤ 0.05, (∗∗) p ≤ 0.01 vs DSS-treated alone group; (#) p < 0.05 vs the control group.
3.3. Magnolol inhibit the pro-inflammatory cytokines in colonic tissues and in MLN
and blood in stool. Furthermore, DSS typically caused colonic shortening in Fig. 2C. However, such changes were also attenuated by magnolol treatments.
As an additional marker of local inflammation, the concentrations of cytokines (TNF-α, IL-1β and IL-12) were further measured by ELISA. The results showed that the expression levels of TNF-α, IL-1β and IL-12 in the DSS group were significantly increased compared with the control group in Fig. 4A. The administration of magnolol inhibited the production of inflammatory cytokines in colonic tissues. To further test the effects of magnolol, mRNA expression levels of pro-inflammatory cytokines were evaluated by qRT-PCR. As shown in Fig. 4B, compared with the control, the expression levels of TNF-α, IL-1β and IL-12 mRNA were also significantly increased by DSS treatment, but were clearly reduced by treatment with magnolol. In addition, to investigate the effects of magnolol on host dependent immune responses, we investigated the cytokine responses of the MLN cells to CBL. The results showed that magnolol could markedly inhibit the elevated expression of these cytokines (Fig. 4C). These results indicated that magnolol suppressed the transcription and final secretion of TNF-α, IL-1β and IL12.
3.2. Magnolol relieved histopathological changes in DSS-induced colitis in mice H&E stained sections of colonic segments were observed under a light microscope. The results showed that there were no histological abnormalities in control group. However, overall damage to the surface epithelium, disruption of cryptal glands, and infiltration of inflammatory cells were observed in the DSS group (Fig. 3A, B). Administration of magnolol significantly improved pathological changes induced by DSS. Furthermore, MPO is a marker of inflammation. In the present study, magnolol with the doses of 25, 50 and 100 mg/kg markedly decreased the level of DSS-induced hyperactivated MPO (Fig. 3C). These results showed that magnolol treatment relieved DSSinduced murine experimental colitis.
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Fig. 4. Magnolol suppressed pro-inflammatory cytokines in colonic tissues and in MLN. (A) Effects of different doses of magnolol (25, 50 and 100 mg/kg) on TNF-α, IL-β and IL-12 levels in colonic tissues. (B) mRNA levels in colonic tissue, The β-actin was used as an internal control (n = 6). (C) TNF-α, IL-1β and IL12 levels in MLN were measured with an ELISA (n = 6). Data are presented as means ± SD (n = 6). (∗) p ≤ 0.05, (∗∗) p ≤ 0.01 vs DSS-treated alone group; (#) p < 0.05 vs the control group.
3.5. Magnolol regulated the expression of PPAR-γ in DSS-induced colitis mice
3.4. Magnolol inhibited NF-κB activity in DSS-induced colitis mice NF-κB plays a pivotal role in regulating cytokines. To explore whether the inhibition of inflammation by magnolol is mediated by NFκB pathway, NF-κB p65 and IκB phosphorylation levels were determined. The results showed that the phosphorylation of p65 and IκB were significantly enhanced in DSS group, but reversed in magnolol treatment groups (Fig. 5).
PPAR-γ, a member of the nuclear hormone receptor family, could regulate NF-κB pathway activation, and thus decreasing the level of pro-inflammatory cytokine. To further explore the anti-inflammatory mechanisms of magnolol, we investigated the possible role of magnolol in the expression of PPAR-γ in DSS-induced mice colitis. The results showed that there was a significant decrease in the expression of PPARγ in the DSS group compared with the control group. However, the treatment with magnolol could regulate the expression of PPAR-γ. The 73
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Fig. 5. Magnolol modulated DSS-induced NF-κB activation in colonic tissue. NF-κB p65 and IκB protein levels in the colonic tissues were analyzed by Western blotting. β-actin was used as a control. Data are presented as means ± SD (n = 3). (∗) p < 0.05 and (∗∗) p < 0.01 vs the group treated with only DSS; (#) p < 0.05 vs the control group.
used for animal experiments in our study were designed in the range of 25 to 100 mg/kg. The therapeutic effect of magnolol was notable, which is in agreement with previous study [27]. It is well-known that UC is characterized by namely diarrhea, bloody feces and body weight loss. In our study, we found that magnolol treatment attenuated body weight loss and DAI scores. In addition, DSS typically caused colonic shortening, while this change was significantly improved by the treatment of magnolol. From histopathological observations, we found the submucosa and crypt structure in the DSS group were irregular, whereas the irregularities of the structures were significantly improved in magnolol treatment groups. Moreover, the treatment with magnolol also significantly reduced the MPO activity which is directly proportional to the number of neutrophils [28]. Many evidences have shown that intense local immune response of UC is associated with release of pro-inflammatory cytokines [29]. Recent genetic and immunological studies have also shown that the involvement of cytokines (TNF-α, IL-1β and IL-12) contributed to IBD perpetuation and tissue destruction [30–32]. TNF-α is the primary and the earliest endogenous mediator in inflammatory diseases and can change the function of the intestinal barrier [33]. IL-1β can also regulate inflammation, and it is necessary in the early stages of the inflammation, which leads to the inflamed colon [34]. Among them, IL12 can not only induce CD4+ T cells to Th1 cells, and promote natural killer cells and T cells to secrete TNF-α cytokines to mediate inflammation [35]. Thus, we assessed the levels of TNF-α, IL-1β and IL-12 by ELISA. The results showed that the levels of TNF-α, IL-1β and IL-12 were remarkably enhanced after DSS challenge. However, the levels of TNF-α, IL-1β and IL-12 were significantly decreased in magnolol treatment groups compared with DSS group. Moreover, the immune responses of the host are closely associated with UC. Therefore, we investigated the secretion of cytokines in MLN cells after stimulation
magnolol (100 mg/kg) treatment group presented more obvious effects than those in the DSS and magnolol (25, 50 mg/kg) treatment groups (Fig. 6). 3.6. Magnolol modulated ZO-1 and occludin expression in DSS-induced colitis mice Epithelial TJ tight junction protein (TJ), an especially important aspect of the mechanical barrier, can prevent harmful substances from breaching the mucosa, maintain cellular integrity and permeability, and thus ensuring a relatively stable internal environment. ZO-1 and occludin are important epithelial TJ proteins. Thus, we detected the effects of magnolol on epithelial TJ proteins (ZO-1 and occludin). As shown in in Fig. 7, the expression of ZO-1 and occludin were downregulated in DSS-induced colitis mice compared to the control group. However, the expression levels of ZO-1 and occludin were significantly improved by the treatment of magnolol, suggesting the importance of magnolol for restoring the integrity of the TJ networks. 4. Discussion UC is a chronic and relapsing inflammatory disease of the gastrointestinal tract. Although the etiology and pathogenesis of UC are complicated and remain uncertain, environmental changes, genetic makeups and gut microbiota are reported to be related to UC [3]. In addition, many therapeutic drugs have been used for this disease, but most of these agents are not very effective and have severe side effects. Magnolol is a natural compound and has been reported to have several pharmacological effects, including anti-allergic [20], anti-oxidant [21], anti-inflammatory [22], anti-arthritic [23] and antimicrobial [10,24]. According to the previous reports [25,26], the doses of magnolol 74
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cytokines, such as TNF-α, IL-1β and IL-12. The activation of NF-ĸB pathway has been observed in IBD patients and DSS-induced murine experimental colitis model [7]. In order to test the mechanism of antiUC, we detected the effect of magnolol on the NF-κB pathway. As expected, the phosphorylations of p65 and IκB which initiate the activations of NF-κB were significantly increased in DSS group, but magnolol markedly down-regulated the increase induced by DSS. In addition, PPAR-γ, a member of the nuclear hormone receptor family, has been reported to negatively regulate inflammation [36]. PPAR-γ has also been implicated in inflammation-related diseases, including colitis [37]. In addition, several studies have also shown that PPAR-γ activation exerts a satisfactory anti-inflammatory effect in IBD patients and experimental colitis models [8,9]. Moreover, it has been reported that PPAR-γ is an up-stream target of NF-κB, and there is a negative correlation between NF-κB and PPAR-γ [38,39]. More importantly, targeted disruption of PPAR-γ expression in macrophages increased the susceptibility to DSS-induced colitis in mice [40]. Therefore, we further detected the protective mechanisms of magnolol on UC. As anticipated, the expression of PPAR-γ was markedly increased in magnolol treated groups compared with DSS group, suggesting PPAR-γ may play an important role in anti-UC of magnolol. Many studies have reported that intestinal barrier plays a key role in maintaining intestinal health [41]. An impaired intestinal barrier results in the increasing permeability of intestinal mucosa to harmful bacteria, leading to intestinal inflammation. TJ proteins have been well documented to play an important role in the maintenance of intestinal mucosal barrier integrity [42]. The structural abnormalities in TJ proteins are the major cause of altered intestinal barrier in IBD patients [43]. Previous study has also showed that structural abnormalities in TJ proteins, including the reduction of ZO-1 and occludin, in IBD patients is a cause of altered intestinal permeability [44]. Therefore, the regulation of TJ proteins may be a highly regarded target for novel therapeutic treatment of UC. In our study, the treatment with magnolol significantly inhibited the reduction of ZO-1 and occludin induced by DSS. Our data showed that magnolol might have a protective effect on barrier integrity by maintaining the expression of ZO-1 and occludin, thereby reducing the severity of colitis.
Fig. 6. Magnolol modulated DSS-induced PPAR-γ activation in colonic tissue. PPAR-γ level in the colonic tissue were analyzed by Western blot. β-actin was used as a control. Data are presented as means ± SD (n = 3). (∗) p < 0.05 and (∗∗) p < 0.01 vs the group treated with only DSS; (#) p < 0.05 vs the control group.
with CBL. We found that magnolol also significantly decreased the expression of TNF-α, IL-1β and IL-12. NF-κB is a transcription factor that promotes transcription of genes encoding pro-inflammatory
Fig. 7. Magnolol modulated ZO-1 and occludin expression in DSS-induced colitis mice. ZO-1 and occludin protein levels in the colonic tissues were analyzed by Western blot. β-actin was used as a control. Data are presented as means ± SD (n = 3). (∗) p < 0.05 and (∗∗) p < 0.01 vs the group treated with only DSS; (#) p < 0.05 vs the control group.
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In conclusion, this study suggested that magnolol treatment attenuates DSS-induced colitis outcomes. The protection associated with magnolol was not only due to the anti-inflammatory effects but also due to the regulation of the gut integrity in mice. Our findings suggested that magnolol might be used for the treatment of UC.
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Conflict of interest policy form and author contribution to study form The authors claim that none of the material in the paper has been published or is under consideration for publication elsewhere. In addition, there is no conflict of interest policy form. Author contribution to study form Concept and design: PS, ZZ, YC, and YH. Acquisition of data: PS, ZZ, SL, CG, and KZ. Analysis and interpretation: PS, ZZ, XL, and JL. Drafting and editing of the manuscript: PS, ZZ, and NZ. All the authors read and approved the final manuscript. Declaration of interest There is no conflict of interest. Acknowledgements This work was supported by the Key Project of Chinese National Programs for Research and Development (no. 2016YFD0501009) and the National Natural Science Foundation of China (nos. 31472248 and 31572582). References [1] J. Cosnes, et al., Epidemiology and natural history of inflammatory bowel diseases, Gastroenterology 140 (6) (2011) 1785–1794. [2] J. Terzic, et al., Inflammation and colon cancer, Gastroenterology 138 (6) (2010) 2101–2114 (e5). [3] H.S. de Souza, C. Fiocchi, Immunopathogenesis of IBD: current state of the art, Nat. Rev. Gastroenterol. Hepatol. 13 (1) (2016) 13–27. [4] H. Vargas Robles, et al., Analyzing beneficial effects of nutritional supplements on intestinal epithelial barrier functions during experimental colitis, J. Vis. Exp. (119) (2017). [5] L. Zuo, et al., Targeting delivery of anti-TNFalpha oligonucleotide into activated colonic macrophages protects against experimental colitis, Gut 59 (4) (2010) 470–479. [6] G. Rogler, et al., Nuclear factor kappaB is activated in macrophages and epithelial cells of inflamed intestinal mucosa, Gastroenterology 115 (2) (1998) 357–369. [7] C.O. Elson, et al., Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota, Immunol. Rev. 206 (2005) 260–276. [8] M. Sanchez-Hidalgo, et al., Rosiglitazone, an agonist of peroxisome proliferatoractivated receptor gamma, reduces chronic colonic inflammation in rats, Biochem. Pharmacol. 69 (12) (2005) 1733–1744. [9] J. Torres, et al., New therapeutic avenues in ulcerative colitis: thinking out of the box, Gut 62 (11) (2013) 1642–1652. [10] G.Y. Zuo, et al., In vitro synergism of magnolol and honokiol in combination with antibacterial agents against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), BMC Complement. Altern. Med. 15 (425) (2015). [11] W. Wei, et al., Magnolol inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model, Inflammation 38 (1) (2015) 16–26. [12] F. Yunhe, et al., The effect of magnolol on the toll-like receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharide-induced acute lung injury in mice, Eur. J. Pharmacol. 689 (1–3) (2012) 255–261. [13] T.C. Yang, et al., Magnolol attenuates sepsis-induced gastrointestinal dysmotility in rats by modulating inflammatory mediators, World J. Gastroenterol. 14 (48) (2008) 7353–7360. [14] W. Tang, et al., Phytochemical profiles and biological activity evaluation of Zanthoxylum bungeanum Maxim seed against asthma in murine models, J. Ethnopharmacol. 152 (3) (2014) 444–450.
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Magnolol treatment attenuates dextran sulphate sodium-induced murine experimental colitis by regulating inflammation and mucosal damage
T
Peng Shen1, Zecai Zhang1, Yue He, Cong Gu, Kunpeng Zhu, Shan Li, Yanxin Li, Xiaojie Lu, ⁎ Jiuxi Liu, Naisheng Zhang, Yongguo Cao College of Veterinary Medicine, Jilin University, Changchun 130062, People's Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords: Magnolol Colitis Dextran sulphate sodium Inflammation Mucosal damage
Magnolol, the main and active ingredient of the Magnolia officinalis, has been widely used in traditional prescription to the human disorders. Magnolol has been proved to have several pharmacological properties including anti-bacterial, anti-oxidant and anti-inflammatory activities. However, the effects of magnolol on ulcerative colitis (UC) have not been reported. The aim of this study was to investigate the protective effects and mechanisms of magnolol on dextran sulphate sodium (DSS)-induced colitis in mice. The results showed that magnolol significantly alleviated DSS-induced body weight loss, disease activities index (DAI), colon length shortening and colonic pathological damage. In addition, magnolol restrained the expression of TNF-α, IL-1β and IL-12 via the regulation of nuclear factor-κB (NF-κB) and Peroxisome proliferator-activated receptor-γ (PPAR-γ) pathways. Magnolol also enhanced the expression of ZO-1 and occludin in DSS-induced mice colonic tissues. These results showed that magnolol played protective effects on DSS-induced colitis and may be an alternative therapeutic reagent for colitis treatment.
1. Introduction Ulcerative colitis (UC), a form of inflammatory bowel disease (IBD), is a type of chronic and relapsing inflammatory of the gastrointestinal tract. Nowadays, millions of patients around the world are suffering with UC, and the risk of colon cancer is also significantly increasing [1,2]. Although multiple factors such as environmental changes, gene variations, gut microbiota, and immunological stimulus are thought to be associated with UC, its etiology and pathogenesis are complicated and remain uncertain [3]. To study this disease, a model of mice colitis has been used by the oral administration of dextran sulphate sodium (DSS), which could research into the pathogenesis of UC and is similar to human UC [4]. Emerging evidences show that the intestinal barrier plays a vital role in intestinal inflammation in experimental colitis [5]. In addition, it has been reported that the damage of intestinal epithelial barrier is regulated by the abnormal activity of some pro-inflammatory signals. Specifically, nuclear factor-κB (NF-κB), a transcription factor, promotes transcription of genes encoding pro-inflammatory cytokines. Its activation induces the production of important immune mediators, such as TNF-α, IL-1β and IL-12 [6]. The activation of NF-κB has been observed in UC patients and DSS-induced colitis mice [7]. Moreover, Peroxisome
⁎
1
proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear hormone receptor family. Its activation could regulate NF-κB activation which decreases the levels of pro-inflammatory cytokines [8,9]. Currently, many therapeutic drugs have been used for UC, but most of these drugs exists many resistances problem, and the therapies for UC have major adverse effects. Therefore, the development of novel therapies for UC is urgently needed. Magnolol (Fig. 1), the main and active ingredient of the Magnolia officinalis, is a traditional Chinese medicine herb which has been widely used in traditional prescription to the human disorders. Magnolol has been proved to have several pharmacological effects including antibacterial, anti-oxidant and anti-inflammatory activities [10]. Previous studies in our laboratory have confirmed that magnolol inhibits the inflammatory response in mouse mastitis and acute lung injury [11,12]. Furthermore, magnolol also has been widely used for treatment of gastrointestinal disorders [13], indicating it may have the potential to be become an available anti-UC agent. Thus, we investigated the potential protective effects and mechanisms of magnolol on DSS induced UC model mice.
Corresponding author. E-mail address: [email protected] (Y. Cao). These authors contributed equally to this work and should be considered co-first authors.
https://doi.org/10.1016/j.lfs.2018.01.016 Received 9 October 2017; Received in revised form 12 January 2018; Accepted 15 January 2018 Available online 02 February 2018 0024-3205/ © 2018 Elsevier Inc. All rights reserved.
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measured on a daily basis. The disease activity index (DAI) was calculated with a previously established scoring system [15]. 2.4. Myeloperoxidase (MPO) assay MPO is an enzyme that is directly proportional to the number of neutrophils in the tissue. The MPO activity was measured according to the manufacturer's instructions. Briefly, colitis tissue, weighing approximately 100 mg, was fixed with phosphate buffered saline (PBS, weight/volume ratio 1:9) and homogenized. The supernatants were analyzed using the MPO kit (Jiancheng biotechnology, China) and detected by spectrophotometer at an absorbance value of 460 nm. Fig. 1. The chemical structure of magnolol.
2.5. Histologic analysis 2. Materials and methods
For the histological analysis, day 5 after colitis induction with 2.5% DSS, the colon was washed in PBS. Then, the colonic tissues were fixed in 10% formalin. The colon specimens were embedded in paraffin and then deparaffinized with xylene and rehydrated for hematoxylin & eosin (H&E). Histological grading was assessed according to a scoring scheme in the H&E-stained sections [16].
2.1. Chemicals Magnolol (> 98% HPLC) was purchased from (Chengdu, China). DSS (molecular weight 36–50 kDa) was obtained from MP Bio medicals, Morgan Irvine, CA. Mouse TNF-α, IL-1β and IL-12 enzyme-linked immunosorbent assay (ELISA) kits were obtained from Biolegend (San Diego, CA, USA). Rabbit monoclonal antibodies IκB, p65, p-IκB, and pp65 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The primary antibody that was raised against zonula occludens-1 (ZO-1) and occludin were obtained from Wanleibio Co., Ltd. (Liaoning, China). Rabbit monoclonal antibody PPAR-γ was purchased from GeneTex (Shanghai, China). β-actin and horseradish peroxidase conjugated goat anti-rabbit antibodies were provided by Tianjin Sungene Biotech Co., Ltd. (Tianjin, China).
2.6. Preparation of cecal bacterial lysates Cecal bacterial lysates (CBLs) were prepared as described by Dieleman et al. [17]. Briefly, the cecal contents in each group were solubilized by vortexing the contents in Roswell Park Memorial Institute (RPMI) 1640 medium and then incubating them with 10 μg/ml DNase and 0.01 M MgCl2. Then, the contents were homogenized for 3 min using 0.1 mm glass beads. The homogenate was centrifuged at 10000g for 10 min. The supernatant was filtered through a 0.45 μm syringe filter.
2.2. Animals and mice model of DSS-induced colitis
2.7. Mesenteric lymph node cell cultures
Male C57BL/6 mice, weighing approximately 21 to 23 g, were obtained from the Center of Experimental Animals of Bethune Medical College of Jilin University (Jilin, China). All mice were housed in a temperature maintained room (24 ± 1 °C). Before experimentation, all mice were adapted to their new environment for a minimum of 1 week, and supplied with standard diet and tap water ad libitum. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Mice were randomly divided into six groups of six mice each: Group I: Control group Group II: DSS group Group III: magnolol (25 mg/kg body weight) + DSS group Group IV: magnolol (50 mg/kg body weight) + DSS group Group V: magnolol (100 mg/kg body weight) + DSS group Group VI: magnolol (100 mg/kg body weight) group. For acute colitis model, the DSS group mice were exposed to 2.5% DSS, which was dissolved in drinking water, continuously for 5 days. In the magnolol + DSS groups, the mice were treated with different doses of magnolol 3 days before and during DSS treatment via oral gavage once per day [14]. Mice in the control group and DSS group were given the same volume of water. On the 5 days of the colitis induced by DSS, mice were sacrificed, samples were collected, and the colon was excised from cecum to 1 cm above the anus. The lengths of the colon were detected, which indirectly stipulated the inflammatory index of the colon.
Mesenteric lymph node (MLN) was harvested from mice of six experimental groups. Single cell suspensions were prepared as described by Ruyssers [18]. 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. 2.8. Cytokine analysis by ELISA The colonic tissues were weighed and homogenized with PBS [1:9 (w/v)] on ice and then centrifuged at 2000g for 40 min at 4 °C. The supernatant was collected and stored at −20 °C. The TNF-α, IL-1β and IL-12 levels were detected by ELISA kits according to the manufacturer's protocol. 2.9. RNA isolation and real-time PCR Total RNA from colonic tissues were extracted using TRIzol (Invitrogen, Carlsbad, CA) as per manufacturer's instructions. The RNA was reverse transcribed into cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Thermo). The quantification of relative mRNA concentrations, including TNF-α, IL-1β, IL-12 and β-actin mRNA, was performed with qRT-PCR using a 7500 Fast Real-Time PCR System (Applied Biosystems) and the SYBR Green Plus reagent kit (Roche), as described elsewhere [19]. The sequences of primers are listed in Table 1.
2.3. Measurements of UC and clinical colitis scoring 2.10. Western blot The baseline clinical disease activity index (DAI) was the sum evaluation of the clinical score [15]. In brief, body weight, feces condition and fecal blood test scores were added together. Body weight was
The colonic tissues were homogenated, then centrifuged for 10 min at 4 °C. The supernatant was collected and its protein concentration was 70
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control of protein loading.
Table 1 Sequence of primers used in current investigation in qRT-PCR. Name
Primer sequence
TNF-α
Sense: 5′-GCCTCCCTCTCATCAGTTCTA-3′ Anti-sense: 5′-GGCAGCCTTGTCCCTTG-3′ Sense: 5′-ACCTGTGTCTTTCCCGTGG-3′ Anti-sense: 5′-TCATCTCGGAGCCTGTAGTG-3′ Sense: 5′-GGTCACACTGGACCAAAGGGACTATG-3′ Anti-sense: 5′-ATTCTGCTGCCGTGCTTCCAAC-3′ Sense: 5′-CTACCGTCGTGACTTCGC-3′ Anti-sense: 5′-GGGTGACATCTCCCTGTT-3′
IL-1β IL-12 β-actin
2.11. Statistical analysis All data are shown as the mean ± standard deviation (SD). Data sets that involved more than two groups were assessed by one-way ANOVA followed by Tukey's multiple-comparison test. All experiments were repeated at least three times. P ≤ 0.05 were considered statistically significant. 3. Results 3.1. Magnolol attenuated colitis induced by DSS
measured using a BCA protein assay kit (23227, Thermo). The total protein was extracted as per the manufacturer's protocol. Protein extracts from colons were analyzed by western blot. Briefly, 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. Finally, protein expression was detected using the Bio-rad Imaging System (Bio-rad Biosciences, USA). The β-actin was used as an internal
It is well known that UC is characterized by namely diarrhea, bloody feces, body weight loss and shortening of the colon length. The results are shown in Fig. 2A. Mice in the DSS group had a significant loss of body weight compared with the control group. However, magnolol treatment significantly attenuated the body weight loss during the progression of murine experimental colitis. Magnolol treatment also markedly decreased the DAI scores induced by DSS (Fig. 2B), which is a clinical parameter reflects the severity of weight loss, stool consistency
Fig. 2. Magnolol treatment improved DSS-induced colitis in mice. (A) Bodyweight 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 ± SD (n = 6 per group). (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01 vs the DSS-treated group on the same day; (#) p < 0.05 vs the control group.
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Fig. 3. Magnolol treatment prevented DSS-induced colon damage in mice (H&E staining ×200). (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. (C) MPO activity in the colonic tissues was detected. Data are presented as means ± SD (n = 6). (∗) p ≤ 0.05, (∗∗) p ≤ 0.01 vs DSS-treated alone group; (#) p < 0.05 vs the control group.
3.3. Magnolol inhibit the pro-inflammatory cytokines in colonic tissues and in MLN
and blood in stool. Furthermore, DSS typically caused colonic shortening in Fig. 2C. However, such changes were also attenuated by magnolol treatments.
As an additional marker of local inflammation, the concentrations of cytokines (TNF-α, IL-1β and IL-12) were further measured by ELISA. The results showed that the expression levels of TNF-α, IL-1β and IL-12 in the DSS group were significantly increased compared with the control group in Fig. 4A. The administration of magnolol inhibited the production of inflammatory cytokines in colonic tissues. To further test the effects of magnolol, mRNA expression levels of pro-inflammatory cytokines were evaluated by qRT-PCR. As shown in Fig. 4B, compared with the control, the expression levels of TNF-α, IL-1β and IL-12 mRNA were also significantly increased by DSS treatment, but were clearly reduced by treatment with magnolol. In addition, to investigate the effects of magnolol on host dependent immune responses, we investigated the cytokine responses of the MLN cells to CBL. The results showed that magnolol could markedly inhibit the elevated expression of these cytokines (Fig. 4C). These results indicated that magnolol suppressed the transcription and final secretion of TNF-α, IL-1β and IL12.
3.2. Magnolol relieved histopathological changes in DSS-induced colitis in mice H&E stained sections of colonic segments were observed under a light microscope. The results showed that there were no histological abnormalities in control group. However, overall damage to the surface epithelium, disruption of cryptal glands, and infiltration of inflammatory cells were observed in the DSS group (Fig. 3A, B). Administration of magnolol significantly improved pathological changes induced by DSS. Furthermore, MPO is a marker of inflammation. In the present study, magnolol with the doses of 25, 50 and 100 mg/kg markedly decreased the level of DSS-induced hyperactivated MPO (Fig. 3C). These results showed that magnolol treatment relieved DSSinduced murine experimental colitis.
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Fig. 4. Magnolol suppressed pro-inflammatory cytokines in colonic tissues and in MLN. (A) Effects of different doses of magnolol (25, 50 and 100 mg/kg) on TNF-α, IL-β and IL-12 levels in colonic tissues. (B) mRNA levels in colonic tissue, The β-actin was used as an internal control (n = 6). (C) TNF-α, IL-1β and IL12 levels in MLN were measured with an ELISA (n = 6). Data are presented as means ± SD (n = 6). (∗) p ≤ 0.05, (∗∗) p ≤ 0.01 vs DSS-treated alone group; (#) p < 0.05 vs the control group.
3.5. Magnolol regulated the expression of PPAR-γ in DSS-induced colitis mice
3.4. Magnolol inhibited NF-κB activity in DSS-induced colitis mice NF-κB plays a pivotal role in regulating cytokines. To explore whether the inhibition of inflammation by magnolol is mediated by NFκB pathway, NF-κB p65 and IκB phosphorylation levels were determined. The results showed that the phosphorylation of p65 and IκB were significantly enhanced in DSS group, but reversed in magnolol treatment groups (Fig. 5).
PPAR-γ, a member of the nuclear hormone receptor family, could regulate NF-κB pathway activation, and thus decreasing the level of pro-inflammatory cytokine. To further explore the anti-inflammatory mechanisms of magnolol, we investigated the possible role of magnolol in the expression of PPAR-γ in DSS-induced mice colitis. The results showed that there was a significant decrease in the expression of PPARγ in the DSS group compared with the control group. However, the treatment with magnolol could regulate the expression of PPAR-γ. The 73
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Fig. 5. Magnolol modulated DSS-induced NF-κB activation in colonic tissue. NF-κB p65 and IκB protein levels in the colonic tissues were analyzed by Western blotting. β-actin was used as a control. Data are presented as means ± SD (n = 3). (∗) p < 0.05 and (∗∗) p < 0.01 vs the group treated with only DSS; (#) p < 0.05 vs the control group.
used for animal experiments in our study were designed in the range of 25 to 100 mg/kg. The therapeutic effect of magnolol was notable, which is in agreement with previous study [27]. It is well-known that UC is characterized by namely diarrhea, bloody feces and body weight loss. In our study, we found that magnolol treatment attenuated body weight loss and DAI scores. In addition, DSS typically caused colonic shortening, while this change was significantly improved by the treatment of magnolol. From histopathological observations, we found the submucosa and crypt structure in the DSS group were irregular, whereas the irregularities of the structures were significantly improved in magnolol treatment groups. Moreover, the treatment with magnolol also significantly reduced the MPO activity which is directly proportional to the number of neutrophils [28]. Many evidences have shown that intense local immune response of UC is associated with release of pro-inflammatory cytokines [29]. Recent genetic and immunological studies have also shown that the involvement of cytokines (TNF-α, IL-1β and IL-12) contributed to IBD perpetuation and tissue destruction [30–32]. TNF-α is the primary and the earliest endogenous mediator in inflammatory diseases and can change the function of the intestinal barrier [33]. IL-1β can also regulate inflammation, and it is necessary in the early stages of the inflammation, which leads to the inflamed colon [34]. Among them, IL12 can not only induce CD4+ T cells to Th1 cells, and promote natural killer cells and T cells to secrete TNF-α cytokines to mediate inflammation [35]. Thus, we assessed the levels of TNF-α, IL-1β and IL-12 by ELISA. The results showed that the levels of TNF-α, IL-1β and IL-12 were remarkably enhanced after DSS challenge. However, the levels of TNF-α, IL-1β and IL-12 were significantly decreased in magnolol treatment groups compared with DSS group. Moreover, the immune responses of the host are closely associated with UC. Therefore, we investigated the secretion of cytokines in MLN cells after stimulation
magnolol (100 mg/kg) treatment group presented more obvious effects than those in the DSS and magnolol (25, 50 mg/kg) treatment groups (Fig. 6). 3.6. Magnolol modulated ZO-1 and occludin expression in DSS-induced colitis mice Epithelial TJ tight junction protein (TJ), an especially important aspect of the mechanical barrier, can prevent harmful substances from breaching the mucosa, maintain cellular integrity and permeability, and thus ensuring a relatively stable internal environment. ZO-1 and occludin are important epithelial TJ proteins. Thus, we detected the effects of magnolol on epithelial TJ proteins (ZO-1 and occludin). As shown in in Fig. 7, the expression of ZO-1 and occludin were downregulated in DSS-induced colitis mice compared to the control group. However, the expression levels of ZO-1 and occludin were significantly improved by the treatment of magnolol, suggesting the importance of magnolol for restoring the integrity of the TJ networks. 4. Discussion UC is a chronic and relapsing inflammatory disease of the gastrointestinal tract. Although the etiology and pathogenesis of UC are complicated and remain uncertain, environmental changes, genetic makeups and gut microbiota are reported to be related to UC [3]. In addition, many therapeutic drugs have been used for this disease, but most of these agents are not very effective and have severe side effects. Magnolol is a natural compound and has been reported to have several pharmacological effects, including anti-allergic [20], anti-oxidant [21], anti-inflammatory [22], anti-arthritic [23] and antimicrobial [10,24]. According to the previous reports [25,26], the doses of magnolol 74
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cytokines, such as TNF-α, IL-1β and IL-12. The activation of NF-ĸB pathway has been observed in IBD patients and DSS-induced murine experimental colitis model [7]. In order to test the mechanism of antiUC, we detected the effect of magnolol on the NF-κB pathway. As expected, the phosphorylations of p65 and IκB which initiate the activations of NF-κB were significantly increased in DSS group, but magnolol markedly down-regulated the increase induced by DSS. In addition, PPAR-γ, a member of the nuclear hormone receptor family, has been reported to negatively regulate inflammation [36]. PPAR-γ has also been implicated in inflammation-related diseases, including colitis [37]. In addition, several studies have also shown that PPAR-γ activation exerts a satisfactory anti-inflammatory effect in IBD patients and experimental colitis models [8,9]. Moreover, it has been reported that PPAR-γ is an up-stream target of NF-κB, and there is a negative correlation between NF-κB and PPAR-γ [38,39]. More importantly, targeted disruption of PPAR-γ expression in macrophages increased the susceptibility to DSS-induced colitis in mice [40]. Therefore, we further detected the protective mechanisms of magnolol on UC. As anticipated, the expression of PPAR-γ was markedly increased in magnolol treated groups compared with DSS group, suggesting PPAR-γ may play an important role in anti-UC of magnolol. Many studies have reported that intestinal barrier plays a key role in maintaining intestinal health [41]. An impaired intestinal barrier results in the increasing permeability of intestinal mucosa to harmful bacteria, leading to intestinal inflammation. TJ proteins have been well documented to play an important role in the maintenance of intestinal mucosal barrier integrity [42]. The structural abnormalities in TJ proteins are the major cause of altered intestinal barrier in IBD patients [43]. Previous study has also showed that structural abnormalities in TJ proteins, including the reduction of ZO-1 and occludin, in IBD patients is a cause of altered intestinal permeability [44]. Therefore, the regulation of TJ proteins may be a highly regarded target for novel therapeutic treatment of UC. In our study, the treatment with magnolol significantly inhibited the reduction of ZO-1 and occludin induced by DSS. Our data showed that magnolol might have a protective effect on barrier integrity by maintaining the expression of ZO-1 and occludin, thereby reducing the severity of colitis.
Fig. 6. Magnolol modulated DSS-induced PPAR-γ activation in colonic tissue. PPAR-γ level in the colonic tissue were analyzed by Western blot. β-actin was used as a control. Data are presented as means ± SD (n = 3). (∗) p < 0.05 and (∗∗) p < 0.01 vs the group treated with only DSS; (#) p < 0.05 vs the control group.
with CBL. We found that magnolol also significantly decreased the expression of TNF-α, IL-1β and IL-12. NF-κB is a transcription factor that promotes transcription of genes encoding pro-inflammatory
Fig. 7. Magnolol modulated ZO-1 and occludin expression in DSS-induced colitis mice. ZO-1 and occludin protein levels in the colonic tissues were analyzed by Western blot. β-actin was used as a control. Data are presented as means ± SD (n = 3). (∗) p < 0.05 and (∗∗) p < 0.01 vs the group treated with only DSS; (#) p < 0.05 vs the control group.
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In conclusion, this study suggested that magnolol treatment attenuates DSS-induced colitis outcomes. The protection associated with magnolol was not only due to the anti-inflammatory effects but also due to the regulation of the gut integrity in mice. Our findings suggested that magnolol might be used for the treatment of UC.
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Conflict of interest policy form and author contribution to study form The authors claim that none of the material in the paper has been published or is under consideration for publication elsewhere. In addition, there is no conflict of interest policy form. Author contribution to study form Concept and design: PS, ZZ, YC, and YH. Acquisition of data: PS, ZZ, SL, CG, and KZ. Analysis and interpretation: PS, ZZ, XL, and JL. Drafting and editing of the manuscript: PS, ZZ, and NZ. All the authors read and approved the final manuscript. Declaration of interest There is no conflict of interest. Acknowledgements This work was supported by the Key Project of Chinese National Programs for Research and Development (no. 2016YFD0501009) and the National Natural Science Foundation of China (nos. 31472248 and 31572582). References [1] J. Cosnes, et al., Epidemiology and natural history of inflammatory bowel diseases, Gastroenterology 140 (6) (2011) 1785–1794. [2] J. Terzic, et al., Inflammation and colon cancer, Gastroenterology 138 (6) (2010) 2101–2114 (e5). [3] H.S. de Souza, C. Fiocchi, Immunopathogenesis of IBD: current state of the art, Nat. Rev. Gastroenterol. Hepatol. 13 (1) (2016) 13–27. [4] H. Vargas Robles, et al., Analyzing beneficial effects of nutritional supplements on intestinal epithelial barrier functions during experimental colitis, J. Vis. Exp. (119) (2017). [5] L. Zuo, et al., Targeting delivery of anti-TNFalpha oligonucleotide into activated colonic macrophages protects against experimental colitis, Gut 59 (4) (2010) 470–479. [6] G. Rogler, et al., Nuclear factor kappaB is activated in macrophages and epithelial cells of inflamed intestinal mucosa, Gastroenterology 115 (2) (1998) 357–369. [7] C.O. Elson, et al., Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota, Immunol. Rev. 206 (2005) 260–276. [8] M. Sanchez-Hidalgo, et al., Rosiglitazone, an agonist of peroxisome proliferatoractivated receptor gamma, reduces chronic colonic inflammation in rats, Biochem. Pharmacol. 69 (12) (2005) 1733–1744. [9] J. Torres, et al., New therapeutic avenues in ulcerative colitis: thinking out of the box, Gut 62 (11) (2013) 1642–1652. [10] G.Y. Zuo, et al., In vitro synergism of magnolol and honokiol in combination with antibacterial agents against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), BMC Complement. Altern. Med. 15 (425) (2015). [11] W. Wei, et al., Magnolol inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model, Inflammation 38 (1) (2015) 16–26. [12] F. Yunhe, et al., The effect of magnolol on the toll-like receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharide-induced acute lung injury in mice, Eur. J. Pharmacol. 689 (1–3) (2012) 255–261. [13] T.C. Yang, et al., Magnolol attenuates sepsis-induced gastrointestinal dysmotility in rats by modulating inflammatory mediators, World J. Gastroenterol. 14 (48) (2008) 7353–7360. [14] W. Tang, et al., Phytochemical profiles and biological activity evaluation of Zanthoxylum bungeanum Maxim seed against asthma in murine models, J. Ethnopharmacol. 152 (3) (2014) 444–450.
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