Abstract
Inflammatory bowel disease (IBD) is chronic inflammatory and relapsing disease of the gut. It has been known that activation of nuclear factor-κB (NF-κB) and production of proinflammatory cytokines play important roles in the pathogenesis of IBD. In this study, the effect of vanillin (4-hydroxy-3-methoxybenzaldehyde), a potent nuclear factor-κB (NF-κB) inhibitor, was evaluated in mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis. Oral administration of vanillin improved macroscopic and histological features of TNBS-induced colitis in a dose-dependent manner. Vanillin not only prevented TNBS-induced colitis but also ameliorated the established colitis. By in vivo NF-κB bioluminescence imaging, electrophoretic mobility shift assay, and Western blot, we found that vanillin suppressed in vivo NF-κB activities through the inhibition of p65 translocation, inhibitor of nuclear factor-κB(IκB)-α phosphorylation, and IκB kinase activation. Furthermore, vanillin reduced the expressions of proinflammatory cytokines [interleukin (IL)-1β, IL-6, interferon-γ, and tumor necrosis factor-α] and stimulated the expression of anti-inflammatory cytokine (IL-4) in colonic tissues. In conclusion, this work identified vanillin as an anti-inflammatory compound with the capacity to prevent and ameliorate TNBS-induced colitis. Due to its safety, vanillin could be a potent candidate for the treatment of IBD.
A sharp rise in the incidence of inflammatory bowel disease (IBD) has been observed in the Western world since the early 1950s. At present, IBD is fairly common in northern countries, such as the United States and United Kingdom, affecting 0.5 to 1.0% of the population during their lifetime (Russel, 2000). Although IBD is more common in developed countries, there are indications that more cases are being seen lately in low-incidence areas, such as southern Europe, the Middle East, Eastern Asia, the Indian subcontinent, Latin America, and Eastern Europe (Economou and Pappas, 2008).
IBD, including Crohn's disease (CD) and ulcerative colitis in a clinical setting, is chronic intestinal inflammatory disease characterized by frequent relapsing with clinical manifestations, such as diarrhea, blood in the stool, abdominal pain, and weight loss. The etiology of IBD is still unknown, but the imbalance of mucosal homeostasis plays an important role in the pathogenesis of IBD (Cho, 2008). Several murine models producing the main symptoms and molecular mechanisms of CD have been established and have been used to explore new therapeutics for these diseases (Strober et al., 2002). The murine model that is frequently used is based on the intrarectal administration of trinitrobenzene sulfonic acid (TNBS), which randomly haptenates the proteins in the colonic mucosa and triggers an inflammatory response similar to that in CD (Neurath et al., 1995). In the TNBS-induced colitis model, increases in mucosal proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-12, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, have been shown to play important roles in sustained inflammatory responses (Neurath et al., 1995). It has been shown that the expressions of these proinflammatory cytokine genes are mainly regulated by the transcription factor nuclear factor-κB (NF-κB) (Barnes and Karin, 1997). In addition, activation of NF-κB plays a central role in initiating the inflammatory process in IBD (Schreiber et al., 1998). Thus, inhibition of NF-κB activation may be a promising target for the treatment of patients with IBD.
At present, the mainstay of therapy for IBD is anti-inflammatory drugs, such as 5-aminosalicylic acid (5-ASA), and immunosuppressants, such as glucocorticoids, azathioprine, and 6-mercaptopurine. These drugs have been shown to inhibit NF-κB activities; however, these drugs often trigger undesirable side effects, such as pneumonitis, pancreatitis, hepatitis, bone marrow suppression, and risk of infection (Bantel et al., 2000). Therefore, development of natural anti-inflammatory medicines with high efficacy, low toxicity, and low price is desired for the treatment of patients with IBD.
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a widely used flavor compound in food and cosmetics, with an estimated annual worldwide consumption of more than 2000 tons (Walton et al., 2003). Vanillin has been reported to inhibit mutagenesis induced by chemical and physical mutagens and to suppress the invasion and migration of cancer cells (Lirdprapamongkol et al., 2005; Cheng et al., 2008; Liang et al., 2009). It also displays chemopreventive effects in multiorgan carcinogenesis and hepatocarcinogenesis models in rats (Akagi et al., 1995). Moreover, vanillin displays antimicrobial and antioxidative properties and is used as a food preservative and for medicinal purposes (Fitzgerald et al., 2004; Santosh Kumar et al., 2004). Recently, vanillin has been shown to inhibit lipopolysaccharide-stimulated NF-κB activation and cyclooxygenase-2 gene expression in murine macrophages (Murakami et al., 2007). The in vitro inhibitory ability of vanillin on NF-κB activation implied that vanillin might be effective in mice with colitis. Therefore, we administered TNBS-induced mice with vanillin to evaluate the beneficial effects of vanillin on intestinal inflammation. Our data showed that oral administration of vanillin prevented colitis and ameliorated established colitis via the NF-κB signaling pathway. These findings suggested that vanillin exhibited therapeutic value for patients with IBD.
Materials and Methods
Induction of Colitis and Vanillin Treatment. Female BALB/c mice (6–8 weeks old; 21 ± 2 g) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Mouse experiments were conducted under ethics approval from the China Medical University Animal Ethics Committee.
To induce colitis, 5 mg of TNBS (Sigma-Aldrich, St. Louis, MO) in 0.1 ml of 50% ethanol was slowly administered into the colon of a lightly anesthetized mouse via a thin catheter (polyethylene-50; BD Biosciences, Heidelberg, Germany) attached to a 0.5-ml syringe. The catheter tip was inserted 3 cm proximal to the anal verge, and the mouse was held in a vertical position for 30 s after instillation. Control mice received the same volume of 50% ethanol alone. If a mouse excreted this solution quickly (<10 min), it was rejected for further analysis.
To investigate the dosage effect of vanillin, various amounts of vanillin (Sigma-Aldrich) and 5 mg of TNBS in 0.1 ml of 50% ethanol were administered into the colon. To examine the preventive effect of vanillin, 50 mM vanillin (equivalent to 36.3 mg/kg body weight) was administered orally for three consecutive days before TNBS administration. To study the therapeutic effect of vanillin, 50 mM vanillin (equivalent to 36.3 mg/kg body weight) was orally administered for seven consecutive days starting 1 day after TNBS administration. Animals were monitored daily for appearance of diarrhea, loss of body weight, and survival. All of the mice were sacrificed 7 days after administration of TNBS.
Macroscopic and Microscopic (Histological) Assessment. Colonic weight and length were measured as gross indicators of colitis. Colons were also examined under a dissecting microscope and graded for macroscopic lesions. Macroscopic assessment of the colitis severity was scored according to a previously established scoring system as follows: 0, no ulcer and no inflammation; 1, local hyperemia without ulceration; 2, ulceration without hyperemia; 3, ulceration and inflammation at one site only; 4, two or more sites of ulceration and inflammation; and 5, ulceration extending more than 2 cm (Wallace and Keenan, 1990). For histological analysis, colons were fixed, sectioned, and stained with hematoxylin and eosin. Histological changes were graded semiquantitatively from 0 to 4 according to previously described criteria as follows and in a blinded manner: 0, no sign of inflammation; 1, very low level of leukocyte infiltration; 2, low level of leukocyte infiltration; 3, high level of leukocyte infiltration, high vascular density, and thickening of the colon wall; and 4, transmural infiltration, loss of goblet cells, high vascular density, and thickening of the colon wall (Neurath et al., 1995). The macroscopic and microscopic scores were assessed by three experts.
In Vivo and ex Vivo Imaging of NF-κB Activity. Transgenic mice, which carried the luciferase gene driven by a NF-κB-responsive element, were used as described previously (Ho et al., 2007). Transgenic mice were orally administered 50 mM vanillin, equivalent to 36.3 mg/kg vanillin body weight, for seven consecutive days after TNBS administration. One week later, mice were anesthetized with isoflurane, injected intraperitoneally with 150 mg/kg d-luciferin body weight, placed face up in the chamber, and imaged for 5 min with the camera (IVIS Imaging System 100 Series; Xenogen, Alameda, CA) set at the highest sensitivity. Photons emitted from tissues were quantified using Living Image software (Xenogen). For ex vivo imaging, mice were sacrificed and entire colons were rapidly removed. Colons were imaged with the same setting used for in vivo studies. Signal intensity was quantified as the sum of all detected photon counts per second within the region of interest after subtracting of background luminescence and is presented as photons per second per square centimeter per steradian (sr).
Luciferase Activity Assay in Organ Homogenates. Colons were removed, homogenized in 1 ml of Triton lysis buffer (50 mM Tris-HCl, pH 7.8, 1% Triton X-100, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride), centrifuged at 12,000 rpm for 15 min at 4°C, and stored at -20°C. Protein concentration was quantified with the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA), based on the method of Bradford (1976). Luciferase activity was measured as relative light units as described previously (Hsiang et al., 2005) and normalized for total protein content.
Nuclear Extraction and Biotinylated Electrophoretic Mobility Shift Assay. The excised colons were homogenized in ice-cold lysis buffer (10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.1% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin). The homogenates were then incubated on ice for 10 min and centrifuged at 15,000 rpm for 10 min at 4°C. The supernatants were stored at -70°C as the cytosolic extracts, and the pellets were incubated on ice for 1 h with nuclear extraction buffer (20 mM HEPES-KOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.2% Nonidet P-40, 1 μg/ml leupeptin, and 1 μg/ml pepstatin). The resultant homogenates were centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatants were collected and stored at -70°C as nuclear extracts. Protein concentration was quantified with the Bio-Rad protein assay (Bio-Rad Laboratories), based on the method of Bradford (1976). EMSA was performed as described previously (Lee et al., 2008)
Western Blot Assay. The cytosolic or nuclear proteins (10 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis, and the protein bands were then transferred electrophoretically to nitrocellulose membranes. Membranes were blocked in blocking buffer (20 mM Tris-HCl, pH 7.6, 140 mM NaCl, 0.1% Tween 20, and 5% skim milk powder) and probed with polyclonal antibodies against IκB kinase (IKK), phosphorylated IKK, IκB-α, phosphorylated IκB-α, and p65 (Cell Signaling Technology Inc., Danvers, MA). The bound antibody was detected with peroxidase-conjugated anti-rabbit antibody followed by enhanced chemiluminescence system (Amersham, Chalfont St. Giles, Buckinghamshire, UK) and exposed by autoradiography.
Reverse Transcription-Polymerase Chain Reaction. Total RNAs were extracted from colonic tissues using RNeasy Mini kit (QIAGEN, Valencia, CA). One microgram of total RNA was reverse-transcribed using oligo(dT)15 primer and SuperScript III (Invitrogen, Carlsbad, CA) in a total volume of 20 μl. Two microliters of reverse transcription mixture was subjected to PCR to measure the mRNAs of cytokines and β-actin. PCR amplification was performed with Taq polymerase (Promega, Madison, WI) for 26 cycles at 92°C for 45 s, 55°C for 45 s, and 72°C for 2 min. PCR primers for cytokines and β-actin were as follows: IL-1β sense, 5′-CATCCTCTGTGACTCATGGG-3′; IL-1β antisense, 5′-CTTCTTCTTGGGTATTGCTTG-3′; IL-4 sense, 5′-TCGGCATTTTGAACGAGGTC-3′; IL-4 antisense, 5′-GAAAAGCCCGAAAGAGTCTC-3′; IL-6 sense, 5′-TATGAAGTTCCTCTCTGCAA-3′; IL-6 antisense, 5′-CTTTGTATCTCTGGAAGTTTCAG-3′; IFN-γ sense, 5′-GCTCTGAGACAATGAACGCT-3′; IFN-γ antisense, 5′-AAAGAGATAATCTGGCTCTGC-3′; TNF-α sense, 5′-TAGCCCACGTCGTAGCAAAC-3′; TNF-α antisense, 5′-CACCCATTCCCTTCACAGAG-3′; β-actin sense, 5′-GGAGAAGATCTGGCACCACACC-3′; and β-actin antisense, 5′-CCTGCTTGCTGATCCACATCTGCTGG-3′.
Statistics Analysis. Data are presented as mean ± S.E.M. Student's t test was used for a comparison between two experiments. A value of P < 0.05 was considered statistically significant.
Results
Vanillin Suppressed TNBS-Induced Colitis in Mice. Previous studies showed that intrarectal administration of TNBS induced colitis resembling human IBD (Neurath et al., 1995). Therefore, we initially tested the dosage effect of TNBS in mice. Colitis severity was evaluated by macroscopic and histological features. In control mice, no sign or a very low level of injury in the colon was observed (Fig. 1). TNBS significantly induced macroscopic and microscopic damage in colons in a dose-dependent manner. The entire colonic wall became thick due to edema. The major injury of colitis was observed in the distal half of the colon, and focal ulcers were detected in approximately 63% of colonic tissues treated with 5 mg of TNBS.
Next, we evaluated the effect of vanillin on TNBS-induced colitis. Various amounts of vanillin and 5 mg of TNBS were coadministered into the colon. As shown in Fig. 2, vanillin improved the macroscopic and histological features of TNBS-induced colitis in a dose-dependent manner. Macroscopic examination of colons after TNBS induction showed inflammation compared with controls (Fig. 2A). In contrast, colons from vanillin-treated mice showed mild inflammation. Histological examination of colons from TNBS-induced mice showed mucosal thickening and an increase in lymphoid follicle size (Fig. 2B). In contrast, vanillin significantly ameliorated the signs of colitis compared with TNBS-induced mice. Macroscopic score, colonic weight/length ratio, and microscopic score were significantly lower in the group treated with 50 mM vanillin. These findings suggested the therapeutic potential of vanillin in mice with TNBS-induced colitis.
Vanillin Prevented Colitis and Ameliorated Established Colitis. We further tested whether vanillin was able to prevent TNBS-induced colitis or ameliorate established colitis. Vanillin (50 mM, equivalent to 36.3 mg/kg) was orally administered for three consecutive days before TNBS treatment. 5-ASA has been used over decades for the treatment of IBD (Hanauer and Meyers, 1997). Therefore, 5-ASA was used as a positive control for the comparison. As shown in Fig. 3, pretreatment of 5-ASA slightly decreased the macroscopic damage and histological features of colons in mice with TNBS-induced colitis. However, pretreatment of vanillin significantly reduced the weight/length ratio and improved the macroscopic and histological damage of TNBS-induced colitis. These data suggested that preventive treatment with vanillin was capable of protecting colonic injuries in mice after the administration of TNBS.
The therapeutic effect of vanillin was then assessed by oral administration of vanillin for seven consecutive days after TNBS treatment. As expected, oral administration of 5-ASA significantly reduced the colonic weight/length ratio (Fig. 4). Vanillin also significantly reduced the weight/length ratio. In addition, vanillin significantly improved the distortion of crypts, loss of goblet cells, and infiltration of mononuclear cells in mice with TNBS-induced colitis. These findings suggested that vanillin was capable of ameliorating established colitis in mice.
Vanillin Suppressed NF-κB Activities in Colons of Mice with TNBS-Induced Colitis. NF-κB is a critical molecule involved in the regulation of inflammation (Barnes and Karin, 1997). We wondered whether vanillin improved TNBS-induced colitis via the suppression of NF-κB activity. Therefore, in vivo bioluminescence was used to monitor the NF-κB activity in NF-κB transgenic mice. As shown in Fig. 5A, a strong luminescence was observed in the lower abdominal region of TNBS-treated mice. Ex vivo imaging showed that a bioluminescent signal was detected in the colon. These data suggested that the lower abdominal luminescence originated from the colon. Oral administration of vanillin in mice with established colitis led to a decrease in bioluminescent signals in the lower abdominal region and colonic tissues, suggesting that vanillin inhibited NF-κB activities of colons in mice with colitis.
To demonstrate a correlation between imaging, actual luciferase enzyme activity, and NF-κB activity, we excised the colons, prepared the organ extracts, and performed the luciferase activity assays and biotinylated EMSAs. Luminescence imaged with the camera correlated highly with luciferase enzyme activity (Fig. 5B). Luciferase activity assay showed that TNBS induced luciferase activity in the colon, whereas vanillin suppressed TNBS-induced luciferase activity. Biotinylated EMSA showed that TNBS increased the DNA-binding ability of NF-κB in the colon, whereas vanillin reduced the TNBS-induced DNA-binding ability of NF-κB (Fig. 5C). These findings suggested that vanillin improved TNBS-induced colitis through the inhibition of NF-κB activity.
Vanillin Inhibited NF-κB Signal Transduction Pathways of Colons in Mice with TNBS-Induced Colitis. The activation of NF-κB is preceded by translocation of NF-κBto the nucleus after phosphorylation and degradation of IκB-α (Karin and Ben-Neriah, 2000). Western blot was therefore performed to analyze the signaling pathway involved in the regulation of NF-κB activity by vanillin. As shown in Fig. 6, administration of TNBS elevated the translocation of p65 to the nucleus, stimulated the phosphorylation of IκB-α, and induced the phosphorylation of IKK. However, vanillin inhibited p65 translocation and suppressed the levels of phosphorylated IκB-α and IKK in the colon. These data suggested that vanillin inhibited TNBS-induced NF-κB activation in colons via the inhibition of IκB-α phosphorylation and IKK activation.
Vanillin Reduced the Expressions of Cytokine Genes of Colonic Tissues in Mice with TNBS-Induced Colitis. We further evaluated the effect of vanillin on the productions of inflammatory cytokines that are linked to TNBS-induced colitis. As shown in Fig. 7, TNBS induced mRNA expressions of proinflammatory cytokines, such as IL-1β, IL-6, IFN-γ, and TNF-α, in colonic mucosa, whereas vanillin suppressed the mRNA expressions of these cytokines. The expression of anti-inflammatory cytokine IL-4 was down-regulated in TNBS-treated colonic tissues, whereas vanillin stimulated the expression of IL-4 gene. These findings suggested that oral administration of vanillin suppressed proinflammatory cytokine production and stimulated anti-inflammatory cytokine production in mice with TNBS-induced colitis.
Discussion
TNBS-induced colitis is widely used to evaluate the effects of drugs because of its similarity to CD and the availability of a quantitative scoring system (Neurath et al., 1995). Our data showed that vanillin was capable of decreasing the colonic weight/length ratio and improving macroscopic and microscopic damage in a concentration-related manner. Oral administration of vanillin not only prevented TNBS-induced colitis but also ameliorated established colitis. Therefore, these findings suggested that vanillin could be a potent therapeutic agent for the treatment of patients with IBD.
NF-κB is the key transcription factor for proinflammatory responses in IBD. It is thought to be important in the initiation and progression of human IBD and animal models of colitis (Schreiber et al., 1998). Disease activity in mice with TNBS-induced colitis is inhibited by antisense oligonucleotides for p65 subunit of NF-κB, also suggesting the critical role of NF-κB in mediating inflammatory responses of IBD (Neurath et al., 1996). NF-κB bioluminescence imaging was used to monitor the in vivo NF-κB activities of transgenic mice with TNBS-induced colitis in this study. NF-κB transgenic mice model has been used to monitor the chronic inflammation induced by ultraviolet light, TNF-α, IL-1α, or lipopolysaccharide (Carlsen et al., 2002, 2004). Our data showed that TNBS induced a strong NF-κB-driven luminescent signal in the lower abdominal region and colonic tissues of mice with colitis. Moreover, macroscopic and histological analyses indicated that inflammation was evoked in the same region. Therefore, the correlation between the NF-κB bioluminescence imaging and histological changes enforced the critical role of NF-κB in TNBS-induced colitis. Oral administration of vanillin led to a marked decrease in bioluminescent signals in the lower abdominal region and colonic tissues of mice with established colitis. Furthermore, biotinylated EMSA and Western blotting showed that vanillin inhibited NF-κB activity via the inhibition of IκB-α phosphorylation and IKK activation. Therefore, our results demonstrated that noninvasive NF-κB imaging system can be used to assess the inflammation induced by TNBS in living animals. In addition, our data demonstrated the feasibility of NF-κB bioluminescence imaging for the discovery of agents against TNBS-induced colitis.
Homeostasis of T-helper (Th) 1/Th2 cytokines is important in intestinal mucosal immunity (Strober et al., 2002). T-helper cells are a subgroup of lymphocytes that activate and direct other immune cells through release of cytokines. An imbalance between Th1 and Th2 cytokines has been implicated in the pathogenesis of IBD, particularly CD (Holland et al., 2008). Moreover, an increase in proinflammatory cytokines, mainly Th1 cytokines, is observed in patients with CD, also suggesting that CD is a Th1-mediated disease (Neurath et al., 1997). TNBS-induced colitis has been reported to be associated with Th1 cell responses, requiring T-cell activation as the central initiating event that subsequently leads to macrophage recruitment and activation (Elson et al., 1996). The bias toward Th1 cytokines, such as IL-1β, IL-6, TNF-α, and IFN-γ, is critical in the establishment of chronic inflammation (Cho, 2008). In contrast, administration of anti-inflammatory cytokine IL-4, the principal effector molecule of Th2 cells, has led to therapeutic potentials in TNBS-induced colitis (Hogaboam et al., 1997). These results suggested the beneficial effects of Th2 cytokines in the treatment of IBD. Our data showed that vanillin down-regulated the expression of Th1 proinflammatory cytokines in mice with TNBS-induced colitis. In contrast to Th1 cytokines, vanillin increased the mRNA expression of anti-inflammatory cytokines in vivo. Vanillin inhibiting a Th1 response and favoring a Th2 response might explain why vanillin was effective in preventing and ameliorating the Th1-mediated disease TNBS-induced colitis.
Increased levels of inflammatory cytokines were secreted in the colons of IBD patients, leading to the production of other inflammatory mediators, such as NO and reactive oxygen species (Reinecker et al., 1993). In patients with IBD, NO overproduction results in mucosal injury (Ischiropoulos et al., 1992). The massive infiltration of leukocytes in IBD is thought to produce large amounts of reactive oxygen species that would participate in intestinal damage (Kruidenier et al., 2003). These findings clearly indicate that these proinflammatory mediators are involved in the pathogenesis of IBD. Vanillin is a potent antioxidant. It inhibits protein oxidation and lipid peroxidation by quenching singlet oxygen (Kamat et al., 2000). It scavenges free radicals. It also protects DNA and mitochondrial membrane against oxidative stress in vitro (Santosh Kumar et al., 2004). These findings implied that, in addition to the regulation of Th1 cytokines, the antioxidative potential of vanillin might also contribute to its therapeutic effect on TNBS-induced colitis. In addition, it is known that the antioxidant properties, such as free radical scavenging activity, of 5-ASA may be a potential mechanism for its protective effect in IBD (Joshi et al., 2005). Vanillin was more effective than 5-ASA in prevention or therapy of TNBS-induced colitis in this study, suggesting that the antioxidant activity of 5-ASA might be less than that of vanillin.
Oral or colonic administration of 5-ASA has been used for decades for the treatment of IBD. However, patients administered 5-ASA or its derivatives suffer several serious adverse conditions, such as anorexia, dyspepsia, nausea/vomiting, hemolysis, neutropenia, agranulocytosis, folate malabsorption, reversible, male infertility, and neuropathy (Sands, 2000). Vanillin is generally regarded as safe by the Flavor and Extract Manufacturers Association and is recognized as suitable for food use by the Food and Drug Administration (Opdyke, 1977). Vanillin displays no genetic toxic effect in the Salmonella mutagenicity test and in human lymphocytes (Mortelmans et al., 1986; Jansson and Zech, 1987). In addition, oral chronic toxicity studies with rats fed diets containing 1000 mg/kg/day vanillin for 2 years show no effect on growth or hematology and no macroscopic or microscopic changes in the tissues (Hagan et al., 1967). Moreover, no adverse effect is observed when the diet contains 150 mg/kg vanillin body weight (Kirwin and Galvin, 1993). In our study, vanillin at 50 mM (equivalent to 36.3 mg/kg) was sufficient to prevent and improve IBD in mice. Therefore, due to its safety, vanillin seems to be a potent drug candidate for the treatment of IBD.
In conclusion, our work identified vanillin as an anti-inflammatory compound with the capacity to prevent and ameliorate TNBS-induced colitis. Vanillin inhibited in vivo NF-κB activity via the inhibition of IκB-α phosphorylation and IKK activation; reduced the production of Th1 cytokines; and in turn, suppressed TNBS-induced colitis in mice. In addition to the inhibition of NF-κB activity, vanillin is able to scavenge free radicals and quench singlet oxygen, the important risk factors of CD. Therefore, these results suggested that vanillin may be a potent therapeutic agent for IBD with a broad spectrum of therapeutic mechanisms.
Footnotes
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This study was supported in part by the National Science Council [Grant NSC 97-2320-B-039-012-MY3]; the Committee on Chinese Medicine and Pharmacy at the Department of Health [Grants CCMP 96-RD-201, CCMP 97-RD-201]; and China Medical University [Grant CMU97-CMC-004].
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T.-Y.H. and C.-Y.H. contributed equally to this work.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.109.152835.
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ABBREVIATIONS: IBD, inflammatory bowel disease; CD, Crohn's disease; TNBS, trinitrobenzene sulfonic acid; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; NF-κB, nuclear factor-κB; 5-ASA, 5-aminosalicylic acid; sr, series resistance; EMSA, electrophoretic mobility shift assay; IKK, IκB kinase; IκB, inhibitor of nuclear factor-κB; PCR, polymerase chain reaction; Th, T-helper.
- Received February 26, 2009.
- Accepted May 6, 2009.
- The American Society for Pharmacology and Experimental Therapeutics