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DOI 10.1002/mnfr.201400614

RESEARCH ARTICLE

Citrus nobiletin ameliorates experimental colitis by reducing inflammation and restoring impaired intestinal barrier function Yongjian Xiong, Dapeng Chen, Changchun Yu, Bochao Lv, Jinyong Peng, Jingyu Wang and Yuan Lin Department of Pharmacology, Dalian Medical University, Dalian, P. R. China Scope: Inflammatory bowel disease is a chronic inflammatory disorder of the gastrointestinal tract. Citrus nobiletin can exert robust anti-inflammatory effects in vivo and in vitro. We evaluated the impact of nobiletin on the excessive inflammatory response and impaired barrier function in a rodent colitis model. Methods and results: Colitis was established by infusion with 1 mL 2,4,6-trinitrobenzene sulfonic acid (TNBS) dissolved in ethanol (40% v/v) in rats at a 125 mg/kg dose. Caco-2 cell monolayer exposed to LPSs is used as a culture model for intestinal permeability measurements. Nobiletin decreased rat epithelial proinflammatory cytokines and mediators production. Nobiletin restored impaired barrier function in colitic rats and Caco-2 monolayer. Nobiletin decreased protein expressions of Akt, nuclear factor-kappa B (NF-␬B), and myosin light chain kinase (MLCK) isolated from rat intestinal epithelial tissue and Caco-2 cell, respectively. PI3K inhibitor or siRNA targeting of either Akt or NF-␬B mitigated the impact of nobiletin on MLCK expression and barrier function in Caco-2 monolayer, respectively. Conclusion: Nobiletin exerted anti-inflammatory effects in TNBS-induced colitis through the downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression. Nobiletin restored barrier function, which had been damaged after TNBS administration, through the inhibition of the Akt–NF-␬B–MLCK pathway.

Received: September 3, 2014 Revised: November 6, 2014 Accepted: December 18, 2014

Keywords: Akt / Colitis / Impaired barrier function / NF-␬B / Nobiletin

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Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction

Inflammatory bowel disease (IBD) is characterized by severe inflammation of the small bowel and/or colon, resulting in Correspondence: Professor Yuan Lin, Department of Pharmacology, Dalian Medical University, 9 West Section, Lvshun South Road, Dalian 116044, P. R. China Fax: +86-411-86110409 E-mail: [email protected] Abbreviations: COX-2, cyclooxygenase 2; DAI, disease activity index; IBD, inflammatory bowel disease; iNOS, inducible nitric oxide synthase; MLCK, myosin light chain kinase; MPO, myeloperoxidase; NF-␬B, nuclear factor kappa B; NO, nitric oxide; PI3K, phosphatidylinositol 3-kinase; SASP, sulfasalazine; siRNA, small interfering RNA; TER, transepithelial electrical resistance; TNBS, 2,4,6-trinitrobenzene sulfonic acid; TNF-␣, tumor necrosis factoralpha  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

recurrent diarrhea and abdominal pain. Crohn’s disease and ulcerative colitis are the two major forms of IBD [1–3]. The pathogenesis of IBD is complex and multifactorial, as environmental, microbial, genetic, and immune influences lead to relapsing acute inflammation and intestinal tissue damage. [4] A defective intestinal epithelial barrier, leading to the increased intestinal penetration of luminal bacterial Ags, has been postulated to be an important pathogenic factor that contributes to the development of intestinal inflammation. [5] The involvement of myosin light chain kinase (MLCK) in barrier function regulation has been solidly established by other researchers [5–8]. It has also been shown that the overexpression of constitutively active MLCK results in increased intestinal permeability [9]. Additionally, MLCK was shown Colour online: See the article online to view Fig. 2 in colour. www.mnf-journal.com

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temperature (23 ± 2⬚C). Rats were acclimatized for 1 wk before entering the study.

2.2 Reagents

Figure 1. Chemical formula of nobiletin.

to play a central role in barrier function mediated by Caco-2 cells [10]. Caco-2 cell barrier dysfunction is associated with increased MLCK activity, and MLCK inhibitors can prevent barrier impairment [11,12]. The Akt and nuclear factor kappa B (NF-␬B) pathways are involved in MLCK-dependent barrier regulation [5, 13, 14]. The current therapies used to manage human IBD are very expensive and, importantly, are not effective in all patients. Therefore, effective and safe new therapies for the management of IBD are needed [15]. Dietary polyphenolic chemicals, especially polymethoxylated flavonoids, have long been suggested to have attractive functions [16]. Nobiletin (Fig. 1), a widely distributed citrus polymethylated flavonoid, has attracted much attention recently for its beneficial effects on human health [17]. From 10 kg of a citrus peel, 84 mg of nobiletin can be extracted [18]. The absolute bioavailability of nobiletin after oral administration in rats is about 56% [19]. Nobiletin shows anti-inflammatory, anticancer, and antiinsulin resistance activities [20–22]. Recent studies showed that nobiletin, at a low concentration, exerted significant antiinflammatory effects via the downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression [23]. However, the effects of nobiletin on IBD have not been reported. The present study was performed to evaluate the effects of nobiletin on 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced colitis and to characterize the therapeutic mechanism involved. Here, we tested the suppressive properties of nobiletin on TNBS-induced colitis. Antiinflammatory effects, restoration of barrier function, and the underlying mechanisms were assessed.

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Materials and methods

2.1 Animals Sprague–Dawley male rats (age 5–7 wk, weighing 140–160 g) were obtained from the Experimental Animal Center at Dalian Medical University (Certificate of Conformity: No. SCXK 2008-0002). The experimental protocol was approved by the Dalian Medical University Animal Care and Ethics Committee, and all animals used were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication no. 85-23, revised 1985). All rats were given free access to drinking water and standard AIN93M diet under controlled conditions of humidity (50 ± 10%), light (12/12-h light/dark cycle), and  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Nobiletin was purified by Chengdu Must Bio-Technology Co. (Chengdu, China). MLCK antibody was obtained from Abcam (Cambridge, UK). Unless otherwise indicated, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.3 Experimental design Rats were randomly divided into five groups, each consisting of ten rats. The group I served as normal control group; groups II, III, IV, and V were subjected for the induction of colitis. The group II served as colitis control group. Groups III, IV, and V received treatment, respectively, of anticolitic agent sulfasalazine (SASP; 100 mg/kg b.wt, intragastric, dissolved in saline), nobiletin (L; 20 mg/kg b.wt, intragastric, dissolved in saline), nobiletin (H; 40 mg/kg b.wt, intragastric, dissolved in saline) after 1 day of the inflammation induction for 7 consecutive days. Colitis control rats were similarly treated, but were instead infused with 1 mL saline. Nobiletintreated normal rats (no TNBS) group was also established by oral administration of nobiletin (20, 40 mg/kg) for 7 consecutive days, respectively. Rats were housed in plastic cages (two rats per cage) and given free access to drinking water and standard AIN93M diet.

2.4 Experimental colitis induced by TNBS Acute colitis was induced according to previously established protocols with slight modifications [5–7]. Briefly, after a fasting period of 24 h with free access to drinking water, a catheter was inserted through the anus so that its terminus reached approximately to the level of the splenic flexure (8 cm proximal to the anal verge) under urethane anesthesia. Subsequently, the colon was infused with 1 mL TNBS dissolved in ethanol (40% v/v) at a dose of 125 mg/kg. The distal colonic samples (the full thickness of the intestinal wall was prepared) were harvested for biochemical studies.

2.5 Monitoring TNBS-induced colitis Inflammation was evaluated using routine hematoxylin and eosin (H&E) stained colon sections according to previously described morphological criteria [24]. Animal body weights, diarrhea incidence, and total food intake for each group were recorded daily. The colon was scored for macroscopically visible damage on a 0–10 scale [25]. The disease activity index (DAI) was determined as previously reported [15, 26]. www.mnf-journal.com

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2.6 Cell cultures Caco-2 cells were purchased from the ATCC (Rockville, MD, USA) and maintained at 37°C in a 5% CO2 environment. The culture medium was composed of DMEM with 4.5 mg/mL glucose, 50 U/mL penicillin, 50 U/mL streptomycin, 4 mM glutamine, 25 mM HEPES, and 10% FBS.

sis factor-alpha (TNF-␣), IL-1␤, IL-6, nitric oxide (NO), and prostaglandin E2 (PGE2 ), were determined using double-antibody sandwich ELISAs (R&D Systems, USA), according to the manufacturer’s instructions. Colonic NO level was assayed by Griess reaction method using commercial kit (R&D Systems). 2.10 Western blotting assays

2.7 Evaluation of barrier function As previously described, intestinal permeability (barrier function) was measured in vivo as follows. Rats were denied access to food, but allowed water for 3 h. Then, 150 ␮L 80 mg/mL fluorescein isothiocyanate 4 kD dextran (Sigma) was gavaged and serum was harvested 1 and 3 h later. Serum recovery was measured using a Synergy HT plate reader (BioTek, Winooski, VT, USA) [27]. Transepithelial electrical resistance (TER) and inulin flux level were measured to detect barrier function in vitro. Reduced TER and elevated inulin flux levels are two parameters of barrier dysfunction [28]. An epithelial voltohmmeter was used for measurement of TER of filter-grown Caco-2 intestinal monolayers, as previously reported. Caco-2 paracellular permeability was determined using an established paracellular marker, inulin, with a molar mass of 5000 g/mol. A known concentration (2 mM) of radiolabeled inulin (14 C) was added to the apical solution [29, 30]. Caco-2 cells (4 × 105 ) were seeded in the upper chamber of a transwell filter. TER of Caco-2-plated filters was measured every day. About 5 days later, TER could rise to a steady state (always 540 ± 12 ⍀/cm2 ), then barrier function model was established and Caco-2-plated filters could be used. Caco-2 monolayers were treated with LPS or nobiletin. Nontreated Caco-2 monolayers served as normal control. TER always decreased to 310 ± 7 ⍀/cm2 after LPS treatment. Nobiletin could increase the decreased TER level to 400 ± 9 ⍀/cm2 . The relative TER in various treatment groups was calculated as a percentage of normal control. TER in normal control was set to 100%. TER determination was repeated for at least three times.

Proteins were separated by SDS-PAGE (12%) and transferred to polyvinylidene difluoride membranes. Membranes were blotted for specific antibodies. The blots were developed using an enhanced chemiluminescence method (GE Healthcare). Quantification was performed by densitometric analysis of specific bands on the immunoblots using a Multi Spectral imaging system (UVP, Cambridge, UK). 2.11 Real-time PCR analysis of MLCK mRNA expression As previously described [33], total RNA isolation and real-time PCR for RNA quantification were performed. The housekeeping gene GAPDH served as an internal control. The mRNA levels of MLCK were normalized to the mRNA levels of the housekeeping gene GAPDH. The comparative Ct method (2−⌬⌬Ct ) was used to analyze differences in the levels of MLCK mRNA between groups [34]. 2.12 Statistical analysis One-way ANOVA was used where three or more groups of data were compared. Student’s t-test was used to compare two groups of data. Data were expressed as the mean ± SD. The data followed a normal distribution and each group had equal variances. To further evaluate the data, Kruskal–Wallis rank sum test was also used. All experiments were repeated for at least three times and a p value of less than 0.05 was considered statistically significant.

2.8 Cell transfection

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Caco-2 cells were transfected with Lipofectamine 2000 (Invitrogen) and Akt-targeted (sense sequence, TTT GTA GTC ATT GTC CTC CAG CAC AGC TGG AGG ACA ATG ACT ACT TTT T), or NF-␬B p65-targeted (sense sequence GAT TGA GGA GAA ACG TAA A), or a control small interfering RNA (siRNA) oligos (Dharmacon, Lafayette, CO, USA) as previously described [31, 32]. Confluent monolayers were treated with LPS 3 days later. The efficiency of gene silencing was confirmed by Western blotting.

3.1 Nobiletin treatment ameliorated intestinal inflammation in rats in the TNBS-induced colitis model

2.9 ELISA assays In colon, levels of serum proinflammatory cytokines and mediators, including myeloperoxidase (MPO), tumor necro C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Results

Body weight and food intake were significantly reduced compared to those in normal control rats after the induction of colitis (Fig. 2B and C). Biochemical and macroscopic analysis confirmed the successful establishment of colitis. Macroscopically visible damage, DAI, and the colon weight-to-length ratio (Fig. 2 D–F) were significantly increased compared to those in normal control rats. Nobiletin (20 or 40 mg/kg) and SASP ameliorated the inflammation symptoms induced by TNBS, including increasing the body weight and food intake, and decreasing macroscopically visible damage, DAI, and the www.mnf-journal.com

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Figure 2. Nobiletin suppressed inflammatory symptoms in 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced colitis. Nobiletin (L, 20 mg/kg) and nobiletin (H, 40 mg/kg) increased the decreased body weight (B) and food intake (C), and decreased the increased macroscopically visible damage (D), disease activity index (E), and colon weight/length ratio (F) induced by TNBS. **p < 0.01 versus control group; ## p < 0.01 versus TNBS-treated group. SASP, sulfasalazine.

colon weight-to-length ratio. Together, these data show that nobiletin can suppress inflammation in the TNBS-induced colitis model.

3.2 Nobiletin reduced neutrophil infiltration and levels of proinflammatory cytokines and mediators The enhancement of neutrophil infiltration and levels of proinflammatory cytokines and mediators are associated with the initiation of intestinal inflammation [35–37]. MPO activity is measured based on neutrophil infiltration into the damaged tissue [38]. MPO activity and levels of proinflammatory cytokines (TNF-␣, IL-1␤, and IL-6) and mediators (NO and PGE2 ) in tissues from colitic rats were significantly increased compared to those in the control rats (Fig. 3A). SASP and nobiletin (20 or 40 mg/kg) reversed the increased MPO activity and changes in cytokines levels. Expression levels of iNOS and COX-2 proteins were significantly higher in colitic rats compared to the control group; SASP and nobiletin (20 or 40 mg/kg) reversed the changes in iNOS and COX-2 expression (Fig. 3B). Nobiletin (20 or  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

40 mg/kg) exerted no significant effects on iNOS and COX-2 expressions in normal rats (Supporting Information Fig. 1).

3.3 Nobiletin treatment reduces intestinal permeability via Akt and NF-␬B inhibition Intestinal homeostasis requires an epithelial barrier that regulates interactions between luminal material and the interstitium, which include the gut microbiota and mucosal immune cells, respectively [4]. Thus, it is not surprising that mild or severe intestinal barrier disruption can enhance or directly trigger experimental IBD, and increased intestinal permeability leads to intestinal barrier dysfunction [39]. In the colitic rats, intestinal permeability was significantly increased compared to control rats; SASP or nobiletin (20 or 40 mg/kg) reversed the change in intestinal permeability (Fig. 4A). MLCK activity is increased in the intestinal epithelia of patients with active IBD [40]. Activation of the Akt and NF-␬B pathways has been implicated in the pathogenesis of IBD [41, 42]. The protein expression levels of MLCK and NF-␬B were higher than those in the control group; SASP and nobiletin (20 or 40 mg/kg) treatment significantly reversed the change in MLCK and NF-␬B expression levels www.mnf-journal.com

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Figure 3. Nobiletin suppressed proinflammatory cytokines and mediators level. (A) Nobiletin (L, 20 mg/kg) and nobiletin (H, 40 mg/kg) decreased the high myeloperoxidase (MPO) activity, tumor necrosis factor alpha (TNF-␣) level, IL-1␤ level, IL-6 level, nitric oxide level, and prostaglandin E2 (PGE2 ) level induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS); (B) nobiletin reduced the increased inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expression induced by TNBS. **p < 0.01 versus control group; ## p < 0.01 versus TNBS-treated group. SASP, sulfasalazine.

(Fig. 4B). Additionally, phosphatidylinositol 3-kinase (PI3K) and Akt levels were increased in the colitic rat group; nobiletin (20 or mg/kg) treatment reversed these changes in PI3K expression and Akt phosphorylation levels (Fig. 4C). Nobiletin (20 or 40 mg/kg) exerted no significant effects on MLCK, NF-␬B, PI3K expressions and Akt phosphorylation in normal rats (Supporting Information Fig. 2).

3.4 Nobiletin increased relative epithelial resistance and suppressed inulin flux in the Caco-2 cell barrier dysfunction model Guo et al. showed that at a low concentration, LPS treatment induced high TER levels and relative inulin flux levels, indicating barrier dysfunction at the cellular level [43]. Thus, Caco-2 monolayers treated with 0.5 ng/mL of LPS for 3 days were used to establish an in vitro model of barrier dysfunction. The effects of nobiletin (0, 10, 20, 40, or 80 ␮M) treatment on TER levels and inulin flux were measured, and we found that at concentrations of 20 ␮M or greater, nobiletin significantly increased TER levels and suppressed inulin flux. At incubation times of 12 h or longer, nobiletin showed signifi C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

cant stimulatory effects on TER levels (from 310 ± 12 to 400 ± 9 ⍀/cm2 ) or inhibitory effects on inulin flux (Fig. 5F–I).

3.5 Nobiletin suppresses Akt phosphorylation in an LPS-induced barrier dysfunction model Nobiletin inhibited Akt expression in TNBS-induced colitic rats, implicating Akt in the suppression of colitis in rats by nobiletin (Fig. 4C). To further characterize this finding, we studied the effects of nobiletin on LPS-induced barrier dysfunction. LPS exerted significant stimulatory effects on Akt phosphorylation (Fig. 6A and B). At doses of 0–80 ␮M, and at incubation times of 0–36 h, nobiletin exerted dose- and time-dependent inhibitory effects on Akt phosphorylation, further implicating Akt in nobiletin-induced suppression of LPS-induced barrier dysfunction. LPS significantly increased Akt phosphorylation levels, which could be prevented by nobiletin treatment (Fig. 6C). In the presence of the PI3K inhibitor wortmannin, Akt phosphorylation was abolished and neither LPS nor nobiletin treatment could alter Akt phosphorylation levels. Furthermore, wortmannin prevented nobiletin-dependent inhibitory www.mnf-journal.com

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Figure 4. Nobiletin decreased intestinal permeability in colitis group and possible mechanisms. (A) Nobiletin (L, 20 mg/kg) and nobiletin (H, 40 mg/kg) reduced the increased intestinal permeability induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). (B) Nobiletin reversed the increased myosin light chain kinase (MLCK) and nuclear factor kappa-B (NF-␬B) p65 protein level induced by TNBS. (C) Nobiletin reduced the increased phosphoinositide 3kinase (PI3K) protein level and Akt phosphorylation triggered by TNBS. **p < 0.01 versus control group; ## p < 0.01 versus TNBS-treated group. SASP, sulfasalazine.

effects on Akt in this barrier dysfunction model. Similarly, siRNA-induced knockdown of Akt in Caco-2 cells also prevented nobiletin-dependent inhibitory effects on Akt in the barrier dysfunction model, which also indicated that the cell transfection successfully targeted Akt (Fig. 6D). 3.6 Nobiletin-dependent increased TER levels and reduced inulin flux in a barrier dysfunction model mediated by Akt activation TER level was measured at 72 h following LPS treatment. PI3K inhibitor wortmannin (100 nM) was added to Caco-2 monolayers 1 h prior to LPS treatment. LPS treatment was performed at 72 h after cell transfection of siRNA targeting of Akt. Nobiletin was added to Caco-2 monolayers at 60 h after LPS treatment. TER level was measured at 12 h following nobiletin treatment. In the absence of wortmannin, LPS significantly reduced TER levels (from 500 ± 12 to 310 ± 12 ⍀/cm2 ), whereas nobiletin could reverse this effect (from 310 ± 12 to 400 ± 9 ⍀/cm2 ) (Fig. 7A). The presence of wortmannin partially blocked LPS-induced upregulation of TER levels, which was  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

not reversed by nobiletin treatment, suggesting that wortmannin could prevent nobiletin-dependent stimulatory effects on TER levels in this barrier dysfunction model. Wortmannin also prevented nobiletin-dependent inhibitory effects on inulin flux in this barrier dysfunction model (Fig. 7C). Similarly, siAkt prevented nobiletin-dependent upregulation of TER levels (Fig. 7B), and also mitigated the nobiletindependent inhibitory effects on inulin flux (Fig. 7D) in this barrier dysfunction model.

3.7 Nobiletin treatment suppresses MLCK expression in our barrier dysfunction model LPS significantly increased the mRNA and protein expression levels of MLCK in Caco-2 cell monolayers (Fig. 8A and B). At doses of 0–80 ␮M, and at incubation time of 0–36 h, nobiletin showed dose- and time-dependent inhibitory effects on MLCK mRNA and protein expression levels. Pretreatment with the PI3K inhibitor wortmannin prevented nobiletindependent inhibitory effects on MLCK expression in our barrier dysfunction model (Fig. 8C). www.mnf-journal.com

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Figure 5. Nobiletin restored impaired barrier function in vitro. (A) Effect of increasing concentrations of nobiletin (0, 10, 20, 40, 80 ␮M) on increased transepithelial electrical resistance (TER) induced by LPSs. (B) Time-course effect of nobiletin (40 ␮M) on increased TER induced by LPS. (C) Effects of increasing concentrations of nobiletin (0, 10, 20, 40, 80 ␮M) on decreased inulin flux induced by LPS. (D) Time-course effect of nobiletin (40 ␮M) on decreased inulin flux induced by LPS. **p < 0.01 versus control group; ## p < 0.01 versus LPS-treated group.

Similarly, siRNA-induced knockdown of Akt in Caco2 cells also inhibited the nobiletin-dependent reduction of MLCK mRNA and protein levels in our barrier dysfunction model (Fig. 8D). Taken together, these data suggest that the Akt signaling pathway mediates nobiletin-dependent modulation of MLCK expression. 3.8 Nobiletin-dependent inhibitory effects on NF-␬B protein levels in our barrier dysfunction model LPS significantly increased NF-␬B p65 protein expression levels, nobiletin treatment exerted dose- and time-dependent inhibitory effects on NF-␬B p65 protein levels (Fig. 9A and B). Nobiletin treatment significantly reduced NF-␬B p65 protein expression levels in our barrier dysfunction model. Pretreatment with the PI3K inhibitor wortmannin prevented nobiletin-dependent inhibitory effects on NF-␬B p65 expression levels in our barrier dysfunction model (Fig. 9C). Similarly, siRNA knockdown of Akt in Caco-2 cells also inhibited the nobiletin-dependent reduction in NF-␬B p65 protein levels in our barrier dysfunction model (Fig. 9D). 3.9 Nobiletin-dependent inhibition of modulating MLCK levels, TER levels, and inulin flux depends upon NF-␬B Nobiletin exerted inhibitory effects on NF-␬B p65 protein levels in our barrier dysfunction model (Fig. 10A). Additionally, siRNA-induced knockdown of NF-␬B p65 in Caco-2 cells abolished NF-␬B p65 protein expression, and neither LPS nor nobiletin treatment altered NF-␬B p65 protein levels, which  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

further indicated that cell transfection of siRNA targeting NF␬B p65 was effective. LPS significantly increased MLCK mRNA and protein expression levels, whereas nobiletin treatment reversed the change in MLCK levels (Fig. 10B). Furthermore, siRNAmediated knockdown of NF-␬B p65 prevented nobiletindependent inhibitory effects on MLCK expression in our barrier dysfunction model. TER level was measured at 72 h following LPS treatment. LPS treatment was performed at 72 h after cell transfection of siRNA targeting of NF-␬B p65. Nobiletin was added to Caco-2 monolayers at 60 h after LPS treatment. TER level was measured at 12 h following nobiletin treatment. In nontransfected Caco-2 monolayers, LPS significantly reduced TER levels (from 500 ± 12 to 310 ± 12 ⍀/cm2 ), whereas nobiletin could reverse this effect (from 310 ± 12 to 400 ± 9 ⍀/cm2 ) (Fig. 7A). However, siRNA targeting NF␬B p65 blocked nobiletin-dependent effects on TER levels. Similarly, siRNA-mediated knockdown of NF-␬B p65 also prevented nobiletin-dependent inhibitory effects on inulin flux in our barrier dysfunction model (Fig. 10C and D). Taken together, these data show that nobiletin-dependent inhibitory effects on MLCK expression, TER levels, and inulin flux were mediated through the inhibition of the Akt–NF-␬B pathway.

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Discussion

Nobiletin, a polymethoxylated flavone found exclusively in citrus fruit peels, has been recognized to be a promising antiinflammatory agent. Here, the immunosuppressive effects of www.mnf-journal.com

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Figure 6. Nobiletin suppressed the increased Akt phosphorylation induced by LPSs in vitro. (A) Effects of increasing concentrations of nobiletin (0, 10, 20, 40, 80 ␮M) on the increased Akt phosphorylation induced by LPS in Caco-2 monolayer. (B) Time-course effects of nobiletin (40 ␮M) on the increased Akt phosphorylation by LPS in Caco-2 monolayer. (C) PI3K inhibitor wortmannin (100 nM) prevented the inhibitory effects of nobiletin on Akt phosphorylation. (D) siRNA-induced knockdown of Akt prevented the inhibitory effects of nobiletin on Akt phosphorylation. **p < 0.01 versus control group; ## p < 0.01 versus LPS-treated group.

citrus nobiletin in TNBS-induced colitis were performed. At a dose of 20 or 40 mg/kg, nobiletin exerted anti-inflammatory effects and reduced inflammation. Furthermore, nobiletin could also restore epithelial barrier function. As expected, results showed that TNBS induced inflammation, including reduced body weight and food intake, and increased macroscopically visible damage, DAI, and colon weight-to-length ratio. Nobiletin treatment increased body weight and food intake, and reduced macroscopically visible damage, DAI, and the colon weight-to-length ratio, implicating that nobiletin suppressed elevated inflammation response triggered by TNBS. Enhanced neutrophil infiltration and increased levels of proinflammatory cytokines and mediators have been associated with the initiation of intestinal inflammation [35–37]. MPO activity was measured based on neutrophil infiltration into damaged tissues [38]. Results showed that nobiletin treatment reduced the high levels of MPO activity induced by TNBS. Furthermore, nobiletin treatment suppressed the secretion of proinflammatory cytokines (TNF-␣, IL-1␤, and IL6) and mediators (NO and PGE-2). Nobiletin also inhibited the expression of the upstream proteins iNOS and COX-2. Our data indicated that nobiletin could exert anti-inflammatory effects through the inhibition of neutrophil infiltration, re C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ducing the secretion of cytokines and downregulating iNOS and COX-2 expression. Defective intestinal epithelial barrier function is characterized by increased intestinal permeability, which is an important pathogenic factor that contributes to the development of intestinal inflammation [5, 44]. Therapeutic restoration of barrier function can improve pathophysiological and clinical outcomes in both Crohn’s disease and ulcerative colitis [39]. Both in vitro and in vivo studies have demonstrated that intestinal barrier regulation requires intestinal epithelial MLCK activation [45–47], and MLCK activity is increased in intestinal epithelia from IBD patients. Thus, the effects of nobiletin on intestinal permeability and MLCK protein expression were measured. Results showed that TNBS significantly increased intestinal permeability, implicating the loss of intestinal barrier function. Nobiletin treatment suppressed the increased intestinal permeability and MLCK expression, suggesting that nobiletin is capable of restoring intestinal barrier function. Transcription factor NF-␬B activation is reported to a pivotal role in regulating intestinal epithelial barrier function [48, 49]. The PI3K–Akt pathway has been shown to be crucial for increasing intestinal permeability [50, 51]. Therefore, the effects of nobiletin on NF-␬B, PI3K expression and Akt phosphorylation were also measured. Results www.mnf-journal.com

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Figure 7. Wortmannin or siAkt prevented LPSs-induced barrier dysfunction model in vitro. (A) PI3K inhibitor wortmannin (100 nM) prevented inhibition of nobiletin on Caco-2 transepithelial electrical resistance (TER) in barrier dysfunction model. (B) siRNA-induced knockdown of Akt prevented inhibition of nobiletin on Caco-2 TER barrier dysfunction model. (C) PI3K inhibitor wortmannin (100 nM) prevented inhibition of nobiletin on Caco-2 inulin flux in barrier dysfunction model. (D) siRNA-induced knockdown of Akt prevented inhibition of nobiletin on Caco-2 inulin flux barrier dysfunction model. **p < 0.01 versus control group; ## p < 0.01 versus LPS-treated group; && p < 0.01 versus TER or inulin flux before the treatment of LPS in the presence of wortmannin or siRNA-induced knockdown of Akt, respectively.

showed that nobiletin treatment suppressed the increased levels of NF-␬B, PI3K, and Akt that were triggered by TNBS. Together, our data showed that nobiletin bolstered barrier function by decreasing Akt phosphorylation, and downregulating NF-␬B expression and MLCK expression. However, the relationship between these pathways remains unclear. To further verify the restoration effects of nobiletin and the underlying mechanisms, Caco-2 cell monolayers exposed to LPSs were used as a culture model for intestinal barrier function measurements in vitro. TER levels and inulin flux were measured as parameters of barrier dysfunction [43]. Guo et al. showed that LPS could induce barrier loss at doses lower than 10 ng/mL [43]. Dose- and time-dependent curves showed that Caco-2 cell monolayers treated with 0.5 ng/mL LPS for 3 days could be used as a barrier dysfunction model in vitro. At a dose of 20–80 ␮M, nobiletin increased TER levels and decreased inulin flux level upon stimulation with LPS, suggesting a restorative effect of nobiletin on barrier function. At doses of 10–80 ␮M, nobiletin suppressed Akt phosphorylation levels after stimulation with LPS, implying that no C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

biletin exerted inhibitory effects on Akt phosphorylation levels in the barrier dysfunction model. Either pretreatment with the PI3K inhibitor wortmannin (100 ng/mL) or cell transfection of siRNA targeting Akt prevented nobiletin-dependent effects on TER levels and inulin flux. These data showed that the restorative effect of nobiletin was mediated by a decrease in Akt phosphorylation, consistent with in vivo results that a decrease in Akt phosphorylation is followed by nobiletin treatment in colitic rats. Taking into account the importance of MLCK in intestinal barrier regulation, the effects of nobiletin on the expression of MLCK in our barrier dysfunction model were also measured. At doses of 10–80 ␮M, nobiletin decreased the increased MLCK mRNA and protein level in Caco-2 cell model of barrier dysfunction, indicating that nobiletin decreased MLCK expression, consistent with the decrease in MLCK expression followed by nobiletin treatment in rat colitis model. To further detect the involvement of Akt pathway in modulating of MLCK expression, PI3K inhibitor wortmannin and siRNA targeting Akt were used in the present study. Either pretreatment with the PI3K inhibitor wortmannin or cell transfection www.mnf-journal.com

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Figure 8. Wortmannin or siRNAinduced knockdown of Akt prevented myosin light chain kinase (MLCK) expression in LPSs-induced barrier dysfunction model. (A) Effects of increasing concentrations of nobiletin (0, 10, 20, 40, 80 ␮M) on the increased MLCK mRNA and protein expression induced by LPS in Caco-2 monolayer. (B) Time-course effects of nobiletin (40 ␮M) on the increased MLCK mRNA and protein expression by LPS in Caco-2 monolayer. (C) PI3K inhibitor wortmannin prevented the inhibitory effects of nobiletin on MLCK mRNA and protein expression in vitro. (D) siRNA-induced knockdown of Akt prevented the inhibitory effects of nobiletin on MLCK mRNA and protein expression in vitro. **p < 0.01 versus control group; ## p < 0.01 versus LPS-treated group; && p < 0.01 versus MLCK expression before the treatment of LPS in the presence of wortmannin or siRNA-induced knockdown of Akt.

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Figure 9. Wortmannin or siRNAinduced knockdown of Akt prevented inhibitory effects of nobiletin on NF-␬B expression in LPSs-induced barrier dysfunction model. (A) Effects of increasing concentrations of nobiletin (0, 10, 20, 40, 80 ␮M) on the increased NF-␬B protein expression induced by LPS in Caco-2 monolayer. (B) Timecourse effects of nobiletin (40 ␮M) on the increased NF-␬B protein expression by LPS in Caco-2 monolayer. (C) PI3K inhibitor wortmannin prevented inhibitory effects of nobiletin on NF-␬B protein expression in vitro. (D) siRNAinduced knockdown of Akt prevented inhibitory effects of nobiletin on NF-␬B protein expression in vitro. **p < 0.01 versus control group; ## p < 0.01 versus LPS-treated group; && p < 0.01 versus NF-␬B protein expression before the treatment of LPS in the presence of wortmannin or siRNA-induced knockdown of Akt.

of siRNA targeting Akt prevented the nobiletin-dependent effects on MLCK expression. These data showed that nobiletin exerted inhibitory effects on MLCK expression through the inhibition of Akt phosphorylation. Previous studies have shown that NF-␬B activation is involved in barrier function disruption in proinflammatory cytokine-treated intestinal epithelial cells [48,49]. NF-␬B pathway can be activated by molecules upstream of PI3K/Akt [52, 53], the effect of nobiletin on NF-␬B expression and the involvement of Akt phosphorylation in nobiletin-dependent effects of NF-␬B expression were then measured. Results showed that at doses ranging from 10 to 80 ␮M, nobiletin inhibited NF-␬B expression in Caco-2 cell monolayers model of barrier dysfunction, consistent with the decrease in NF␬B expression followed by nobiletin treatment in vivo. Either pretreatment with the PI3K inhibitor wortmannin or cell transfection of siRNA to target Akt prevented the nobiletindependent effects on NF-␬B p65 expression. These data showed that the inhibitory effect of nobiletin on NF-␬B expression was mediated through the inhibition of Akt phosphorylation. Research showed that cytokines-induced stimulation in MLCK promoter activity could be mediated by NF-␬B activation, and inhibition of NF-␬B activation could prevent the cytokines-induced increase in promoter activity and the subsequent increase in MLCK protein expression [54]. Therefore, the possible role of NF-␬B modulation on MLCK expression was measured in Caco-2 cell. The delivery of siRNA  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

targeting NF-␬B p65 prevented the nobiletin-dependent inhibitory effects on MLCK levels, showing that the nobiletindependent effects on MLCK levels were mediated by the inhibition of NF-␬B. Similarly, siRNA targeting NF-␬B p65 prevented the effects of nobiletin on TER levels and inulin flux in our barrier dysfunction model. These data showed that the restorative effects of nobiletin were related to a decrease in NF-␬B expression. Taken together, our data show that nobiletin can restore epithelial barrier dysfunction through the inhibition of Akt phosphorylation and the subsequent inactivation of NF-␬B and downregulation of MLCK gene expression levels. Drug permeation across the intestinal membrane is described by the effective permeability coefficient (Peff ) [55]. Yao and co-workers showed that Peff value of nobiletin (15 ␮g/mL) for each intestinal segment was described as follows: duodenum: 43.5 × 10−6 cm/s; jejunum: 44.5 × 10−6 cm/s; ileum: 47.5 × 10−6 cm/s; colon: 62.5 × 10−6 cm/s [56]. Peff value >20 × 10−6 cm/s means high absorptive capacity in small intestine [57]. These findings implicate that nobiletin could be absorpted well in colon. Nobiletin (100 ␮M) shows antiangiogenic activity in endothelial cells [17], oral administration of nobiletin (200 mg/kg) exerts beneficial effects in alleviating insulin resistance in rats [58], and nobiletin at a dose of 40 ␮M in vitro or 40 mg/kg in vivo in our study restored barrier function and suppressed inflammation symptom, suggesting its potential value of using relative low doses of nobiletin in treating colitis. www.mnf-journal.com

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Figure 10. siRNA-induced knockdown of NF-␬B prevented the inhibitory effects of nobiletin on LPSs-induced barrier dysfunction in vitro. (A) siRNA-induced knockdown of NF-␬B prevented the inhibitory effects of nobiletin on NF-␬B expression in barrier dysfunction. (B) siRNA-induced knockdown of NF-␬B prevented the inhibitory effects of nobiletin on myosin light chain kinase (MLCK) mRNA and protein expression in barrier dysfunction. (C) siRNA-induced knockdown of NF-␬B prevented the stimulatory effects of nobiletin on transepithelial electrical resistance (TER) level in barrier dysfunction. (D) siRNA-induced knockdown of NF-␬B prevented the stimulatory effects of nobiletin on inulin flux level in barrier dysfunction. **p < 0.01 versus control group; ## p < 0.01 versus LPS-treated group; && p < 0.01 versus MLCK expression or TER level or inulin flux level before the treatment of LPS in the presence of siRNA-induced knockdown of NF-␬B.

In conclusion, nobiletin exerted anti-inflammatory effects in the TNBS-induced colitis model through the inhibition of neutrophil infiltration, reduction of cytokines secretion, and the downregulation of iNOS and COX-2 expression. Nobiletin could also restore barrier function in vivo and in vitro. This restoration effect was mediated by the inhibition of Akt phosphorylation and the subsequent inactivation of NF-␬B and downregulation of MLCK gene expression. To the best of our knowledge, this is the first report of the suppressive effects of nobiletin on IBD. Nobiletin can reduce the excessive inflammatory response in colitis and restore epithelial barrier function. Such data may provide new insights that might help to inform therapies to alleviate IBD symptoms. This study was supported by University Innovation Team Project Foundation of Education Department of Liaoning Province (grant number LT2013019); and National Natural Science Foundation of China (grant number 30772601). The authors have declared no conflict of interest.

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