Neomangiferin modulates the Th17/Treg balance and ameliorates colitis in mice.
Background: Anemarrhena asphodeloides (Liliaceae family) and Mangifera indica L (Anacardiaceae family) contain neomangiferin as the main active constituent and have been used to treat inflammation, asthma, and pain.
Purpose: A preliminary study found that neomangiferin inhibited splenic T cell differentiation into Th17 cells and promoted Treg cell production in vitro. Therefore, we examined its anti-colitic effects in vitro and in vivo.
Methods: Splenocytes isolated from C57BL/6J mice were treated with neomangiferin. Colitis was either induced in vivo by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to C57BL/6J mice or occurred spontaneously in colitis caused by interleukin (IL)-10 knockout at age of 13 weeks. Mice were treated daily with neomangiferin or sulfasalazine. Inflammatory markers, cytokines, enzymes and transcription factors were measured by enzyme-linked immunosorbent assay, immunoblot, and flow cytometry.
Results: Neomangiferin suppressed retinoic acid receptor-related orphan receptor gamma t (ROR[gamma]t) and 1L-17 expression in IL-6/transforming growth factor [beta]-stimulated Th17 splenocytes and increased IL-10 expression in vitro. Mouse TNBS-induced colon shortening, macroscopic score, and myeloperoxidase activity were inhibited by neomangiferin, which also reduced TNBS-induced activation of nuclear factor-[kappa]B and extracellular signal-regulated kinases, as well as expression of inducible nitric oxide synthase and cyclooxygenase-2. In addition, neomangiferin inhibited TNBS-induced expression of tumor necrosis factor-[alpha], IL-17, IL-6, and 1L-1[beta], and increased IL-10 expression. Neomangiferin inhibited TNBS-induced differentiation to Th17 cells and promoted the development of Treg cells. Moreover, in [IL-10.sup.-/-] mice, neomangiferin inhibited colonic myeloperoxidase activity, suppressed Th17 cell differentiation, and reduced levels of TNF-[alpha] and IL-17.
Conclusion: Neomangiferin may restore the balance between Th17/Treg cells by suppressing IL-17 and ROR[gamma]t expression and inducing IL-10 and forkhead box P3 expression, thus ameliorating colitis.
Inflammatory bowel disease (1BD) including Crohn's disease and ulcerative colitis is a chronically relapsing condition associated with overly aggressive T-cell responses to host gut microbiota in the gastrointestinal tract (Corridoni et al. 2014; Maloy and Powrie 2011). The gastrointestinal tract is continuously exposed to a variety of foreign antigens, including those expressed by gut microbiota, and the immune response to these leads to chronic gastrointestinal inflammation (McLean et al. 2015; Wang and Achkar 2015). Experimental colitis can be induced in conventional animals, but not in germ-free animals (Eastaff-Leung et al. 2010; Mayne and Williams 2013). Therefore, gut microbiota are a prerequisite for IBD. The lamina propria of patients with IBD contains higher levels of interleukin (IL)-17 and Thl7 cells than those of control subjects (Galzez 2014; Kanai et al. 2012; Yang et al. 2014). Therefore, the adaptive response of immune cells, including Th17 and Treg cells, might be involved in the pathogenesis of IBD. Th17 cells produce IL-17, IL-21, and IL-22 (Leppkes et al. 2009). IL-17 cytokine acts as a potent mediator of immune reactions by increasing chemokine production in various tissues, facilitating recruitment of monocytes and neutrophils to inflammation sites (Monteleone et al. 2012). IL17 acts synergistically with proinflammatory cytokines such as tumor necrosis factor (TNF)-[alpha] and IL-1[beta] (Granet and Miossec 2004). Th17-based inflammation plays an important role in the development of 1BD. Therefore, inhibition of Th17 cell differentiation has been identified as a potential therapeutic target in 1BD (Gizinski et al. 2010).
Anemarrhena asphodeloides (Liliaceae family) and Mangifera indica L. (Anacardiaceae family) contain mangiferin and neomangiferin as major active constituents and have been frequently used as herbal medicines and functional foods to treat inflammation, asthma, and pain (Wang et al. 2014; Xu et al. 2011; Zhou et al. 2015). Mangiferin has shown central nervous system-stimulating, anti-oxidant, analgesic, anti-inflammatory, anti-colitic, and anti-diabetic activities (Jung et al. 2009; Garrido et al. 2004; Mckay and Blumberg 2007; Pinto et al. 2005; Jeong et al. 2014; Marquez et al. 2012). Neomangiferin (mangiferin-7-0-/3-D-glucoside), which is more hydrophilic than mangiferin, exhibits anti-osteoporotic and anti-lipidemic properties (Zhou et al. 2015; Qin et al. 2008). However, the anti-inflammatory effect of neomangiferin has not been studied thoroughly.
In a preliminary in vitro study, neomangiferin potently suppressed transforming growth factor (TGF)[beta]/IL-6-induced differentiation of splenic T cells into Th17 cells. It also increased granulocyte macrophage colony stimulating factor (GM-CSF)-induced differentiation of splenic T cells to Treg cells. The present study extended this work by investigating the anti-inflammatory effects of neomangiferin in vivo by using mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis or with colitis induced by 1L-10-knockout ([IL-10.sup.-/-]).
Materials and methods
Chemicals and reagents
TNBS, gentamycin, collagenase type VIII, and RPM1 1640 were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Antibodies for [beta]-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Antibodies for 1[kappa]B kinase alpha (1[kappa]B[alpha]), p-1[kappa]B[alpha], extracellular signal-regulated kinases (ERK), p-ERK, p65, and p-p65 were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, U.S.A.). Pan T Cell Isolation Kit II, anti-IL-17A, and-FoxP3, anti-CD3, anti-CD28, GM-CSF, recombinant IL-6, and recombinant TGF[beta] were purchased from MiltenyiBiotec (Bergisch Gladbach, Germany). Neomangiferin was purchased from Carbosynth ltd (Berkshire, UK).
Male C57BL/6 (20-23 g, 6 weeks old) and Male C57BL/6 [IL-10.sup.-/-] mice (18-21 g, 6 weeks-old) obtained from Jackson (Indianapolis, IN, U.S.A.) were supplied from RaonBio (Seoul, Korea), were provided with water and food ad libitum, and maintained in a ventilated room at an ambient temperature of 22[degrees]C [+ or -] 1[degrees]C with 50% [+ or -] 10% humidity and a 12-h diurnal light cycle (lights on 07:0019:00) for 1 weeks before the experiment.
All animal experiments were approved by the Committee for the Care and Use of Laboratory Animals in the Kyung Hee University and performed in accordance with the Kyung Hee University guidelines for Laboratory Animals Care and Usage (IRB No. KHUASP (SE)-14-057).
Preparation of splenocytes
The spleens were aseptically taken out from male C57BL/6 mice, gently crushed, and were lysed with tris-buffered ammonium chloride (Lee et al. 2015). The cell suspension was prepared in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum and T cells were isolated using magnetic cell sorting with Pan T Cell Isolation Kit II (MiltenyiBiotec).
For Th17 cell differentiation, purified T cells were stimulated with anti-CD3 (1 [micro]g/ml) and anti-CD28 (1 [micro]g/ml) in the presence of recombinant IL-6 (20 ng/ml) and recombinant TGF-[beta] (1 ng/ml) for 5 days (Okamoto et al. 2012). For Treg cell differentiation, purified T cells were stimulated with anti-CD3 (1 [micro]g/ml) and anti-CD28 (1 [micro]g/ml) for 5 days (Fantini et al. 2007). T cells were fixed and stained with anti-CD4, anti-CD25, anti-FoxP3 or anti-IL-17A antibodies and then analyzed by flow cytometry.
Induction of colitis in wild-type and [IL-10.sup.-/-] mice
To prepare colitis in wild-type mice, the mice were randomly divided into 5 groups: normal control and TNBS-induced colitic control groups treated with vehicle, neomangiferin (10 or 20 mg/kg), or sulfasalazine (50 mg/kg). Each group consisted of six mice. Colitis was induced by intra-rectal injection of 0.1 ml of 2.5% (w/v) TNBS solution dissolved in 50% ethanol into the colon of anesthetized mice (Joh et al. 2011). TNBS solution was injected 3.5-4 cm proximal to the anus. The mice were held in a vertical position for 0.5 min to allow distribution of TNBS solution in the entire colon. If the injected TNBS solution was excreted into the anus, the mouse was excluded from the experiment. Normal group was treated with saline instead of TNBS solution. Neomangiferin (10 or 20 mg/kg) or sulfasalazine (50 mg/kg) dissolved in 2% tween 80 were orally administered once daily for 3 days from 24 h after TNBS treatment.
To prepare spontaneous chronic colitis in [IL-10.sup.-/-] mice, the [IL-10.sup.-/-] mice (7 weeks old) were housed for 6 weeks and then neomangiferin (20 mg/kg) or sulfasalazine (50 mg/kg) dissolved in 2% tween 80 were orally administered once a day for 3 weeks, as previously reported (Ung et al. 2010).
The mice were sacrificed 20 h after the final administration of test agents and the colon was quickly removed, opened longitudinally, and gently washed with PBS. Macroscopic evaluation scored the severity of colitis according to the method of Joh et al. (2011) (0, no ulcer and no inflammation; 1, no ulceration and local hyperemia; 2, ulceration with hyperemia; 3, ulceration and inflammation at one site only; 4, two or more sites of ulceration and inflammation; 5, ulceration extending more than 2 cm). Then the colons were stored at -80[degrees]C until usage for immunoblotting and ELISA.
For the histological exam, colons were fixed in 10%-buffered formalin solution, cut into 10-|xm sections, stained with hematoxylineosin, and observed under a light microscopy. For the immunostaining for antigen-presenting cells (APC) such as macrophages, the sections were detected using anti-CD86 antibody and DAB substrate kit (Thermo Scientific, Rockford, IL, USA) (Kim et al. 2014). Horseradish peroxidase activity was visualized with 3-amino-9-ethylcarbazole.
Assay of myeloperoxidase activity
The colons isolated from the mice were homogenized in a solution containing 0.5% hexadecyl trimethyl ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7.0), and then centrifuged for 20 min at 20,000 x g and 4[degrees]C. A 50 [micro]l aliquot of the supernatant was added to the reaction mixture consisting of 1.6 mM tetramethyl benzidine and 0.1 mM H202, incubated at 37[degrees]C for 3 min, and then monitored the absorbance at 650 nm. The myeloperoxidase activity was calculated as the quantity of enzyme degrading 1 [micro]mol/ml of peroxide at 37[degrees]C, and expressed in unit/mg protein (Joh et al. 2011). The protein content was determined by the method of Bradford (1976).
Flow cytometry of lamina propria cells
To assay Th17 and Treg cells in the lamina propria, mouse colons were cut into small pieces, incubated with 2.5 mM EDTA at 37[degrees]C with agitation for 20 min, minced and digested for 20 min with 1 mg/ml collagenase type VIII at 37[degrees]C. The cells were then filtered, and the T cells were then isolated using a Pan T cell Isolation Kit II. The isolated T cells were fixed and stained with anti-CD4, anti-CD25, anti-FoxP3, or anti-lL-17A antibodies and then analyzed by the flow cytometry.
The colonic tissues of mice were homogenized in radio immunoprecipitation assay (R1PA) lysis buffer and centrifuged (Kim et al. 2014). The supernatant proteins (30 [micro]g) were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidenedifluoride membranes, and then hybridized overnight, at 4[degrees]C with 1:1000 diluted antibody of COX-2, iNOS, p65, p-p65, TAK1, p-TAK1, p38, p-p38, JNK, p-JNK, ERK, p-ERK, occludin, claudin-1, ZO-1 or [beta]-actin, then incubated with 1:2000 diluted anti-rabbit or anti-mouse IgG secondary antibody for 1 h at room temperature, and washed 3 times with PBS containing 0.1% Tween 20 for 10 min. Bands were visualized with the enhanced chemiluminescence reagent.
Enzyme-linked immunosorbent assay (ELISA)
For the cytokine assay, the colon tissues were homogenized in a 600 [micro]l of ice-cold RIPA lysis buffer containing 0.1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail (Kim et al. 2014). The lysate was centrifuged (10,000 xg, 4[degrees]C) for 20 min, and the supernatant was transferred to 96-well ELISA plates. TNF-[alpha], IL-1[beta], IL-10, and IL-17 concentrations were determined using commercial ELISA kits.
Quantitative real time - polymerase chain reaction (qRT-PCR)
Real Time PCR for IL-5, IL-10, IL-17, interferon (IFN)-[gamma], T-bet, GATA binding protein 3 (GATA3), retinoic acid receptor-related orphan receptor gamma t (ROR[gamma]t), forkhead P3 (Foxp3), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed with 2[micro]g of total RNA isolated from the colon, utilizing Takara thermal cycler, which used SYBER premix agents, as described previously (Gu et al. 2013). Thermal cycling conditions were as follows: activation of DNA polymerase for 5 min at 95[degrees]C, followed by 38 cycles of amplification for 10 s at 95[degrees]C and for 30 s at 60[degrees]C. The normalized expression of the assayed genes, with respect to GAPDH, was computed for all samples using the Microsoft Excel data spreadsheet. Primers were used as follows: IL-5 for, 5'-AAAGAGAAGTGTGGCGAGGA GAGAC-3', rev, 5'-CCTTCCATTGCCCACTCTGTACTCATC-3'; IL-10 for, 5'-ATG CTG CCT GCT CTT ACT GAC TG-3', rev, 5'-CCC AAG TAA CCC TTA AAG TCC TGC-3'; IL-17 for, 5'-TTT AAC TCC CTT GGC GCA AAA-3' rev, 5'-CTT TCC CTC CGC ATT GAC AC-3'; IFN-[gamma] for, 5'-TCAAGTGGCATAGATGTGGAAGAA3', rev, 5'-TGGCTCTGCAGGATTTTCATG-3'; T-bet for, 5'-CCTCTTCTATCCAACCAGTATC-3', rev, 5'-CTCCGCTTCATAACTGTGT3'; GATA3 for, 5'-GAAGGCATCCAGACCCGAAAC-3', rev, 5'-ACCCATGGCGGTGACCATGC-3'; ROR[gamma]t for, 5'-ACAGCCACTGCATTCCCA GTTT-3', rev, 5'-TCTCGGAAGGACTTGCAGACAT-3'; Foxp3 for, 5'-CCC ATC CCC AGG AGT CTT-3', rev, 5'-ACC ATG ACT AGG GGC ACT GTA-3'; and GAPDH for, 5'-TGC AGT GGC AAA GTG GAG AT-3', rev, 5'-TIT GCC GTG AGT GGA GTC AT-3'.
Data were expressed as the mean [+ or -] standard deviation (SD). The data of animal experiments were analyzed by one-way analysis of variance followed by the student-Newman-Keuls test for multiple comparisons. P values of 0.05 or less were considered statistically significant.
Effect of neomangiferin on splenic T cell differentiation
In a preliminary study, neomangiferin strongly inhibited TGF[beta]/IL-6-induced differentiation of splenic CD4+ T cells to Th17 cells. The simulation of IL-6 and TGF[beta] with anti-CD3 and antiCD28 in splenic CD[4.sup.+] T cells significantly induced a population of Th17 cells (25.8%). However, neomangiferin treatment significantly suppressed this TGF[beta]/IL-6-mediated induction of the Th17 cell population, as compared with non-treated cells. Neomangiferin also inhibited the expression of IL-17 and its transcription factor ROR[gamma]t (Fig. 1A). Moreover, neomangiferin increased anti-CD3 and anti-CD28-induced differentiation of splenic T cells to Treg cells and induced the expression of IL-10, whereas it did not influence Foxp3 expression (Fig. IB). Neomangiferin did not exhibit cytotoxicity against splenocytes that were treated for 5 days during the differentiation experiment.
Anti-inflammatory effect of neomangiferin in mice with TNBS-induced colitis
To examine the anti-inflammatory effect of neomangiferin in vivo, we administered it to mice with TNBS-induced colitis. TNBS caused colon shortening and severe inflammation, as evidenced by thickened, shortened, erythematous, and macrophage-infiltrated colons (Fig. 2A-C). TNBS-induced colon shortening was proportional to the infiltration of APC into the colon. Macroscopic scores and the activity of myeloperoxidase also increased (Fig. 2D). Neomangiferin treatment inhibited TNBS-induced colon shortening, epithelial cell disruption, APC infiltration, and myeloperoxidase activity. Neomangiferin (20 mg/kg) inhibited myeloperoxidase activity by 73.9%, compared with that of mice treated with TNBS alone (p < 0.05). TNBS treatment markedly decreased the expression of the tight junction proteins ZO-1, occludin, and claudin-1 in the colon (Fig. 2E), as well as the infiltration of APC by immunostaining using anti-CD86 antibody (Fig. 2F). Oral administration of neomangiferin inhibited TNBS-induced suppression of tight junction proteins. In addition, neomangiferin treatment inhibited TNBS-induced ERK phosphorylation, NF-[kappa]B activation, and COX-2 and iNOS expression (Fig. 3A-C). Neomangiferin (20 mg/kg) inhibited the expression of TNF-[alpha], 1L-1[beta], IL-17, and IL-6 by 45.5%, 78.1%, 63.6%, and 85.5%, respectively (Fig. 3D). Furthermore, neomangiferin increased IL-10 expression. These anti-inflammatory effects of neomangiferin were comparable to those of sulfasalazine.
Next, we examined the effect of neomangiferin on T cell differentiation in the colonic lamina propria of mice with TNBS-induced colitis (Fig. 4A). TNBS treatment significantly increased differentiation to Th17 cells and inhibited differentiation to Treg cells. However, treatment with neomangiferin inhibited TNBS-induced Th17 cell differentiation and increased Treg cell differentiation. We also measured the levels of T cell differentiation markers using qRT-PCR (Fig. 4B). TNBS significantly induced expression of IL-17, IFN-[gamma], ROR[gamma]t, and T-bet and suppressed expression of IL-10, IL-5, Foxp3, and GATA3. Treatment with neomangiferin significantly attenuated TNBS-mediated induction of IL-17, IFN-[gamma], ROR[gamma]t, and T-bet expression and increased expression of TNBS-suppressed IL-10, IL-5, Foxp3, and GATA3.
Anti-inflammatory effect of neomangiferin in IL-[10.sup.-/-] mice
IL-[10.sup.-/-] mice maintained under specific pathogen-free condition for >6 weeks develop spontaneous chronic enterocolitis that is phenotypically similar to IBD in humans (Ung et al. 2010). In this study, IL-[10.sup.-/-] mice were maintained for 13 weeks and then treated with neomangiferin for 3 weeks. These mice showed spontaneous colitis associated with rectal prolapse and diarrhea. However, the histopathology was significantly less severe in the neomangiferin-treated IL-[10.sup.-/-] mice, as compared with the vehicle-treated control. Neomangiferin treatment inhibited epithelial cell disruption, as evidenced by the infiltration of mononuclear and polymorphonuclear cells and reduced myeloperoxidase activity (Fig. 5A-D). Neomangiferin (20 mg/kg) inhibited myeloperoxidase activity by 52.6%, as compared with vehicle-treated IL-[10.sup.-/-] mice (p < 0.05). Neomangiferin treatment increased the expression of tight junction proteins (Fig. 5E). These were supported that neomangiferin inhibited the infiltration of APC by immunostaining using anti-CD86 antibody (Fig. 5F). Moreover, ERK phosphorylation, NF-[kappa]B activation, and COX-2 and iNOS expression were inhibited by neomangiferin (Fig. 6A-C). Neomangiferin (20 mg/kg) inhibited TNF-[alpha], IL-1[beta], IFN-[gamma], and 1L-17 expression by 17.3%, 54.5%, 27.8%, and 52.8%, respectively (Fig. 6D). These anti-inflammatory effects of neomangiferin were comparable to those of sulfasalazine.
Next, we investigated the effect of neomangiferin on T cell differentiation in the colonic lamina propria of IL-10-mice (Fig. 7A). Th17 cell differentiation was induced in IL-[10.sup.-/-] mice and Treg cell differentiation was suppressed, as previously reported (Eastaff-Leung et al. 2010). However, treatment of IL-[10.sup.-/-] mice with neomangiferin suppressed differentiation to Th17 cells and increased differentiation to Treg cells. We also measured T cell differentiation markers using qRT-PCR (Fig. 7B). Treatment with neomangiferin significantly inhibited IL-17 and ROR[gamma]t expression, but did not inhibit IFN-[gamma], IL-10, IL-5, Foxp3, GATA3, or T-bet expression. The inhibitory effects of neomangiferin on IL-17 and ROR[gamma]t expression were more potent than those of sulfasalazine.
Inflammatory mediators such as TNF-[alpha], IL-1[beta], IL-10, and IL-17 are secreted by immune cells (neutrophils, monocytes, and T lymphocytes) and regulate IBD (Geremia et al. 2014; Walsh et al. 2013). While excessive stimulation by TNF-a and IL-17 causes colitis, IL-10 is an anti-inflammatory cytokine that attenuates colitis (Liu et al. 2011). IL-10 deficiency induces differentiation to form Th1 and Th17 cells and reduces Treg cell formation, leading to autoimmune diseases such as IBD (Chaudhry et al. 2011). Therefore, inhibitors of Th17 differentiation and activators of Treg cell differentiation play a pivotal role in the initiation and development of these chronic inflammatory diseases.
Aminosalicylates and TNF-[alpha] antibodies, including infliximab, are prominently used to control IBD (Lopez and Peyrin-Biroulet 2013). These drugs suppress the inflammatory responses of macrophages and Th1 cells rather than those of Th17 cells. Additionally, methotrexate upregulates IL-10 expression and downregulates expression of pro-inflammatory cytokines and has been used as an anti-colitic drug (Robinson 1998). Some molecules isolated from natural products, including ocotillol, icariin and and rographolide regulate T cell differentiation and exhibit anti-colitic effects (Lee et al. 2015; Liu et al. 2014; Tao et al. 2013). Ocotillol inhibits Th17 cell formation and ROR[gamma]t and IL-17 expression in the lamina propria of mice with TNBS-induced colitis, while increasing differentiation to Treg cells and inducing Foxp3 and IL-10 expression.
Icariin ameliorates dextran sulfate sodium-induced colitis in mice by inhibiting Th1/Th17 responses via suppression of signal transducer and activator transcription (STAT) (1) and STAT3 activation (Tao et al. 2013). Andrographolide sulfonate might suppress the differentiation of Th1 and Th17 subsets by inhibiting the activation of p38 mitogen-activated protein kinase and NF-[kappa]B, leading to an anti-inflammatory effect in mice with TNBS-induced colitis (Liu et al. 2014). Quercetin also exerted intestinal antiinflammatory activity in chronic T lymphocyte-dependent colitis (Mascaraque et al. 2014). Resveratrol attenuated colitic inflammation in IL-[10.sup.-/-] mice by down-regulating Th1 responses (Singh et al. 2012). However, the anti-inflammatory mechanisms underlying these effects of natural product constituents on adaptive immune cells have not been fully elucidated.
In this study, neomangiferin suppressed the differentiation of splenic T cells to Th17 cells and reduced the expression of IL-17 and ROR[gamma]t in vitro. Neomangiferin also inhibited NF-[kappa]B activation in Th17 cells and induced Treg cell differentiation, increasing expression of IL-10 and Foxp3 in these cells. Additionally, neomangiferin is more hydrophilic than mangiferin. This means that neomaniferin is more readily delivered to the colon following oral administration to mice and humans, as compared to mangiferin.
Therefore, we investigated its anti-inflammatory effects in mice with TNBS-induced colitis. This investigation showed than neomangiferin inhibited Th17 cell differentiation and increased Treg cell differentiation. In addition, it ameliorated TNBS-induced expression of proinflammatory cytokines (TNF-[alpha], IL-1[beta], and IL-6) and the inflammatory markers, COX-2 and iNOS, in these mice. These results suggest that neomangiferin inhibits IL-17 expression and Th17 cell differentiation by suppressing ROR[gamma]t and NF-[kappa]B activation via the inhibition of ERK phosphorylation, while increasing Treg differentiation by inducing the expression of Foxp3 and IL10. Moreover, treatment with neomangiferin significantly inhibited TNBS-induced IFN-[gamma] and T-bet expression and recovered TNBS-induced suppression of IL-5 and GATA3 expression.
Neomangiferin also ameliorated colitic inflammation in IL-[10.sup.-/-] m1ice. Moreover, neomangiferin inhibited IL-17 and ROR[gamma]t expression an1d Th17 cell differentiation. However, it did not influence expression of Foxp3, T-bet, or GATA3, and did not affect Treg cell differentiation. Compared to ocotillol (Lee et al. 2015), neomangiferin more potently inhibited Th17 cell differentiation and IL17 and RORyt expression, whereas ocotillol more potently increased Treg cell differentiation and Foxop3 expression. These results suggest that neomangiferin may inhibit Th17 cell differentiation by suppressing IL-17 and ROR[gamma]t expression. The intestinal inflammatory response in 1BD is initiated by activation of innate immune cells such as macrophages and dendritic cells by foreign antigens, while the inflammatory response is maintained by stimulation of the adaptive immune response, which is mainly mediated by Th cells. The Th1- and Th2-specific transcription factors, T-bet and GATA3, participate in this process: T-bet activation suppresses GATA-3 expression (Foersch et al. 2013; Shen et al. 2014). In human IBD, Crohn's disease has been reported to involve Th1 cells and the related cytokines, IL-12 and IFN-[gamma], while ulcerative colitis involves Th2 cells and the cytokines, IL-5 and IL-13. Recently, Th17 cells capable of producing IL-17 and Treg cells, which produce 1L-10, were reported to have a key role in the pathogenesis of IBD (Shen et al. 2014). These findings suggest that the balance between Th17 and Treg cells must be maintained in order to cure and/or prevent IBD. Neomangiferin potently inhibited Th17 cell differentiation and expression of IL-17 and ROR[gamma]t, while increasing Treg cell differentiation and expression of 1L-10 and Foxp3. These results suggest that neomangiferin can modulate the Th17/Treg balance and ameliorate colitis.
Conflict of interest
The authors declare no conflict of interest in this paper.
Received 3 August 2015
Revised 29 December 2015
Accepted 3 January 2016
Abbreviations: COX-2, cyclooxygenase 2; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinases; Foxp3, forkhead box P3; GAT3, GATA binding protein 3; IBD, inflammatory bowel disease; 1L, interleukin; iNOS, inducible nitric oxide synthase; NF-[kappa]B, nuclear [kappa]B; qRT-PCR, quantitative real time -polymerase chain reaction; ROR[gamma]t, retinoic acid receptor-related orphan receptor gamma t; STAT, signal transducer and activator of transcription; TGF, transforming growth factor; TNBS, 2,4,6-trinitrobenzene sulfonic acid; TNF, tumor necrosis factor.
Su-Min Lim (a), Geum-Dan Kang (a), Jin-Ju Jeong (a), Hyun Sik Choi (b), Dong-Hyun Kim (a), *
(a) Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, 1, Hoegi. Dongdaemun-gu, Seoul 130-701, South Korea
(b) DongWha Pharm Research Institute, 35-71, Topsil-ro, Giheung-gu, Yongin-Shi, Cyeonggi, 46-902 South Korea
* Corresponding author. Tel.: +82 2 961 0357; fax: +82 2 957 5030.
E-mail address: email@example.com (D.-H. Kim).
This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C1020).
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
Chaudhry, A., Samstein, R.M., Treuting, P., Liang, Y., Pils, M.C., Heinrich, J.M., Jack, R.S., Wunderlich, F.T., Bruning, J.C., Muller, W., Rudensky, A.Y., 2011. Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation. Immunity 34, 566-578.
Corridoni, D., Arseneau, K.O., Cominelli, F., 2014. Inflammatory bowel disease. Immunol. Lett. 161, 231-235.
Eastaff-Leung, N., Mabarrack, N., Barbour, A., Cummins, A.. Barry, S., 2010. Foxp3+ regulatory T cells, Th17 effector cells, and cytokine environment in inflammatory bowel disease. J. Clin. Immunol. 30, 80-89.
Fantini, M.C., Dominitzki, S., Rizzo, A., Neurath, M.F., Becker, C., 2007. In vitro generation of CD4+ CD25+ regulatory cells from murine naive T cells. Nat. Protoc. 2, 1789-1794.
Foersch, S., Waldner, M.J., Neurath, M.F., 2013. Innate and adaptive immunity in inflammatory bowel diseases. Dig. Dis. 31, 317-320.
Galvez, J., 2014. Role of Th17 cells in the pathogenesis of human IBD. ISRN Inflamm., 928461.
Garrido, G., Delgado, R., Lemus, Y., Rodriguez, J., Garda, D., Nunez-Selles, A.J., 2004. Protection against septic shock and suppression of tumor necrosis factor alpha and nitric oxide production on macrophages and microglia by a standard aqueous extract of Mangifera indica L. (VIMANG[R]) Role of mangiferin isolated from the extract. Pharmacol. Res. 50, 165-172.
Geremia, A., Biancheri, P, Allan, P., Corazza, G.R., Di Sabatino, A., 2014. Innate and adaptive immunity in inflammatory bowel disease. Autoimmun. Rev. 13, 3-10.
Gizinski, A.M., Fox, DA., Sarkar, S., 2010. Pharmacotherapy, concepts of pathogenesis and emerging treatments. Co-stimulation and T cells as therapeutic targets. Best Pract. Res. Clin. Rheumatol. 24, 463-477.
Granet, C., Miossec, P., 2004. Combination of the pro-inflammatory cytokines IL-1, TNF-alpha and IL-17 leads to enhanced expression and additional recruitment of AP-1 family members, Egr-1 and NF-kappaB in osteoblast-like cells. Cytokine 26, 169-177.
Gu, W., Kim, KA., Kim, D.H., 2013. Ginsenoside Rhl ameliorates high fat diet-induced obesity in mice by inhibiting adipocyte differentiation. Biol. Pharm. Bull. 36, 102-107.
Jeong, J.J., Jang, S.E., Hyam, S.R., Han, M.J., Kim, D.H., 2014. Mangiferin ameliorates colitis by inhibiting IRAKI phosphorylation in NF-xB and MAPK pathways. Eur. J. Pharmacol. 740, 652-661.
Joh, E.H., Lee, I.A., Jung, I.H., Kim, D.H., 2011. Ginsenoside Rbl and its metabolite compound K inhibit IRAK-1 activation-the key step of inflammation. Biochem. Pharmacol. 82, 278-286.
Jung, K., Lee, B., Han, S.J., Ryu, J.H., Kim, D.H., 2009. Mangiferin ameliorates scopolamine-induced learning deficits in mice. Biol. Pharm. Bull. 32, 242-246.
Kanai, T., Mikami, Y., Sujino, T., Hisamatsu, T., Hibi, T., 2012. ROR[gamma]t-dependent IL-17A-producing cells in the pathogenesis of intestinal inflammation. Mucosal. Immunol. 5 pp. 240-224.
Kim, K.A., Lee, I.A., Gu, W., Hyam, S.R., Kim, D.H., 2014. [beta]-Sitosterol attenuates high-fat diet-induced intestinal inflammation in mice by inhibiting the binding of lipopolysaccharide to toll-like receptor 4 in the NF-[kappa]B pathway. Mol. Nutr. Food.
Res. 58, 963-972.
Lee, S.Y., Jeong, J.J., Le, T.H.V., Eun, S.H., Nguyen, M.D., Park, J.H., Kim, D.H., 2015. Ocotillol, a majonoside R2 metabolite, ameliorates 2,4,6-trinitrobenzenesulfonic acid-induced colitis in mice by restoring the balance of Th17/Treg cells. J. Agric. Food Chem. accepted.
Leppkes, M., Becker, C., Ivanov, I.I., Hirth, S., Wirtz, S., Neufert, C., Pouly, S., Murphy, A.J., Valenzuela, D.M., Yancopoulos, G.D., Becher, B., Littman, D.R., Neurath, M.F., 2009. RORgamma-expressing Th17 cells induce murine chronic intestinal inflammation via redundant effects of IL-17A and IL-17F. Gastroenterology 136, 257-267.
Liu, B., Tonkonogy, S.L, Sartor, R.B., 2011. Antigen-presenting cell production of IL10 inhibits T-helper 1 and 17 cell responses and suppresses colitis in mice. Gastroenterology 141. 653-662.
Liu, W., Guo, W., Guo, L, Gu, Y., Cai, P., Xie, N., Yang, X., Shu, Y., Wu, X., Sun, Y., Xu, Q,, 2014. Andrographolide sulfonate ameliorates experimental colitis in mice by inhibiting Thl/Th17 response. Int. Immunopharmacol. 20, 337-345.
Lopez, A., Peyrin-Biroulet, L., 2013. 5-Aminosalicylic acid and chemoprevention: does it work? Dig. Dis. 31, 248-253.
Maloy, K.J., Powrie, F., 2011. Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature 474, 298-306.
Marquez, L., Garda-Bueno, B., Madrigal, J.L., Leza, J.C., 2012. Mangiferin decreases inflammation and oxidative damage in rat brain after stress. Eur. J. Nutr. 51. 729-739.
Mascaraque, C., Aranda, C., Ocon, B., Monte, M.J., Suarez, M.D., Zarzuelo, A., Marin, J.J., Martlnez-Augustin, O., de Medina, F.S., 2014. Rutin has intestinal antiinflammatory effects in the CD[4.sub.+] CD62[L.sub.+] T cell transfer model of colitis. Pharmacol. Res. 90, 48-57.
Mayne, C.G., Williams, C.B., 2013. Induced and natural regulatory T cells in the development of inflammatory bowel disease. Inflamm. Bowel Dis. 19, 1772-1788.
McKay, D.L, Blumberg, J.B., 2007. A review of the bioactivity of South African herbal teas: rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia). Phytother. Res. 21, 1-16.
McLean, M.H., Dieguez, D. Jr, Miller, L.M., Young. HA., 2015. Does the microbiota play a role in the pathogenesis of autoimmune diseases? Gut 64, 332-341.
Monteleone, I., Sarra, M., Pallonem, F., Monteleone, G., 2012. Th17-related cytokines in inflammatory bowel diseases: friends or foes? Curr. Mol. Med. 12, 592-597.
Okamoto, Y., Tanaka, M., Fukui, T., Masuzawa, T., 2012. Brazilian propolis inhibits the differentiation of Th17 cells by inhibition of interleukin-6-induced phosphorylation of signal transducer and activator of transcription 3. Immunopharmacol. Immunotoxicol. 34, 803-809.
Pinto, M.M.M., Sousa, M.E., Nascimento. M.S.J., 2005. Xanthone derivatives: new insights in biological activities. Curr. Med. Chem. 12, 2517-2538.
Qin, L., Han, T., Zhang, Q., Cao, D., Nian, H., Rahman, K., Zheng, H., 2008. Antiosteoporotic chemical constituents from Er-Xian Decoction, a traditional Chinese herbal formula. J. Ethnopharmacol. 118, 271 -279.
Robinson, M., 1998. Medical therapy of inflammatory bowel disease for the 21st century. Eur. J. Surg. Suppl. 582, 90-98.
Shen, X., Du, J., Guan, W., Zhao, Y., 2014. The balance of intestinal Foxp3+ regulatory T cells and Th17 cells and its biological significance. Expert Rev. Clin. Immunol. 10, 353-362.
Singh, U.P., Singh, N.P., Singh, B., Hofseth, L.J., Taub, D.D., Price, R.L., Nagarkatti, M., Nagarkatti, P.S., 2012. Role of resveratrol-induced CD11b(+) Gr-1(+) myeloid derived suppressor cells (MDSCs) in the reduction of CXCR3(+) T cells and amelioration of chronic colitis in IL-10(-/-) mice. Brain Behav. Immun. 26, 72-82.
Tao, F., Qian, C., Guo, W., Luo, Q., Xu, Q,, Sun, Y., 2013. Inhibition of Th1/Th17 responses via suppression of STAT1 and STAT3 activation contributes to the amelioration of murine experimental colitis by a natural flavonoid glucoside icariin. Biochem. Pharmacol. 85, 798-807.
Ung, V.Y., Foshaug, R.R., MacFarlane, S.M., Churchill, T.A., Doyle, J.S., Sydora, B.C., Fedorak, R.N., 2010. Oral administration of curcumin emulsified in carboxymethyl cellulose has a potent anti-inflammatory effect in the IL-10 gene-deficient mouse model of IBD. Dig. Dis. Sci. 55, 1272-1277.
Walsh, D., McCarthy, J., O'Driscoll, C., Melgar, S., 2013. Pattern recognition receptors-molecular orchestrators of inflammation in inflammatory bowel disease. Cytokine Growth Factor Rev 24, 91-104.
Wang, M.H., Achkar, J.P., 2015. Gene-environment interactions in inflammatory bowel disease pathogenesis. Curr. Opin. Gastroenterol. 31, 277-282.
Wang, Y., Dan, Y., Yang, D., Hu, Y., Zhang, L., Zhang, C., Zhu, H., Cui, Z., Li, M., Liu, Y., 2014, The genus Anemarrhena Bunge: A review on ethnopharmacology, phytochemistry and pharmacology. J. Ethnopharmacol, 153, 42-60.
Xu, M., Zhang, M., Wang, D., Yang, C.R., Zhang, Y.J., 2011. Phenolic compounds from the whole plants of Gentiana rhodantha (Gentianaceae). Chem. Biodivers. 8, 1891-1900.
Yang, J., Sundrud, M.S., Skepner, J., Yamagata, T., 2014. Targeting Th17 cells in autoimmune 1diseases. Trends Pharmacol. Sci. 35, 493-500.
Zhou, C., Zhou, J., Han, N., Liu, Z., Xiao, B., Yin, J., 2015. Beneficial effects of neomangiferin on high fat diet-induced nonalcoholic fatty liver disease in rats. Int. Immunopharmacol 25, 218-228.
|Printer friendly Cite/link Email Feedback|
|Author:||Lim, Su-Min; Kang, Geum-Dan; Jeong, Jin-Ju; Choi, Hyun Sik; Kim, Dong-Hyun|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Feb 15, 2016|
|Previous Article:||Inhibition of pulmonary metastasis by Emilia sonchifolia (L.) DC: an in vivo experimental study.|
|Next Article:||Mitraphylline inhibits lipopolysaccharide-mediated activation of primary human neutrophils.|