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doi:10.1111/jgh.12710
GASTROENTEROLOGY
Oridonin’s therapeutic effect: Suppressing Th1/Th17 simultaneously in a mouse model of Crohn’s disease Shubei Wang,* Yong Zhang,† Philippe Saas,‡ Haili Wang,* Ying Xu,* Ke Chen,* Jie Zhong,* Yaozong Yuan,* Ying Wang† and Yunwei Sun* *Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, †Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiaotong University School of Medicine, Shanghai, China; and ‡INSERM, UMR645, Besançon, France
Key words animal models, cytokines, oridonin. Accepted for publication 2 August 2014. Correspondences Professor Yunwei Sun, Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China. Email: [email protected]; Professor Ying Wang, Department of Immunology, Shanghai Institute of Immunology, Institute of Medical Sciences, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China. Email: [email protected]
Abstract Background and Aim: Crohn’s disease is a chronic inflammatory bowel disease. Oridonin is an effective component isolated from Rabdosia rubescens. It can inhibit the activation of transcription factor nuclear factor-kappa B and suppress the over expression of cytokines. We postulated that oridonin may be a potential therapeutic candidate for Crohn’s disease. Methods: To confirm the postulation, we investigated clinical and immunologic modulations of oridonin in a mouse model of trinitrobenzene sulfonic acid-induced colitis. Results: It was found that oridonin attenuated trinitrobenzene sulfonic acid-induced colitis as represented by a reduction in colonic interferon-γ/inteleukin-17 secretion and a decrement in splenic Th1/Th17 cells and effector memory CD4+ T cells. Oridonin treatment inhibited the proliferation of CD4+ T cells and upregulated the apoptosis of lymphocytes by inhibiting nuclear translocation of transcription factor nuclear factor-kappa B. Conclusions: Oridonin is a potential modulator for trinitrobenzene sulfonic acid-induced colitis and other Th1/Th17 mediated inflammatory diseases.
Shubei Wang and Yong Zhang contributed equally to this study. The last two authors contributed equally to the work. Declaration of conflicts of interest: The authors declare no conflict of interest.
Introduction Inflammatory bowel disease (IBD) is a functional disorder characterized by a loss of structural integrity of the gastrointestinal track and colonic mucosa, affecting 1–2% of the western population.1 Crohn’s disease (CD) and ulcerative colitis (UC) are two typical clinical types of IBD. Recent studies2,3 have shown that IBD has been appreciated to have a genetic basis and involves abnormal immune responses to multiple host and environmental factors. Systemic immune responses are considered to be the main factors for IBD development.3 Although CD and UC share many clinical and pathological characteristics, they have considerably different immunopathologies. CD is characterized by concomitant hyper-T helper cell (Th) and Th17 responses with increased secretion of interleukin (IL)-2, IL-17 and interferon (IFN)-γ, whereas UC displays an atypical Th2-mediated response.4–7 504
Nuclear factor kappa B (NF-κB) strongly influences the mucosal inflammation in IBD patients,8 accompanied by the enhanced recruitment of inflammatory cells and production of pro-inflammatory cytokines.9 Corticosteroids, sulfasalazines, antitumor necrosis factor (TNF)-α antibodies and other immunosuppressive drugs in IBD treatment are known to mediate their antiinflammatory effects, at least in part, via inhibition of NF-κB activity.10 NF-κB decoy oligodeoxynucleotides attenuate the severity of colitis in CD animal models.11 Oridonin, a tetracycline diterpenoid compound, is isolated from traditional Chinese herb medicine Rabdosia rubescens to treat tumors and inflammatory diseases. Oridonin can suppress the proliferation of lymphocytes, impair the secretion of Th1 cytokine,12,13 indicating that it has immunosuppressive properties. Further studies indicate that NF-κB-signaling activation is involved in multiple modulation effects induced by oridonin.14 In this study, we have investigated the effects of oridonin in a
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2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis mouse model and delineate underlying molecular mechanisms.
Methods Materials. Oridonin, dexamethasone and TNBS were purchased from Sigma-Aldrich (St Louis, MO, USA). Magnetic beads were purchased from Miltenyi Biotec (Bergisch Gladbach, RBK, Germany). Dynabeads CD3/CD28 T-cell activator and carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation kit was purchased from Invitrogen (Carlsbad, CA, USA). The fluorescent antibodies were obtained from eBioscience (San Diego, CA, USA) or BD Biosciences (San Jose, CA, USA). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) apoptosis assay kit was obtained from Roche (Lewes, UK). Antibodies against β-actin, histone 3, and NF-κB p65 were from Cell Signaling Technology (Beverly, MA, USA). Induction and evaluation of colitis. Male BALB/c mice aged 6–8 weeks were purchased from Shanghai Laboratory Animal Center (Shanghai, China). Food was withdrawn for 24 h before administration of TNBS. Mice were anesthetized, and a 3.5-F catheter was inserted intrarectally (i.r.) to 4 cm proximal to the anus. TNBS of 2.5 mg in 50% ethanol was injected slowly into the lumen.15 Oridonin was dissolved in dimethylsulfoxide (DMSO) at a concentration of 20 mg/mL. Then, the solution was further diluted with physiological saline to yield final concentrations (DMSO diluted in physiological saline, 1% v/v). The mice were treated intraperitoneally (i.p.) daily with the vehicle, oridonin, or dexamethasone. Weight was recorded daily in the experimental animals until they were sacrificed on day 7. After the mice were sacrificed, specimens from the colon were fixed in 4% paraformaldehyde. Hematoxylin–eosin staining was performed. The histology activity index (HAI) was assessed:16 the extent of destruction to the mucosal architecture (0, normal; 1, mild; 2, moderate; and 3, extensive damage), cellular infiltration (0, normal; 1, mild; 2, moderate; and 3, transmural infiltration), mice muscle thickening (0, normal; 1, mild; 2, moderate; and 3, marked thickening), crypt abscess (0, absent; 1, present), and goblet cell depletion (0, absent; 1, present). The scores for each feature were summed up. Detection of IL-2, IFN-γ and IL-17 by flow cytometry. Splenocytes were isolated by Ficoll-hypaque gradient centrifugation. Single cell suspensions were subjected to IL-2, IFN-γ, and IL-17A detection by intracellular cytokine analysis. Cells were acquired by using a BD FACScalibur flow cytometer (BD Biosciences), and data analysis was performed by using FlowJo software (Tree Star, San Carlos, CA, USA). Immunohistochemistry staining. Paraffin slides of the colon were rehydrated and washed in phosphate-buffered saline (PBS). Slides were microwaved in sodium citrate buffer and then incubated with 10% goat serum. Sections were incubated overnight (4°C) with antibodies. Slides were washed again, incubated
with goat antirabbit immunoglobulin G (IgG) conjugated to peroxidase (POD)-labeled polymer, developed using a diaminobenzidine (DAB) reaction, and counterstained with hematoxylin. In each group, the positive cells were counted. In vitro proliferative suppression assay. Mesenteric lymph nodes (MLNs) were removed and single-cell suspensions were prepared. Cells were washed with PBS, resuspended in PBS containing 5-μM CFSE, incubated for 10 min and washed with RPMI 1640 (Invitrogen, Carlsbad, CA, USA). CFSE-labeled cells were cultured for 48 h in the absence or presence of dynabeads CD3/CD28 T-cells activator. Oridonin (0.5 μg/mL and 1.0 μg/mL) was included in the culture, and then cells were collected. TUNEL assay. After cytospin preparation of suspension cells, TUNEL assay was performed. Terminal deoxynucleotidyl transferase with fluorescein was used to label the apoptotic cells. Fluorescein was detected by antifluorescein antibody conjugated with POD and then developed using a DAB reaction. Slides were counterstained with hematoxylin and analyzed by microscopy. Western blot. Equal amounts of proteins (20 μg) were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then electrotransferred to nitrocellulose filter membranes. Membranes were blocked with 5% bovine serum albumin, and then incubated with antibodies against β-actin (1:1000), histone 3 (1:1000) and p65 (1:1000). After washes with tris buffered saline with tween-20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated goat antirabbit IgG at room temperature for 1 h. Protein bands were visualized by enhanced chemiluminescence reagent. The intensities of bands were quantified by BioRad-Image (BioRad, Hercules, CA, USA). Statistical analysis. Data were represented as mean ± standard error of mean. All statistical analyses were performed using GraphPad prism software (GraphPad Software, San Diego, CA, USA). The log–rank test was used to assess the significance of differences in mortality rates among the various groups. For histological score, Mann–Whitney test was employed. Other statistical differences were assessed with Student’s t-test. P values < 0.05 were considered statistically significant.
Results Oridonin halts the development of TNBS-induced colitis. The effect of oridonin on the development and severity of TNBS colitis was evaluated. Oridonin was administered i.p. daily, starting from the second day of TNBS instillation. Dexamethasone, which is known as the most important drug in CD therapy, was used as a positive control. Mice responded to TNBS treatment with a significant mortality, which reached 65% at day 7 (P < 0.05). Administration of 50-μg oridonin improves the survival rate to 60% (P < 0.05 compared with TNBS mice). But treatment of 100-μg oridonin did not significantly improve the survival rate (40.6%, P > 0.05) (Fig. 1a). In addition, mice with TNBS-induced colitis exhibited weight loss
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Figure 1 Oridonin or dexamethasone treatment prevented 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis from progressing. BALB/c mice were treated i.p. with oridonin or dexamethasone 24 h after TNBS injection. This was repeated daily until day 7. (a and b) Survival and body weight changes in BALB/c mice with TNBS-induced colitis either untreated (TNBS) or treated with 50-μg/mouse/day oridonin (50 μg),100-μg/mouse/day orindonin (100 μg), and 2.5-μg/mouse/day dexamethasone (DX) (as positive control). Oridonin and dexamethasone improved the survival rate and recovered lost body weight of TNBS colitis mice. (c) Macroscopic images of colons from oridonin- and dexamethasone-treated mice with TNBS-induced colitis. (d) Histologic findings with hematoxylin–eosin stained sections of colons, made at an original magnification of × 10. Colonic inflammation in TNBS colitis was improved in mice treated with oridonin or dexamethasone. (e) Histopathologic analysis of the colonic inflammation: the histology activity index (HAI) values were computed as described before. Oridonin and dexamethasone treatment decreased HAI, protected the colons against TNBS-induced , Control; , TNBS; , 50-μg Ori; , 100-μg Ori; , DX. inflammation. There were 12–35 mice/group. *P < 0.05 versus TNBS-treated mice. **P < 0.01.
▶ Figure 2 Oridonin and dexamethasone treatment decreased systemic immune responses in mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS)induced colitis. Mice were treated i.p. with 50-μg/mouse/day oridonin (50 μg), 100-μg/mouse/day oridonin (100 μg) or dexamethasone (as positive control) 24 h after TNBS administration. At day 7, splenic mononuclear cells were isolated for flow cytometry (FCM) assays, as detailed in Methods. (a) Dot blots for the effector memory T cells (CD44hiCD62Llow) in CD4+ T cells. Cells were stained with CD4, CD44, CD62L mAb and analyzed by FCM. Oridonin and dexamethasone inhibited the over expression of effector memory T cells in TNBS mice. (b) The percentage of CD25+Foxp3+ Treg cells in CD4+ T cells. (c) Production of interferon (IFN)-γ, interleukin (IL)-2, and IL-17 in CD4+ T cells. (d) The results are expressed as the percentage of effector memory T cells, Treg cells, or IFN-γ+, IL-2+, IL-17+ cells in CD4+ T cells. Cytokine production was decreased in TNBS mice after oridonin and , Control; ■, TNBS; , 50 μg; , 100 μg; , dexamethasone treatment. ns P > 0.05; *P < 0.05 versus control mice; **P < 0.01; ***P < 0.001. DX.
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when compared with control mice, as less as 23.6%. Oridonin (either 50 μg/day or 100 μg/day) treatment rapidly recovered body weight loss efficiently (96.9% and 100.4% of the initial body weight, P < 0.05 compared with TNBS mice), which was better than dexamethasone treatment (Fig. 1b). This may be due to the high metabolism rate of dexamethasone in vivo.17 Oridonin treatment also ameliorated hyperemia and inflammation (Fig. 1c), reduced infiltration of lymphocytes and inflammatory activity (Fig. 1d). Compared with that from colitis mice, HAI value in the colon from 50 μg and 100-μg oridonin-treated mice was also decreased (6.500 ± 0.327 vs 3.600 ± 1.288 and 4.429 ± 0.896, respectively, P < 0.05) (Fig. 1e). Oridonin alters peripheral CD4+ T-cell subsets and functionality in TNBS-induced colitis mice. As is well known, the spleen is the principal peripheral immune organ. To evaluate the variety of CD4+ T cells in the periphery, the expression of splenic CD4+ T-cell subsets was further determined. The frequency of naïve (CD44lowCD62Lhi) and effector/memory (CD44hiCD62Llow) T cells in the spleen were compared. The percentage of effector/memory T cells increased significantly after TNBS treatment (18.4 ± 1.9% vs 7.7 ± 1.0%, P < 0.05). Both doses of oridonin were able to downregulate the percentage of effector/memory CD4+ T cells (50-μg oridonin: 11.3 ± 0.9%; 100-μg oridonin: 9.4 ± 2.3%; TNBS: 18.4 ± 1.9%, respectively, P < 0.05) (Fig. 2a,d). In our study, the percentage of CD4+CD25+Foxp3+ Treg in the spleen of colitis mice was decreased. However, oridonin treatment only slightly increased the frequency of Treg cells in 50-μg oridonin-treated group (P > 0.05) (Fig. 2b,d). Th17 cells were also detected to evaluate the immunological effect of oridonin. In TNBS mice, IL-17-producing CD4+ T cells dramatically elevated (CD4+IL-17+ T cells: 2.002 ± 0.277% vs 0.663 ± 0.034%, P < 0.05). Oridonin reduced the expansion of Th17 in TNBStreated mice (50-μg oridonin: 1.106 ± 0.172%; 100-μg oridonin: 1.191 ± 0.242%; TNBS: 2.002 ± 0.277%, respectively, P < 0.05) at a comparable level in colitis mice with dexamethasone treatment (Fig. 2c,d). These data suggest that oridonin and dexamethasone could directly decrease the level of IL-17 in splenic CD4+ T cells of mice with TNBS-induced colitis. To determine the effect of oridonin on peripheral Th1 subset of TNBS mice, IFN-γ- and IL-2-secreting cells were also determined. Our results indicated that IL-2-, IFN-γ-producing CD4+ T cells dramatically elevated (CD4+IL-2+ T cells: 51.1 ± 2.8% vs 42.4 ± 2.3%; CD4+IFN-γ+ T cells: 13.1 ± 1.2% vs 8.2 ± 1.0%, P < 0.05) in TNBS mice. Treatment with both doses of oridonin strongly suppressed IFN-γ expression (50-μg oridonin: 5.156 ± 0.717%; 100-μg oridonin: 6.564 ± 1.083%; TNBS: 13.1 ± 1.2%, respectively, P < 0.05) (Fig. 2c,d), which was similar to dexamethasone treatment. However, only 100-μg oridonin could significantly inhibit the over expression of IL-2 in CD4+ T cells (51.1 ± 2.8% vs 56.6 ± 4.1%, P < 0.05), whereas IL-2 was not significantly reduced in 50-μg oridonin-treated mice (P > 0.05). Dexamethasone did not affect the expression of IL-2 (Fig. 2c,d). Like dexamethasone, oridonin also had a therapeutic effect in improving TNBS-induced colitis by restraining exaggerated Th1 and Th17 responses and over-homeostasis of effector/memory cells. 508
Oridonin selectively modulates cytokine and Foxp3 expression in the colon of colitis mice. As shown in Figure 3, IFN-γ and IL-17 level in the colon increased markedly (8.4- and 11.8-fold, respectively, P < 0.05) in colitis mice. However, local IL-2 level remained unchanged significantly (P > 0.05). Oridonin treatment significantly inhibited IL-17 expression in the colon of mice with TNBS-induced colitis (50-μg oridonin: 1.168 ± 0.209%; 100-μg oridonin: 1.294 ± 0.322%; TNBS: 4.729 ± 0.707%, P < 0.05). Similar effects were observed for IFN-γ production (50-μg oridonin: 1.730 ± 0.318%; 100-μg oridonin: 1.778 ± 0.369%; TNBS: 6.400 ± 0.979%, P < 0.05). IL-2 expression remained unaltered in treated groups (P > 0.05) (Fig. 3a,b). Unexpectedly, there was 30-fold more Foxp3 expression in colonic tissues of TNBS-treated mice (P < 0.05). Treg proportions were also decreased in oridonin-treated colitis mice (50-μg oridonin: 0.227 ± 0.075%; 100-μg oridonin: 0.430 ± 0.112%; TNBS: 1.590 ± 0.394%, respectively, P < 0.05). Thus, oridonin exhibited similar effects to dexamethasone to ameliorate TNBS-induced colitis with a reduction of IL-17 and IFN-γ production in colon tissues. Oridonin impairs the proliferation and cytokine secretion of T cells in vitro. T-cell proliferation was measured by CFSE. Oridonin inhibited CD4+ T cell proliferation in a dose-dependent fashion (Fig. 4a). And 23.3% of CD4+ T cells divided three times and 41.7% of cells divided two times after stimulation. In the presence of 0.5-μg/mL oridonin, the percentage of T cells dividing three times decreased to 16.7%. The inhibitory effect was more pronounced after 1.0-μg/mL oridonin treatment. Only 5.07% of T cells subdivided three times and 37.9% cells subdivided two times. The apoptotic effect upon T-cell receptor stimulation was determined by TUNEL assay. There was no detectable apoptotic cell in unstimulated and stimulated MLN cells in the absence of oridonin. The percentage of apoptotic cells significantly increased to 48.4% after 0.5-μg/mL oridonin treatment, and 63.6% in the presence of 1.0-μg/mL oridonin (Fig. 4b), suggesting that CD4+ T cells from colitic pathology were more susceptible to apoptosis upon outside activation signaling. As was the case with in vivo experiment, the presence of oridonin did not change the percentage of Tregs (Fig. 4c). IL-17 and IFN-γ secretion of CD4+ T cells was blocked dramatically in the presence of both 0.5 and 1.0 μg/mL oridonin (CD4+ IFN-γ+ T cells: 9.868 ± 2.528% vs 7.372 ± 2.134% and 5.157 ± 1.760%; CD4+ IL-17+ T cells: 2.462 ± 0.671% vs 2.027 ± 0.464% and 1.260 ± 0.226%, P < 0.05). IL-2 expression only moderately decreased (1.14-fold, P > 0.05) in the presence of 1.0-μg/mL oridonin (Fig. 4c). NF-κB nuclear translocation is impeded in response to oridonin treatment. NF-κB is critical in inflammatory response and pathogenesis of IBD.18,19 As oridonin had been demonstrated to inhibit NF-κB activation in many other cells, we further verified the inhibitory mechanisms of oridinon on NF-κB-signaling pathways. Oridonin treatment for 48 h did not significantly inhibit NF-κB phosphorylation (Fig. 4e). However, nucleic translocation of p65 was inhibited in a dose-dependent manner (P < 0.05 compared with stimulated cells) (Fig. 4d). Our results demonstrated that oridonin improved the immunological
Journal of Gastroenterology and Hepatology 30 (2015) 504–512 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
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Oridonin modulates CD4+ cells in colitis
Figure 3 Effects of oridonin and dexamethasone treatment on 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis in the expression of Foxp3 and cytokines in colonic tissues. Mice were treated with 50% ethanol (Control), TNBS or with 50-μg/mouse/day oridonin (50 μg), 100-μg/mouse/day orindonin (100 μg), and 2.5-μg/mouse/day dexamethasone (DX) 24 h after TNBS treatment. (a) Colons from mice were subjected to immunohistochemistry staining for Foxp3, interferon (IFN)-γ, interleukin (IL)-2, and IL-17. (b) The results are expressed as the percentage of Foxp3, IFN-γ, IL-2, and IL-17-positive cells in colonic cells. Oridonin decreased the expression of Foxp3, IFN-γ, and IL-17 in colons of TNBS colitis mice, whereas dexamethasone downregulated the expression of IFN-γ and IL-17. IL-2 was not significantly changed after oridonin and dexamethasone treatment. , Control; ■, TNBS; , 50-μg Ori; , 100-μg Ori; , DX. ns P > 0.05; *P < 0.05 versus control mice; **P < 0.01; ***P < 0.001.
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Figure 4 Effects of oridonin on the function of T cells from 2,4,6-trinitrobenzene sulfonic acid (TNBS)immunized mice stimulated with anti-CD3/CD28 beads. The lymphocytes isolated from mesenteric lymph node (MLN) of BALB/c mice with TNBS-induced colitis were cultured for 48 h in growth medium alone (-) or stimulated with anti-CD3/CD28 beads in the absence or presence of different doses of oridonin (0.5 μg/mL, 1.0 μg/ mL). (a) Proliferation of CD4+ T cells were measured by the dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE) after 48-h culture. Oridonin inhibited the proliferation stimulated by anti-CD3/CD28 beads in a dose-dependent manner. (b) Apoptosis of lymphocytes was assessed by transferase deoxytidyl uridine end labeling (TUNEL) assay. Apoptosis was significantly induced after oridonin treatment. (c) The percentage of Treg, Th1, and Th17 cells in CD4+ T cells. Expression of Tregs and IL-2 was not significantly changed, whereas production of interferon (IFN)-γ and interleukin (IL)-17 was effectively affected after oridonin treatment. (d) The expression levels of nuclear and cytosolic p65 protein were determined by Western blot analysis as detailed in Methods. (e) The expression levels of p65 and phosphop65 protein were analyzed by Western blot. Oridonin interfered with the translocation of p65 without altering its phosphorylaion. Results are representative of three independent experiments. ns P > 0.05; *P < 0.05 versus , CD3/CD28; , control mice; **P < 0.01. ■, —; , CD3/CD28 + 1-μg/mL CD3/CD28 + 0.5-μg/mL Ori; Ori.
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pathology and retarded the disease progression of TNBS-induced colitis, partially through interference with NF-κB nuclear translocation.
Discussion The present study has demonstrated that oridonin, an effective component isolated from Rabdosia rubescens, has therapeutic potential on an experimental CD model. This treatment effect of oridonin was accompanied by simultaneous suppression of both local and peripheral Th1/Th17, possibly through mediating and interfering with NF-κB nuclear translocation activity. As the etiology of CD remains unclear, most medical treatments are symptomatic by inhibiting the inflammatory response.20 The role of 5-aminosalicylic acid in inducing remission of active CD and preventing relapse of quiescent CD remains uncertain; it shows only limited value in mild-to-moderate CD.21 When the disease activity of CD is progressing, corticosteroids and immunosuppressive drugs are often added.22 TNF-α blocking agents (infliximab, adalimumab, certolizumab pegol) are effective therapy in CD.23 However, these agents needs to be weighed against the risk of side effects, particularly infection. Research on medications for CD is going on in an attempt to find out the most effective options. In our study, we demonstrated that oridonin had a remarkable treatment effect on the clinical course of CD-like colitis. Oridonin showed similar therapeutic capacity to corticosteroids in improving TNBS-induced colitis. In addition, no severe body weight loss was observed in mice treated with oridonin. Compared with corticosteroids, oridonin seems to be a relatively safe agent. After oridonin treatment, mice with TNBS colitis demonstrated clinical and histological improvement. Oridonin significantly reduced the percentage of both local and peripheral Th1/Th17 cells. On the other hand, oridonin significantly suppressed the over-homeostasis of effector/memory cells participating in the perpetuation of IBD, which were reported to be elevated in CD patients and mice models.24–27 In addition, oridonin acted synergistically on T-cell responses by inhibiting T-cell proliferation, inducing apoptosis of lymphocytes and interfering with T-cell function. Therefore, oridonin could provide a protective effect against the self-reactive immune response in TNBS mice via multiple mechanisms. The detailed mechanism underlying this effect needs to be further investigated. NF-κB-signaling pathway is known to contribute to the production of pro-inflammatory cytokines, which could strongly affect the development of IBD and many other autoimmune responses.10,28 Previous studies14,29 have demonstrated that oridonin could directly interfere with the DNA-binding activity of NF-κB to its response DNA sequences, and inhibit the translocation of NF-κB from cytoplasm to nucleus. It was found in the present study that the immunosuppressive action of oridonin was involved in the NF-κB pathway through its interference with NF-κB translocation. The modification of NF-κB activity might lead to the blockade of cytokine production and increased apoptosis of T cells. Oridonin has been demonstrated to have a broad spectrum of therapeutic actions including anti-inflammatory, antitumor, and anti-immunology activities.12,13,30,31 And several experiment suggest that oridonin is of relatively low in vivo toxicity.32–34 It has
multiple-modulation effects on tumor cells, including inhibiting NF-κB activity and modulating mitogen-activated protein kinase pathways.34 As the pathogenesis of CD involves a complicated network of multiple factors, the use of agents from traditional Chinese medicines such as oridonin should be highlighted, because they can exert immunomodulatory activities through targeting multiple cell subsets and signaling pathways critically related to inflammation. In summary, our data indicate that oridonin has therapeutic effects on TNBS-induced colitis by disturbing pathogenic Th1/ Th17 cells via inhibition of NF-κB signal pathways. The superior efficacy of oridonin in TNBS mice as compared with corticosteroids suggests that oridonin may prove to be a potential candidate for the treatment of colonic inflammation in CD and other autoimmune diseases. However, oridonin is only administered from the initiation of colitis in the present study. The effects of oridonin on established disease will be explored in the near future.
Acknowledgments This research is supported by a grant from Shanghai municipal commission of health and family planning (No. 2013125).
References 1 Satsangi J, Parkes M, Louis E et al. Two stage genome–wide search in inflammatory bowel disease provides evidence for susceptibility loci on chromosomes 3, 7 and 12. Nat. Genet. 1996; 14: 199–202. 2 Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat. Rev. Immunol. 2003; 3: 521–33. 3 Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel disease. Annu. Rev. Immunol. 2010; 28: 573–621. 4 Brand S. Crohn’s disease: Th1, Th17 or both? The change of a paradigm: new immunological and genetic insights implicate Th17 cells in the pathogenesis of Crohn’s disease. Gut 2009; 58: 1152–67. 5 Sartor RB. Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis. Nat. Clin. Pract. Gastroenterol. Hepatol. 2006; 3: 390–407. 6 Karczewski J, Mazur M, Karczewski M. Dual role of Th17 cells in Crohn’s disease. Centr. Eur. J. Immunol. 2012; 37: 286–9. 7 Fuss IJ, Neurath M, Boirivant M et al. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn’s disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J. Immunol. 1996; 157: 1261–70. 8 Billerey-Larmonier C, Uno JK, Larmonier N et al. Protective effects of dietary curcumin in mouse model of chemically induced colitis are strain dependent. Inflamm. Bowel Dis. 2008; 14: 780–93. 9 Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat. Rev. Immunol. 2002; 2: 725–34. 10 Atreya I, Atreya R, Neurath M. NF-κB in inflammatory bowel disease. J. Intern. Med. 2008; 263: 591–6. 11 Fichtner-Feigl S, Fuss IJ, Preiss JC, Strober W, Kitani A. Treatment of murine Th1-and Th2-mediated inflammatory bowel disease with NF-kappaB decoy oligonucleotides. J. Clin. Invest. 2005; 115: 3057–71. 12 Liu J, Yang F, Zhang Y, Li J. Studies on the cell-immunosuppressive mechanism of Oridonin from Isodon serra. Int. Immunopharmacol. 2007; 7: 945–54.
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13 Hu AP, Du JM, Li JY, Liu JW. Oridonin promotes CD4+/CD25+ Treg differentiation, modulates Th1/Th2 balance and induces HO-1 in rat splenic lymphocytes. Inflamm. Res. 2008; 57: 163–70. 14 Leung CH, Grill SP, Lam W, Han QB, Sun HD, Cheng YC. Novel mechanism of inhibition of nuclear factor-κB DNA-binding activity by diterpenoids isolated from Isodon rubescens. Mol. Pharmacol. 2005; 68: 286–97. 15 Bai A, Lu N, Guo Y, Liu Z, Chen J, Peng Z. All-trans retinoic acid down-regulates inflammatory responses by shifting the Treg/Th17 profile in human ulcerative and murine colitis. J. Leukoc. Biol. 2009; 86: 959–69. 16 Inokuchi Y, Morohashi T, Kawana I, Nagashima Y, Kihara M, Umemura S. Amelioration of 2, 4, 6-trinitrobenzene sulphonic acid induced colitis in angiotensinogen gene knockout mice. Gut 2005; 54: 349–56. 17 Shu HJ, Takeda H, Shinzawa H, Takahashi T, Kawata S. Effect of lipopolysaccharide on peptide transporter 1 expression in rat small intestine and its attenuation by dexamethasone. Digestion 2002; 65: 21–9. 18 Schreiber S, Nikolaus S, Hampe J. Activation of nuclear factor κB in inflammatory bowel disease. Gut 1998; 42: 477–84. 19 Tak PP, Firestein GS. NF-kappaB: a key role in inflammatory diseases. J. Clin. Invest. 2001; 107: 7–12. 20 Van Montfrans C, Camoglio L, Van Deventer S. Immunotherapy of Crohn’s disease. Mediators Inflamm. 1998; 7: 149–52. 21 Ford AC, Kane SV, Khan KJ et al. Efficacy of 5-aminosalicylates in Crohn’s disease: systematic review and meta-analysis. Am. J. Gastroenterol. 2011; 106: 617–29. 22 Podolsky DK. Inflammatory bowel disease. N. Engl. J. Med. 1991; 325: 928–37. 23 Behm BW, Bickston SJ. Tumor necrosis factor-alpha antibody for maintenance of remission in Crohn’s disease. Cochrane Database Syst. Rev. 2008; (1): CD006893. 24 Nemoto Y, Kanai T, Kameyama K et al. Long-lived colitogenic CD4+ memory T cells residing outside the intestine participate in the perpetuation of chronic colitis. J. Immunol. 2009; 183: 5059–68.
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25 ten Hove T, Berkhout M, Bruggeman JP et al. Expression of CD45RB functionally distinguishes intestinal T lymphocytes in inflammatory bowel disease. J. Leukoc. Biol. 2004; 75: 1010–5. 26 Roman L, Manzano L, De La Hera A, Abreu L, Rossi I, Alvarez-Mon M. Expanded CD4+ CD45RO+ phenotype and defective proliferative response in T lymphocytes from patients with Crohn’s disease. Gastroenterology 1996; 110: 1008–19. 27 Pasternak BA, D’Mello S, Jurickova II et al. Lipopolysaccharide exposure is linked to activation of the acute phase response and growth failure in pediatric Crohn’s disease and murine colitis. Inflamm. Bowel Dis. 2010; 16: 856–69. 28 Hilliard B, Samoilova EB, Liu TST, Rostami A, Chen Y. Experimental autoimmune encephalomyelitis in NF-κB-deficient mice: roles of NF-κB in the activation and differentiation of autoreactive T cells. J. Immunol. 1999; 163: 2937–43. 29 Hortelano S. Molecular basis of the anti-inflammatory effects of terpenoids. Inflamm. Allergy Drug Targets 2009; 8: 28–39. 30 Zhou GB, Chen SJ, Wang ZY, Chen Z. Back to the future of oridonin: again, compound from medicinal herb shows potent antileukemia efficacies in vitro and in vivo. Cell Res. 2007; 17: 274–6. 31 Xu Y, Xue Y, Wang Y, Feng D, Lin S, Xu L. Multiple-modulation effects of Oridonin on the production of proinflammatory cytokines and neurotrophic factors in LPS-activated microglia. Int. Immunopharmacol. 2009; 9: 360–5. 32 Chen S, Gao J, Halicka HD, Huang X, Traganos F, Darzynkiewicz Z. The cytostatic and cytotoxic effects of oridonin (Rubescenin), a diterpenoid from Rabdosia rubescens, on tumor cells of different lineage. Int. J. Oncol. 2005; 26: 579–88. 33 Zhou G-B, Kang H, Wang L et al. Oridonin, a diterpenoid extracted from medicinal herbs, targets AML1-ETO fusion protein and shows potent antitumor activity with low adverse effects on t (8; 21) leukemia in vitro and in vivo. Blood 2007; 109: 3441–50. 34 Zhen T, Wu C-F, Liu P et al. Targeting of AML1-ETO in t (8; 21) leukemia by oridonin generates a tumor suppressor-like protein. Sci. Transl. Med. 2012; 4: 127ra38–ra38.
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doi:10.1111/jgh.12710
GASTROENTEROLOGY
Oridonin’s therapeutic effect: Suppressing Th1/Th17 simultaneously in a mouse model of Crohn’s disease Shubei Wang,* Yong Zhang,† Philippe Saas,‡ Haili Wang,* Ying Xu,* Ke Chen,* Jie Zhong,* Yaozong Yuan,* Ying Wang† and Yunwei Sun* *Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, †Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiaotong University School of Medicine, Shanghai, China; and ‡INSERM, UMR645, Besançon, France
Key words animal models, cytokines, oridonin. Accepted for publication 2 August 2014. Correspondences Professor Yunwei Sun, Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China. Email: [email protected]; Professor Ying Wang, Department of Immunology, Shanghai Institute of Immunology, Institute of Medical Sciences, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China. Email: [email protected]
Abstract Background and Aim: Crohn’s disease is a chronic inflammatory bowel disease. Oridonin is an effective component isolated from Rabdosia rubescens. It can inhibit the activation of transcription factor nuclear factor-kappa B and suppress the over expression of cytokines. We postulated that oridonin may be a potential therapeutic candidate for Crohn’s disease. Methods: To confirm the postulation, we investigated clinical and immunologic modulations of oridonin in a mouse model of trinitrobenzene sulfonic acid-induced colitis. Results: It was found that oridonin attenuated trinitrobenzene sulfonic acid-induced colitis as represented by a reduction in colonic interferon-γ/inteleukin-17 secretion and a decrement in splenic Th1/Th17 cells and effector memory CD4+ T cells. Oridonin treatment inhibited the proliferation of CD4+ T cells and upregulated the apoptosis of lymphocytes by inhibiting nuclear translocation of transcription factor nuclear factor-kappa B. Conclusions: Oridonin is a potential modulator for trinitrobenzene sulfonic acid-induced colitis and other Th1/Th17 mediated inflammatory diseases.
Shubei Wang and Yong Zhang contributed equally to this study. The last two authors contributed equally to the work. Declaration of conflicts of interest: The authors declare no conflict of interest.
Introduction Inflammatory bowel disease (IBD) is a functional disorder characterized by a loss of structural integrity of the gastrointestinal track and colonic mucosa, affecting 1–2% of the western population.1 Crohn’s disease (CD) and ulcerative colitis (UC) are two typical clinical types of IBD. Recent studies2,3 have shown that IBD has been appreciated to have a genetic basis and involves abnormal immune responses to multiple host and environmental factors. Systemic immune responses are considered to be the main factors for IBD development.3 Although CD and UC share many clinical and pathological characteristics, they have considerably different immunopathologies. CD is characterized by concomitant hyper-T helper cell (Th) and Th17 responses with increased secretion of interleukin (IL)-2, IL-17 and interferon (IFN)-γ, whereas UC displays an atypical Th2-mediated response.4–7 504
Nuclear factor kappa B (NF-κB) strongly influences the mucosal inflammation in IBD patients,8 accompanied by the enhanced recruitment of inflammatory cells and production of pro-inflammatory cytokines.9 Corticosteroids, sulfasalazines, antitumor necrosis factor (TNF)-α antibodies and other immunosuppressive drugs in IBD treatment are known to mediate their antiinflammatory effects, at least in part, via inhibition of NF-κB activity.10 NF-κB decoy oligodeoxynucleotides attenuate the severity of colitis in CD animal models.11 Oridonin, a tetracycline diterpenoid compound, is isolated from traditional Chinese herb medicine Rabdosia rubescens to treat tumors and inflammatory diseases. Oridonin can suppress the proliferation of lymphocytes, impair the secretion of Th1 cytokine,12,13 indicating that it has immunosuppressive properties. Further studies indicate that NF-κB-signaling activation is involved in multiple modulation effects induced by oridonin.14 In this study, we have investigated the effects of oridonin in a
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2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis mouse model and delineate underlying molecular mechanisms.
Methods Materials. Oridonin, dexamethasone and TNBS were purchased from Sigma-Aldrich (St Louis, MO, USA). Magnetic beads were purchased from Miltenyi Biotec (Bergisch Gladbach, RBK, Germany). Dynabeads CD3/CD28 T-cell activator and carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation kit was purchased from Invitrogen (Carlsbad, CA, USA). The fluorescent antibodies were obtained from eBioscience (San Diego, CA, USA) or BD Biosciences (San Jose, CA, USA). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) apoptosis assay kit was obtained from Roche (Lewes, UK). Antibodies against β-actin, histone 3, and NF-κB p65 were from Cell Signaling Technology (Beverly, MA, USA). Induction and evaluation of colitis. Male BALB/c mice aged 6–8 weeks were purchased from Shanghai Laboratory Animal Center (Shanghai, China). Food was withdrawn for 24 h before administration of TNBS. Mice were anesthetized, and a 3.5-F catheter was inserted intrarectally (i.r.) to 4 cm proximal to the anus. TNBS of 2.5 mg in 50% ethanol was injected slowly into the lumen.15 Oridonin was dissolved in dimethylsulfoxide (DMSO) at a concentration of 20 mg/mL. Then, the solution was further diluted with physiological saline to yield final concentrations (DMSO diluted in physiological saline, 1% v/v). The mice were treated intraperitoneally (i.p.) daily with the vehicle, oridonin, or dexamethasone. Weight was recorded daily in the experimental animals until they were sacrificed on day 7. After the mice were sacrificed, specimens from the colon were fixed in 4% paraformaldehyde. Hematoxylin–eosin staining was performed. The histology activity index (HAI) was assessed:16 the extent of destruction to the mucosal architecture (0, normal; 1, mild; 2, moderate; and 3, extensive damage), cellular infiltration (0, normal; 1, mild; 2, moderate; and 3, transmural infiltration), mice muscle thickening (0, normal; 1, mild; 2, moderate; and 3, marked thickening), crypt abscess (0, absent; 1, present), and goblet cell depletion (0, absent; 1, present). The scores for each feature were summed up. Detection of IL-2, IFN-γ and IL-17 by flow cytometry. Splenocytes were isolated by Ficoll-hypaque gradient centrifugation. Single cell suspensions were subjected to IL-2, IFN-γ, and IL-17A detection by intracellular cytokine analysis. Cells were acquired by using a BD FACScalibur flow cytometer (BD Biosciences), and data analysis was performed by using FlowJo software (Tree Star, San Carlos, CA, USA). Immunohistochemistry staining. Paraffin slides of the colon were rehydrated and washed in phosphate-buffered saline (PBS). Slides were microwaved in sodium citrate buffer and then incubated with 10% goat serum. Sections were incubated overnight (4°C) with antibodies. Slides were washed again, incubated
with goat antirabbit immunoglobulin G (IgG) conjugated to peroxidase (POD)-labeled polymer, developed using a diaminobenzidine (DAB) reaction, and counterstained with hematoxylin. In each group, the positive cells were counted. In vitro proliferative suppression assay. Mesenteric lymph nodes (MLNs) were removed and single-cell suspensions were prepared. Cells were washed with PBS, resuspended in PBS containing 5-μM CFSE, incubated for 10 min and washed with RPMI 1640 (Invitrogen, Carlsbad, CA, USA). CFSE-labeled cells were cultured for 48 h in the absence or presence of dynabeads CD3/CD28 T-cells activator. Oridonin (0.5 μg/mL and 1.0 μg/mL) was included in the culture, and then cells were collected. TUNEL assay. After cytospin preparation of suspension cells, TUNEL assay was performed. Terminal deoxynucleotidyl transferase with fluorescein was used to label the apoptotic cells. Fluorescein was detected by antifluorescein antibody conjugated with POD and then developed using a DAB reaction. Slides were counterstained with hematoxylin and analyzed by microscopy. Western blot. Equal amounts of proteins (20 μg) were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then electrotransferred to nitrocellulose filter membranes. Membranes were blocked with 5% bovine serum albumin, and then incubated with antibodies against β-actin (1:1000), histone 3 (1:1000) and p65 (1:1000). After washes with tris buffered saline with tween-20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated goat antirabbit IgG at room temperature for 1 h. Protein bands were visualized by enhanced chemiluminescence reagent. The intensities of bands were quantified by BioRad-Image (BioRad, Hercules, CA, USA). Statistical analysis. Data were represented as mean ± standard error of mean. All statistical analyses were performed using GraphPad prism software (GraphPad Software, San Diego, CA, USA). The log–rank test was used to assess the significance of differences in mortality rates among the various groups. For histological score, Mann–Whitney test was employed. Other statistical differences were assessed with Student’s t-test. P values < 0.05 were considered statistically significant.
Results Oridonin halts the development of TNBS-induced colitis. The effect of oridonin on the development and severity of TNBS colitis was evaluated. Oridonin was administered i.p. daily, starting from the second day of TNBS instillation. Dexamethasone, which is known as the most important drug in CD therapy, was used as a positive control. Mice responded to TNBS treatment with a significant mortality, which reached 65% at day 7 (P < 0.05). Administration of 50-μg oridonin improves the survival rate to 60% (P < 0.05 compared with TNBS mice). But treatment of 100-μg oridonin did not significantly improve the survival rate (40.6%, P > 0.05) (Fig. 1a). In addition, mice with TNBS-induced colitis exhibited weight loss
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Figure 1 Oridonin or dexamethasone treatment prevented 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis from progressing. BALB/c mice were treated i.p. with oridonin or dexamethasone 24 h after TNBS injection. This was repeated daily until day 7. (a and b) Survival and body weight changes in BALB/c mice with TNBS-induced colitis either untreated (TNBS) or treated with 50-μg/mouse/day oridonin (50 μg),100-μg/mouse/day orindonin (100 μg), and 2.5-μg/mouse/day dexamethasone (DX) (as positive control). Oridonin and dexamethasone improved the survival rate and recovered lost body weight of TNBS colitis mice. (c) Macroscopic images of colons from oridonin- and dexamethasone-treated mice with TNBS-induced colitis. (d) Histologic findings with hematoxylin–eosin stained sections of colons, made at an original magnification of × 10. Colonic inflammation in TNBS colitis was improved in mice treated with oridonin or dexamethasone. (e) Histopathologic analysis of the colonic inflammation: the histology activity index (HAI) values were computed as described before. Oridonin and dexamethasone treatment decreased HAI, protected the colons against TNBS-induced , Control; , TNBS; , 50-μg Ori; , 100-μg Ori; , DX. inflammation. There were 12–35 mice/group. *P < 0.05 versus TNBS-treated mice. **P < 0.01.
▶ Figure 2 Oridonin and dexamethasone treatment decreased systemic immune responses in mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS)induced colitis. Mice were treated i.p. with 50-μg/mouse/day oridonin (50 μg), 100-μg/mouse/day oridonin (100 μg) or dexamethasone (as positive control) 24 h after TNBS administration. At day 7, splenic mononuclear cells were isolated for flow cytometry (FCM) assays, as detailed in Methods. (a) Dot blots for the effector memory T cells (CD44hiCD62Llow) in CD4+ T cells. Cells were stained with CD4, CD44, CD62L mAb and analyzed by FCM. Oridonin and dexamethasone inhibited the over expression of effector memory T cells in TNBS mice. (b) The percentage of CD25+Foxp3+ Treg cells in CD4+ T cells. (c) Production of interferon (IFN)-γ, interleukin (IL)-2, and IL-17 in CD4+ T cells. (d) The results are expressed as the percentage of effector memory T cells, Treg cells, or IFN-γ+, IL-2+, IL-17+ cells in CD4+ T cells. Cytokine production was decreased in TNBS mice after oridonin and , Control; ■, TNBS; , 50 μg; , 100 μg; , dexamethasone treatment. ns P > 0.05; *P < 0.05 versus control mice; **P < 0.01; ***P < 0.001. DX.
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when compared with control mice, as less as 23.6%. Oridonin (either 50 μg/day or 100 μg/day) treatment rapidly recovered body weight loss efficiently (96.9% and 100.4% of the initial body weight, P < 0.05 compared with TNBS mice), which was better than dexamethasone treatment (Fig. 1b). This may be due to the high metabolism rate of dexamethasone in vivo.17 Oridonin treatment also ameliorated hyperemia and inflammation (Fig. 1c), reduced infiltration of lymphocytes and inflammatory activity (Fig. 1d). Compared with that from colitis mice, HAI value in the colon from 50 μg and 100-μg oridonin-treated mice was also decreased (6.500 ± 0.327 vs 3.600 ± 1.288 and 4.429 ± 0.896, respectively, P < 0.05) (Fig. 1e). Oridonin alters peripheral CD4+ T-cell subsets and functionality in TNBS-induced colitis mice. As is well known, the spleen is the principal peripheral immune organ. To evaluate the variety of CD4+ T cells in the periphery, the expression of splenic CD4+ T-cell subsets was further determined. The frequency of naïve (CD44lowCD62Lhi) and effector/memory (CD44hiCD62Llow) T cells in the spleen were compared. The percentage of effector/memory T cells increased significantly after TNBS treatment (18.4 ± 1.9% vs 7.7 ± 1.0%, P < 0.05). Both doses of oridonin were able to downregulate the percentage of effector/memory CD4+ T cells (50-μg oridonin: 11.3 ± 0.9%; 100-μg oridonin: 9.4 ± 2.3%; TNBS: 18.4 ± 1.9%, respectively, P < 0.05) (Fig. 2a,d). In our study, the percentage of CD4+CD25+Foxp3+ Treg in the spleen of colitis mice was decreased. However, oridonin treatment only slightly increased the frequency of Treg cells in 50-μg oridonin-treated group (P > 0.05) (Fig. 2b,d). Th17 cells were also detected to evaluate the immunological effect of oridonin. In TNBS mice, IL-17-producing CD4+ T cells dramatically elevated (CD4+IL-17+ T cells: 2.002 ± 0.277% vs 0.663 ± 0.034%, P < 0.05). Oridonin reduced the expansion of Th17 in TNBStreated mice (50-μg oridonin: 1.106 ± 0.172%; 100-μg oridonin: 1.191 ± 0.242%; TNBS: 2.002 ± 0.277%, respectively, P < 0.05) at a comparable level in colitis mice with dexamethasone treatment (Fig. 2c,d). These data suggest that oridonin and dexamethasone could directly decrease the level of IL-17 in splenic CD4+ T cells of mice with TNBS-induced colitis. To determine the effect of oridonin on peripheral Th1 subset of TNBS mice, IFN-γ- and IL-2-secreting cells were also determined. Our results indicated that IL-2-, IFN-γ-producing CD4+ T cells dramatically elevated (CD4+IL-2+ T cells: 51.1 ± 2.8% vs 42.4 ± 2.3%; CD4+IFN-γ+ T cells: 13.1 ± 1.2% vs 8.2 ± 1.0%, P < 0.05) in TNBS mice. Treatment with both doses of oridonin strongly suppressed IFN-γ expression (50-μg oridonin: 5.156 ± 0.717%; 100-μg oridonin: 6.564 ± 1.083%; TNBS: 13.1 ± 1.2%, respectively, P < 0.05) (Fig. 2c,d), which was similar to dexamethasone treatment. However, only 100-μg oridonin could significantly inhibit the over expression of IL-2 in CD4+ T cells (51.1 ± 2.8% vs 56.6 ± 4.1%, P < 0.05), whereas IL-2 was not significantly reduced in 50-μg oridonin-treated mice (P > 0.05). Dexamethasone did not affect the expression of IL-2 (Fig. 2c,d). Like dexamethasone, oridonin also had a therapeutic effect in improving TNBS-induced colitis by restraining exaggerated Th1 and Th17 responses and over-homeostasis of effector/memory cells. 508
Oridonin selectively modulates cytokine and Foxp3 expression in the colon of colitis mice. As shown in Figure 3, IFN-γ and IL-17 level in the colon increased markedly (8.4- and 11.8-fold, respectively, P < 0.05) in colitis mice. However, local IL-2 level remained unchanged significantly (P > 0.05). Oridonin treatment significantly inhibited IL-17 expression in the colon of mice with TNBS-induced colitis (50-μg oridonin: 1.168 ± 0.209%; 100-μg oridonin: 1.294 ± 0.322%; TNBS: 4.729 ± 0.707%, P < 0.05). Similar effects were observed for IFN-γ production (50-μg oridonin: 1.730 ± 0.318%; 100-μg oridonin: 1.778 ± 0.369%; TNBS: 6.400 ± 0.979%, P < 0.05). IL-2 expression remained unaltered in treated groups (P > 0.05) (Fig. 3a,b). Unexpectedly, there was 30-fold more Foxp3 expression in colonic tissues of TNBS-treated mice (P < 0.05). Treg proportions were also decreased in oridonin-treated colitis mice (50-μg oridonin: 0.227 ± 0.075%; 100-μg oridonin: 0.430 ± 0.112%; TNBS: 1.590 ± 0.394%, respectively, P < 0.05). Thus, oridonin exhibited similar effects to dexamethasone to ameliorate TNBS-induced colitis with a reduction of IL-17 and IFN-γ production in colon tissues. Oridonin impairs the proliferation and cytokine secretion of T cells in vitro. T-cell proliferation was measured by CFSE. Oridonin inhibited CD4+ T cell proliferation in a dose-dependent fashion (Fig. 4a). And 23.3% of CD4+ T cells divided three times and 41.7% of cells divided two times after stimulation. In the presence of 0.5-μg/mL oridonin, the percentage of T cells dividing three times decreased to 16.7%. The inhibitory effect was more pronounced after 1.0-μg/mL oridonin treatment. Only 5.07% of T cells subdivided three times and 37.9% cells subdivided two times. The apoptotic effect upon T-cell receptor stimulation was determined by TUNEL assay. There was no detectable apoptotic cell in unstimulated and stimulated MLN cells in the absence of oridonin. The percentage of apoptotic cells significantly increased to 48.4% after 0.5-μg/mL oridonin treatment, and 63.6% in the presence of 1.0-μg/mL oridonin (Fig. 4b), suggesting that CD4+ T cells from colitic pathology were more susceptible to apoptosis upon outside activation signaling. As was the case with in vivo experiment, the presence of oridonin did not change the percentage of Tregs (Fig. 4c). IL-17 and IFN-γ secretion of CD4+ T cells was blocked dramatically in the presence of both 0.5 and 1.0 μg/mL oridonin (CD4+ IFN-γ+ T cells: 9.868 ± 2.528% vs 7.372 ± 2.134% and 5.157 ± 1.760%; CD4+ IL-17+ T cells: 2.462 ± 0.671% vs 2.027 ± 0.464% and 1.260 ± 0.226%, P < 0.05). IL-2 expression only moderately decreased (1.14-fold, P > 0.05) in the presence of 1.0-μg/mL oridonin (Fig. 4c). NF-κB nuclear translocation is impeded in response to oridonin treatment. NF-κB is critical in inflammatory response and pathogenesis of IBD.18,19 As oridonin had been demonstrated to inhibit NF-κB activation in many other cells, we further verified the inhibitory mechanisms of oridinon on NF-κB-signaling pathways. Oridonin treatment for 48 h did not significantly inhibit NF-κB phosphorylation (Fig. 4e). However, nucleic translocation of p65 was inhibited in a dose-dependent manner (P < 0.05 compared with stimulated cells) (Fig. 4d). Our results demonstrated that oridonin improved the immunological
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Figure 3 Effects of oridonin and dexamethasone treatment on 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis in the expression of Foxp3 and cytokines in colonic tissues. Mice were treated with 50% ethanol (Control), TNBS or with 50-μg/mouse/day oridonin (50 μg), 100-μg/mouse/day orindonin (100 μg), and 2.5-μg/mouse/day dexamethasone (DX) 24 h after TNBS treatment. (a) Colons from mice were subjected to immunohistochemistry staining for Foxp3, interferon (IFN)-γ, interleukin (IL)-2, and IL-17. (b) The results are expressed as the percentage of Foxp3, IFN-γ, IL-2, and IL-17-positive cells in colonic cells. Oridonin decreased the expression of Foxp3, IFN-γ, and IL-17 in colons of TNBS colitis mice, whereas dexamethasone downregulated the expression of IFN-γ and IL-17. IL-2 was not significantly changed after oridonin and dexamethasone treatment. , Control; ■, TNBS; , 50-μg Ori; , 100-μg Ori; , DX. ns P > 0.05; *P < 0.05 versus control mice; **P < 0.01; ***P < 0.001.
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Figure 4 Effects of oridonin on the function of T cells from 2,4,6-trinitrobenzene sulfonic acid (TNBS)immunized mice stimulated with anti-CD3/CD28 beads. The lymphocytes isolated from mesenteric lymph node (MLN) of BALB/c mice with TNBS-induced colitis were cultured for 48 h in growth medium alone (-) or stimulated with anti-CD3/CD28 beads in the absence or presence of different doses of oridonin (0.5 μg/mL, 1.0 μg/ mL). (a) Proliferation of CD4+ T cells were measured by the dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE) after 48-h culture. Oridonin inhibited the proliferation stimulated by anti-CD3/CD28 beads in a dose-dependent manner. (b) Apoptosis of lymphocytes was assessed by transferase deoxytidyl uridine end labeling (TUNEL) assay. Apoptosis was significantly induced after oridonin treatment. (c) The percentage of Treg, Th1, and Th17 cells in CD4+ T cells. Expression of Tregs and IL-2 was not significantly changed, whereas production of interferon (IFN)-γ and interleukin (IL)-17 was effectively affected after oridonin treatment. (d) The expression levels of nuclear and cytosolic p65 protein were determined by Western blot analysis as detailed in Methods. (e) The expression levels of p65 and phosphop65 protein were analyzed by Western blot. Oridonin interfered with the translocation of p65 without altering its phosphorylaion. Results are representative of three independent experiments. ns P > 0.05; *P < 0.05 versus , CD3/CD28; , control mice; **P < 0.01. ■, —; , CD3/CD28 + 1-μg/mL CD3/CD28 + 0.5-μg/mL Ori; Ori.
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pathology and retarded the disease progression of TNBS-induced colitis, partially through interference with NF-κB nuclear translocation.
Discussion The present study has demonstrated that oridonin, an effective component isolated from Rabdosia rubescens, has therapeutic potential on an experimental CD model. This treatment effect of oridonin was accompanied by simultaneous suppression of both local and peripheral Th1/Th17, possibly through mediating and interfering with NF-κB nuclear translocation activity. As the etiology of CD remains unclear, most medical treatments are symptomatic by inhibiting the inflammatory response.20 The role of 5-aminosalicylic acid in inducing remission of active CD and preventing relapse of quiescent CD remains uncertain; it shows only limited value in mild-to-moderate CD.21 When the disease activity of CD is progressing, corticosteroids and immunosuppressive drugs are often added.22 TNF-α blocking agents (infliximab, adalimumab, certolizumab pegol) are effective therapy in CD.23 However, these agents needs to be weighed against the risk of side effects, particularly infection. Research on medications for CD is going on in an attempt to find out the most effective options. In our study, we demonstrated that oridonin had a remarkable treatment effect on the clinical course of CD-like colitis. Oridonin showed similar therapeutic capacity to corticosteroids in improving TNBS-induced colitis. In addition, no severe body weight loss was observed in mice treated with oridonin. Compared with corticosteroids, oridonin seems to be a relatively safe agent. After oridonin treatment, mice with TNBS colitis demonstrated clinical and histological improvement. Oridonin significantly reduced the percentage of both local and peripheral Th1/Th17 cells. On the other hand, oridonin significantly suppressed the over-homeostasis of effector/memory cells participating in the perpetuation of IBD, which were reported to be elevated in CD patients and mice models.24–27 In addition, oridonin acted synergistically on T-cell responses by inhibiting T-cell proliferation, inducing apoptosis of lymphocytes and interfering with T-cell function. Therefore, oridonin could provide a protective effect against the self-reactive immune response in TNBS mice via multiple mechanisms. The detailed mechanism underlying this effect needs to be further investigated. NF-κB-signaling pathway is known to contribute to the production of pro-inflammatory cytokines, which could strongly affect the development of IBD and many other autoimmune responses.10,28 Previous studies14,29 have demonstrated that oridonin could directly interfere with the DNA-binding activity of NF-κB to its response DNA sequences, and inhibit the translocation of NF-κB from cytoplasm to nucleus. It was found in the present study that the immunosuppressive action of oridonin was involved in the NF-κB pathway through its interference with NF-κB translocation. The modification of NF-κB activity might lead to the blockade of cytokine production and increased apoptosis of T cells. Oridonin has been demonstrated to have a broad spectrum of therapeutic actions including anti-inflammatory, antitumor, and anti-immunology activities.12,13,30,31 And several experiment suggest that oridonin is of relatively low in vivo toxicity.32–34 It has
multiple-modulation effects on tumor cells, including inhibiting NF-κB activity and modulating mitogen-activated protein kinase pathways.34 As the pathogenesis of CD involves a complicated network of multiple factors, the use of agents from traditional Chinese medicines such as oridonin should be highlighted, because they can exert immunomodulatory activities through targeting multiple cell subsets and signaling pathways critically related to inflammation. In summary, our data indicate that oridonin has therapeutic effects on TNBS-induced colitis by disturbing pathogenic Th1/ Th17 cells via inhibition of NF-κB signal pathways. The superior efficacy of oridonin in TNBS mice as compared with corticosteroids suggests that oridonin may prove to be a potential candidate for the treatment of colonic inflammation in CD and other autoimmune diseases. However, oridonin is only administered from the initiation of colitis in the present study. The effects of oridonin on established disease will be explored in the near future.
Acknowledgments This research is supported by a grant from Shanghai municipal commission of health and family planning (No. 2013125).
References 1 Satsangi J, Parkes M, Louis E et al. Two stage genome–wide search in inflammatory bowel disease provides evidence for susceptibility loci on chromosomes 3, 7 and 12. Nat. Genet. 1996; 14: 199–202. 2 Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat. Rev. Immunol. 2003; 3: 521–33. 3 Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel disease. Annu. Rev. Immunol. 2010; 28: 573–621. 4 Brand S. Crohn’s disease: Th1, Th17 or both? The change of a paradigm: new immunological and genetic insights implicate Th17 cells in the pathogenesis of Crohn’s disease. Gut 2009; 58: 1152–67. 5 Sartor RB. Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis. Nat. Clin. Pract. Gastroenterol. Hepatol. 2006; 3: 390–407. 6 Karczewski J, Mazur M, Karczewski M. Dual role of Th17 cells in Crohn’s disease. Centr. Eur. J. Immunol. 2012; 37: 286–9. 7 Fuss IJ, Neurath M, Boirivant M et al. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn’s disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J. Immunol. 1996; 157: 1261–70. 8 Billerey-Larmonier C, Uno JK, Larmonier N et al. Protective effects of dietary curcumin in mouse model of chemically induced colitis are strain dependent. Inflamm. Bowel Dis. 2008; 14: 780–93. 9 Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat. Rev. Immunol. 2002; 2: 725–34. 10 Atreya I, Atreya R, Neurath M. NF-κB in inflammatory bowel disease. J. Intern. Med. 2008; 263: 591–6. 11 Fichtner-Feigl S, Fuss IJ, Preiss JC, Strober W, Kitani A. Treatment of murine Th1-and Th2-mediated inflammatory bowel disease with NF-kappaB decoy oligonucleotides. J. Clin. Invest. 2005; 115: 3057–71. 12 Liu J, Yang F, Zhang Y, Li J. Studies on the cell-immunosuppressive mechanism of Oridonin from Isodon serra. Int. Immunopharmacol. 2007; 7: 945–54.
Journal of Gastroenterology and Hepatology 30 (2015) 504–512 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
511
Oridonin modulates CD4+ cells in colitis
S Wang et al.
13 Hu AP, Du JM, Li JY, Liu JW. Oridonin promotes CD4+/CD25+ Treg differentiation, modulates Th1/Th2 balance and induces HO-1 in rat splenic lymphocytes. Inflamm. Res. 2008; 57: 163–70. 14 Leung CH, Grill SP, Lam W, Han QB, Sun HD, Cheng YC. Novel mechanism of inhibition of nuclear factor-κB DNA-binding activity by diterpenoids isolated from Isodon rubescens. Mol. Pharmacol. 2005; 68: 286–97. 15 Bai A, Lu N, Guo Y, Liu Z, Chen J, Peng Z. All-trans retinoic acid down-regulates inflammatory responses by shifting the Treg/Th17 profile in human ulcerative and murine colitis. J. Leukoc. Biol. 2009; 86: 959–69. 16 Inokuchi Y, Morohashi T, Kawana I, Nagashima Y, Kihara M, Umemura S. Amelioration of 2, 4, 6-trinitrobenzene sulphonic acid induced colitis in angiotensinogen gene knockout mice. Gut 2005; 54: 349–56. 17 Shu HJ, Takeda H, Shinzawa H, Takahashi T, Kawata S. Effect of lipopolysaccharide on peptide transporter 1 expression in rat small intestine and its attenuation by dexamethasone. Digestion 2002; 65: 21–9. 18 Schreiber S, Nikolaus S, Hampe J. Activation of nuclear factor κB in inflammatory bowel disease. Gut 1998; 42: 477–84. 19 Tak PP, Firestein GS. NF-kappaB: a key role in inflammatory diseases. J. Clin. Invest. 2001; 107: 7–12. 20 Van Montfrans C, Camoglio L, Van Deventer S. Immunotherapy of Crohn’s disease. Mediators Inflamm. 1998; 7: 149–52. 21 Ford AC, Kane SV, Khan KJ et al. Efficacy of 5-aminosalicylates in Crohn’s disease: systematic review and meta-analysis. Am. J. Gastroenterol. 2011; 106: 617–29. 22 Podolsky DK. Inflammatory bowel disease. N. Engl. J. Med. 1991; 325: 928–37. 23 Behm BW, Bickston SJ. Tumor necrosis factor-alpha antibody for maintenance of remission in Crohn’s disease. Cochrane Database Syst. Rev. 2008; (1): CD006893. 24 Nemoto Y, Kanai T, Kameyama K et al. Long-lived colitogenic CD4+ memory T cells residing outside the intestine participate in the perpetuation of chronic colitis. J. Immunol. 2009; 183: 5059–68.
512
25 ten Hove T, Berkhout M, Bruggeman JP et al. Expression of CD45RB functionally distinguishes intestinal T lymphocytes in inflammatory bowel disease. J. Leukoc. Biol. 2004; 75: 1010–5. 26 Roman L, Manzano L, De La Hera A, Abreu L, Rossi I, Alvarez-Mon M. Expanded CD4+ CD45RO+ phenotype and defective proliferative response in T lymphocytes from patients with Crohn’s disease. Gastroenterology 1996; 110: 1008–19. 27 Pasternak BA, D’Mello S, Jurickova II et al. Lipopolysaccharide exposure is linked to activation of the acute phase response and growth failure in pediatric Crohn’s disease and murine colitis. Inflamm. Bowel Dis. 2010; 16: 856–69. 28 Hilliard B, Samoilova EB, Liu TST, Rostami A, Chen Y. Experimental autoimmune encephalomyelitis in NF-κB-deficient mice: roles of NF-κB in the activation and differentiation of autoreactive T cells. J. Immunol. 1999; 163: 2937–43. 29 Hortelano S. Molecular basis of the anti-inflammatory effects of terpenoids. Inflamm. Allergy Drug Targets 2009; 8: 28–39. 30 Zhou GB, Chen SJ, Wang ZY, Chen Z. Back to the future of oridonin: again, compound from medicinal herb shows potent antileukemia efficacies in vitro and in vivo. Cell Res. 2007; 17: 274–6. 31 Xu Y, Xue Y, Wang Y, Feng D, Lin S, Xu L. Multiple-modulation effects of Oridonin on the production of proinflammatory cytokines and neurotrophic factors in LPS-activated microglia. Int. Immunopharmacol. 2009; 9: 360–5. 32 Chen S, Gao J, Halicka HD, Huang X, Traganos F, Darzynkiewicz Z. The cytostatic and cytotoxic effects of oridonin (Rubescenin), a diterpenoid from Rabdosia rubescens, on tumor cells of different lineage. Int. J. Oncol. 2005; 26: 579–88. 33 Zhou G-B, Kang H, Wang L et al. Oridonin, a diterpenoid extracted from medicinal herbs, targets AML1-ETO fusion protein and shows potent antitumor activity with low adverse effects on t (8; 21) leukemia in vitro and in vivo. Blood 2007; 109: 3441–50. 34 Zhen T, Wu C-F, Liu P et al. Targeting of AML1-ETO in t (8; 21) leukemia by oridonin generates a tumor suppressor-like protein. Sci. Transl. Med. 2012; 4: 127ra38–ra38.
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