International Immunopharmacology 20 (2014) 46–53
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Effects and mechanisms of Geniposide on rats with adjuvant arthritis Miao-Miao Dai, Hong Wu ⁎, Hui Li, Jian Chen, Jin-Yun Chen, Shun-Li Hu, Chen Shen College of Pharmacy, Anhui University of Chinese Medicine, Key Laboratory of Modernized Chinese Medicine in Anhui Province, Hefei 230031, Anhui Province, China
a r t i c l e
i n f o
Article history: Received 23 December 2013 Received in revised form 18 January 2014 Accepted 11 February 2014 Available online 26 February 2014 Keywords: Adjuvant arthritis JNK signaling Mesenteric lymph node lymphocytes Peripheral blood lymphocytes Immune tolerance Geniposide
a b s t r a c t Geniposide (GE), an iridoid glycoside compound, is the major active ingredient of Gardenia jasminoides Ellis (GJ) fruit which has anti-inflammatory and other important therapeutic activities. The aim of this study was to investigate the effects of GE on adjuvant arthritis (AA) rats and its possible mechanisms. AA was induced by injecting with Freund's complete adjuvant (FCA). Male SD rats were subjected to treatment with GE at 30, 60 and 120 mg/kg from days 18 to 24 after immunization. Lymphocyte proliferation was assessed by MTT. Interleukin (IL)-6, IL-17, IL-4 and transforming growth factor-beta 1 (TGF-β1) were determined by ELISA. c-Jun N-terminal kinase (JNK) and phospho-JNK (p-JNK) were detected by Western blot. GE (60, 120 mg/kg) significantly relieved the secondary hind paw swelling and arthritis index, along with decreased Th17-cells cytokines and increased Treg-cell cytokines in mesenteric lymph node lymphocytes (MLNL) and peripheral blood lymphocytes (PBL) of AA rats. In addition, GE decreased the expression of p-JNK in MLNL and PBL of AA rats. In vivo study, it was also observed that GE attenuated histopathologic changes of MLN in AA rats. Collectively, GE might exert its antiinflammatory and immunoregulatory effects through inducing Th17 cell immune tolerance and enhancing Treg cell-mediated activities by down-regulating the expression of p-JNK. The mechanisms of GE on JNK signaling in MLNL and PBL may play critical roles in the pathogenesis of rheumatoid arthritis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease, which is characterized by synovial inflammation and joint destruction [1]. It is generally believed that the autoimmune response is associated with a variety of immune cells, especially CD4+ T helper (Th) cells [2]. CD4+ T cells were classically thought to differentiate into two subgroups: Th1 cells and Th2 cells. It has been reported that the imbalance of Th1/Th2 was closely related to RA [3]. However, recent studies have shown that CD4+ T cells could be divided into four subsets, such as pro-inflammatory Th1 cells, anti-inflammatory Th2 cells, and newly defined Th17 cells and regulatory T (Treg) cells. The imbalance of Th17/Treg played an even greater role in the development and pathogenesis of RA [4,5]. The recently identified Th17 cells were CD4+ T cells characterized by the secretion of IL-17. In addition to their key effector cytokine IL-17A (IL-17) which could participate in tissue inflammation and joint destruction of RA by inducing many pro-inflammatory cytokines and chemokines [6,7], they also produced other inflammatory cytokines, including IL-17F, IL-6 and tumor necrosis factor (TNF)-α [8,9]. Furthermore, Th17 cells were crucial for defending against extracellular bacteria and mediating chronic inflammation, autoimmune disease, malignant tumors, and so on [10]. Treg cells, characterized by the expression of the forkhead–winged-helix transcription factor (Foxp3), could suppress immune responses to exert anti-inflammatory ⁎ Corresponding author. Tel.: +86 551 6516 9230; fax: +86 551 6516 9222. E-mail address: [email protected] (H. Wu).
http://dx.doi.org/10.1016/j.intimp.2014.02.021 1567-5769/© 2014 Elsevier B.V. All rights reserved.
effect and maintain unresponsiveness to self-antigens by means of cell–cell contact inhibition and secreting anti-inflammatory cytokines such as IL-10 and TGF-β1 [11]. A previous study has shown that the imbalance of Th17/Treg was existed in the peripheral blood of patients with RA and related to pathogenetic condition [5]. However, how this imbalance is existed in patients with RA has not been reported. AA was chosen as an experimental model for RA, which shares some features with human RA in a number of pathological, histological and immunological aspects. AA was induced by injecting with FCA that containing heat-killed Mycobacterium tuberculosis (MT) by a mechanism involving heat shock proteins (HSPs). Geniposide (GE), an iridoid glycoside compound, is the main bioactive components (structure, see Fig. 1) of Gardenia jasminoides Ellis (GJ) fruit which commonly was used as a traditional medicine in many Asian countries for its antiphlogistic and antipyretic effects [12]. It has been shown that GE exhibited an anti-inflammatory property by downregulating the expression of Toll-like receptor 4 (TLR4) up-regulated by lipopolysaccharide (LPS) in the primary mouse macrophages and mouse models [13]. In addition, recent reports have demonstrated that GE exerted anti-inflammatory activity in the Carrageenan-induced rat paw edema model and displayed inhibitory effects on acetic acid-induced vascular permeability changes [14]. However, the anti-inflammatory effect and the mechanisms of GE on AA rats remain unclear. GE was extracted and purified from GJ by means of solvent extraction and column chromatography. The structure was identified by physicochemical properties and spectroscopic analysis, and the content was determined by Ultra-Performance Liquid Chromatography (UPLC).
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2.2. Materials Mouse anti-JNK monoclonal antibody (D-2, sc-7354) and mouse anti-p-JNK monoclonal antibody (G-7, sc-6254) were obtained from Santa Cruz Biotechnology, Inc. Mouse anti-beta actin monoclonal antibody (TA-09) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (2B-2305) were purchased from ZSGB-BIO. IL-6, IL-17, IL-4 and TGF-β1 ELISA kits were supplied from Elabscience Biotechnology Co., Ltd. The following reagents were offered commercially: Concanavalin A (ConA, stored at − 20 °C), lectin from Phaseolus vulgaris-phytohemagglutinin (PHA-P, stored at 2–8 °C) and 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) from Sigma Chemical Co. (St. Louis, MO, USA); Dulbecco's modified Eagle's medium (DMEM) from Thermo Scientific Co. (USA). Other chemicals used in these experiments were analytical grade from commercial sources. Fig. 1. Structure of Geniposide (formula C17H24O11, molecular weight 404.36).
2.3. Drugs
Total glucosides of paeony (TGP), as the positive control drug, were used for the treatment of RA. The c-Jun N-terminal kinases (JNKs), which belong to the mitogenactivated protein kinase (MAPK) family, play vital roles in the production of cytokines and the degradation of extracellular matrix by regulating matrix metalloproteinase (MMP) gene expression in fibroblast-like synoviocytes (FLSs) and mediating joint destruction in AA rats [15,16]. The JNK signal pathway could be activated by cytokines, growth factors, stresses, and so on. Many studies suggested that the JNK signal pathway plays a pivotal role in cell differentiation, apoptosis, initiation and progress of considerable human diseases as well as in animal models, including ischemia/reperfusion (I/R), RA, diabetes mellitus, and tumor [17,18]. Experience gathered has confirmed that IL-17 could synergize with local inflammatory mediator IL-6 to induce the expression of IL-6 by a transcriptional mechanism that involves JNK signal pathway, and blockage of JNK pathway with JNK inhibitor II could partially inhibit the synergistic effect of IL-17 and IL-6 on the expression of IL-6 [19]. Additionally, it has been shown that the transcription factor Eomesodermin (Eomes), whose expression could substantially suppress Th17 cell induction in primary T cells, was suppressed by TGF-β1 via the JNK-c-Jun signal pathway [20]. We therefore wondered whether the balance of Th17/Treg could be associated with the JNK signal pathway. In this study, we discussed the role of GE in rectifying Th17/Treg balance by inhibiting excessive phosphorylation of JNK. Here, we evaluated the effects and mechanisms of GE on immune balance of Th17/Treg at levels of organ, molecule, and protein, respectively. Our results showed that GE could inhibit the progressive inflammation by inducing Th17 cells tolerance and enhancing Treg-driving effects, suggesting that GE has a significant therapeutic potential in treating autoimmune and other chronic inflammatory disorders. This may provide useful information for both clinical therapy and basic scientific research in RA.
GE is a yellow powder with N94.6% purity (determined by UPLC, ACQuity H-CLASS, Waters Co., USA), which was provided by the Chemistry Lab of Anhui University of Chinese Medicine (Hefei, Anhui Province, China). Standard of TGP (purity N 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). GE and TGP were suspended in water before use. 2.4. Induction of AA AA was induced as previously described [21]. Briefly, rats were immunized on day 0 by a single intradermal injection into the left hind paw with 100 μL of FCA for each rat. 2.5. Treatment of AA Before the onset of arthritis, rats were divided into six groups randomly, in which the AA rats were given intragastrically GE (30, 60, 120 mg/kg) and TGP (50 mg/kg) from days 18 to 24 after immunizations. While in groups of normal and AA model, rats were given an equal volume of water at the same time. 2.6. Arthritis assessment Rats were assessed daily for signs of arthritis by two independent observers who were blinded to the experimental design. Non-injected hind paw volume was determined with YLS-7A volume meter (Shandong Academy of Medical Sciences Equipment Station, China). The severity of arthritis in each paw was graded on a scale of 0–4: 0, no swelling; 1, swelling of finger joints; 2, swelling of phalanx joint and digits; 3, severe swelling of the entire paws; 4, deformity or ankylosis. The maximum arthritis score was 12 including three secondary arthritis paws for each rat [22,23].
2. Materials and methods
2.7. Lymphocyte proliferation assay by MTT
2.1. Animals
MLNs and peripheral blood were removed in sterile condition. MLNL and PBL were isolated by routine method. Then, the cells were cultured in triplicate in a concentration of 1 × 1010 cell/L in 100 μL DMEM containing 10% FBS. First, the cells were stimulated with 5 mg/L ConA for 48 h at 37 °C and 5% of CO2. A 10 μL sample of MTT (5 g/L) was added before the end of stimulation for 4 h and then the cultures were stimulated for 4 h continuously. After incubation, the cultures were centrifuged (760 × g, 10 min) and the supernatants were discarded. A 150 μL of dimethylsulfoxide (DMSO) was added to each well and the absorbance (A) was examined at 490 nm using a MSS ELISA Microwell Reader (Thermo Scientific Co., USA).
Sprague–Dawley (SD) rats (♂, 180 ± 20 g. Grade II, Certificate No. 011) were purchased from the Animal Department of Anhui University of Chinese Medicine (Hefei, Anhui Province, China). All animals were housed under specific pathogen-free conditions with a 12-hour light/dark cycle in a temperature-controlled room at 23 °C (±1) and allowed food and water ad libitum in each group of no more than six. All experiments using rats were performed in accordance with protocols approved by the Ethics Review Committee for Animal Experimentation of Anhui University of Chinese Medicine.
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Levels of IL-6, IL-17, IL-4 and TGF-β1 in MLNL and PBL were evaluated using ELISA kits in accordance with the manufacturer's instructions. 2.9. Western blot Frozen cells were lysed in buffer and the lysates were separated by 10% sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat dry milk in Trisbuffered saline/Tween 20 (TBST) at room temperature for 3 h. Primary antibodies specific for JNK and p-JNK were incubated with the membranes overnight at 4 °C. Membranes were washed with phosphatebuffered saline (PBS) and incubated with the secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-mouse (1:1000) or goat anti-mouse (1:5000) IgG. Immunoreactive proteins were detected with 3, 3′-diaminobenzidine tetrahydrochloride (DAB). All the experiments were performed three times and the results were reproducible. 2.10. Histological analysis The MLNs of rats were removed under sterile conditions, fixed with 10% paraformaldehyde in PBS, and then embedded in paraffin for histological analysis. A series of paraffin sections (5 μm) were stained with hematoxylin and eosin (HE, 200×) and examined by two experienced pathologists in total darkness. 2.11. Statistical analysis Data were expressed as mean ± standard error of the mean (S.E.M.), in triplicate. One-way ANOVA test and Student's t -test (SPSS17.0 Software Products, Chicago, IL, USA) were used to determine significant differences between groups. The histopathologic analysis was analyzed by Ridit procedure. In all tests, P-value b 0.05 was considered statistically significant. 3. Results 3.1. Effect of GE on secondary arthritis in AA rats The onset and peak of inflammatory reaction occurred on day 14 and days 18–21 after immunization, respectively. There was a remarkable secondary inflammatory response in the model, which accompanied with paw swelling, pain, polyarthritis. The administration of GE (30, 60, 120 mg/kg) significantly suppressed the hind paw swelling (P b 0.01) (Fig. 2A). There was no significant reduction in polyarthritis index by treatment with GE at 30 mg/kg (P N 0.05), while a significant decrease in polyarthritis index was observed when the AA rats were treated with GE at 60 and 120 mg/kg (P b 0.01). The efficacy was similar to that of TGP (50 mg/kg) (Fig. 2B). 3.2. Effect of GE on lymphocyte proliferation of MLNL and PBL in AA rats As shown in Fig. 3A, the proliferation of MLNL in AA group decreased significantly compared with normal group. GE (60 and 120 mg/kg) could apparently increase the cell proliferation in AA. TGP (50 mg/kg) had similar effects as GE. However, the results of PBL were just the reverse of that of MLNL (Fig. 3B). 3.3. Effect of GE on production of IL-6, IL-17, IL-4 and TGF-β1 in AA rats The cytokines of AA rats were determined on day 24 after immunization. The production of IL-6 and IL-17 from MLNL and PBL was
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day Fig. 2. Effect of GE on secondary arthritis in AA rats. Rats were immunized on day 0 by a single intradermal injection into the left hind paw with 100 μL of FCA for each rat. GE (30, 60, 120 mg/kg) and TGP (50 mg/kg) were given intragastrically to AA rats from days 18 to 24 after immunization. The paw swelling and polyarthritis were evaluated as described in Materials and methods. A: paw swelling and B: arthritis index. Values are presented as means ± S.E.M. from 6 animals in each group. ##P b 0.01 vs. Normal group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. AA model.
increased, while that of IL-4 and TGF-β1 from MLNL and PBL was decreased when compared with normal group. GE at 30 mg/kg had no effect on the production of IL-17 and TGF-β1 (P N 0.05), while GE at 60, 120 mg/kg dramatically diminished the production of IL-17 and IL-6, and elevated the production of IL-4 and TGF-β1 from MLNL and PBL (P b 0.01), a result consistent with that from TGP (50 mg/kg) (Fig. 4).
3.4. Effect of GE on expression of JNK, p-JNK and β-actin of MLNL and PBL in AA rats The protein expression of JNK, p-JNK and β-actin of MLNL and PBL in AA rats were estimated on day 24 after immunization. It was found that the expression of p-JNK was heightened in AA rats when compared with normal group (P b 0.01). The administration of GE (30, 60, 120 mg/kg) markedly decreased that of p-JNK (P b 0.01), while the protein levels of total JNK had no change (Fig. 5).
3.5. Effect of GE on histopathology of MLNs in AA rats Rats were sacrificed on day 24 after immunization and subjected to histopathological examination. The hyperplasia of lymphatic follicle and the increased number of inflammatory cells were observed in MLN of AA rats. Treatment with GE at 120 mg/kg evidently reduced hyperplastic lymphatic follicle and the infiltration of inflammatory cells in AA rats (Fig. 6).
M.-M. Dai et al. / International Immunopharmacology 20 (2014) 46–53
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Group Fig. 3. Effect of GE on lymphocyte proliferation of MLNL and PBL in AA rats. MLNs and peripheral blood were removed in sterile condition. MLNL and PBL were isolated by routine method. Then, the cells were cultured in triplicate in a concentration of 1 × 1010 cell/L in 100 μL DMEM containing 10% FBS. The cells were stimulated with 5 mg/L ConA for 48 h at 37 °C and 5% of CO2. Lymphocyte proliferation was determined by MTT assay. A: the proliferation of MLNL and B: the proliferation of PBL. Values are presented as means ± S.E.M. from 6 animals in each group. ##P b 0.01 vs. Normal group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. AA model.
4. Discussion RA is a common autoimmune disease, which is characterized by joint swelling and pain, joint stiffness, deformity and serious functional damage. AA could act as an experimental model in our study to demonstrate the effects of GE on human RA because of its similarity to human RA in both clinical and histopathologic features. As shown in Fig. 2, the present study showed that GE significantly relieved the secondary hind paw swelling. GE at a moderate or high dose markedly reduced polyarthritis index, indicating a protective effect on FCA-induced injury in vivo, while GE at low dose did not show a significant inhibitory effect on polyarthritis index in AA rats. Several possibilities could account for this failure. For example, low dose of GE could not be high enough for attenuating polyarthritis index. Or, the treatment time was too short to see improvement in AA rats treated with GE at low dose. If we prolong the time, it could have a dose-dependent effect and time-dependent effect. Besides, it was found that GE improved histological status of
MLNs in AA rats. These findings indicated that GE could alleviate the extent of arthritis in AA rats with FCA-induced. AA was closely related to the hyperfunction of immune function. GE could reduce the proliferation of PBL in AA rats, suggesting that GE could get the immune function back to normal levels by acting on immune cells, so as to relieve AA. Interestingly, in the intestinal mucosal immune system, the proliferation of MLNL was inhibited by AA, while GE could restore and even enhance the proliferation activity of MLNL. This hinted that the effect of GE on AA may also be played by the mucosal immune tolerance mechanisms. Scilicet, in the intestinal immune system, GE stimulated the mucosal immune system and then the intestinal mucosal immunity was enhanced, but the immune response of lesions was suppressed. The balance was skewed toward Th17, resulting in excessive production of IL-17, TNF-α and IL-6, whereas Treg activity and the level of TGF-β1 were deficient in RA patients compared to healthy individuals [24]. One study supported the concept that there was a reciprocal
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Fig. 4. Effect of GE on production of IL-6, IL-17, IL-4 and TGF-β1 in AA rats. MLNL and PBL were collected on day 24 after immunization. Levels of IL-6, IL-17, IL-4 and TGF-β1 were measured by ELISA. A: Levels of IL-6, IL-17, IL-4 and TGF-β1 in MLNL, B: Levels of IL-6, IL-17, IL-4 and TGF-β1 in PBL. Values are presented as means ± S.E.M. from 6 animals in each group. ##P b 0.01 vs. Normal group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. AA model.
relationship between pathogenic Th17 and protective Treg in both developmental pathways and function in the immune system depending on the state of the innate immune system as well as the production of IL-6 [25]. Recent researches have demonstrated that IL-6 was a key factor in determining the balance of Th17/Treg. IL-6 could induce the differentiation of Th17 cells from naive T cells in the presence of TGF-β1; on the contrary, IL-6 could inhibit TGF-β1-induced Treg cell differentiation. Dysregulation or overproduction of IL-6 could result in autoimmune diseases such as systemic lupus erythematous (SLE) and RA, in which Th17 cells were considered to be the leading cause of pathology [26]. Besides, evidence indicated that TGF-β1 was required for the generation of Th17 and Treg. TGF-β1 could induce both anti-inflammatory Treg and pro-inflammatory Th17 depending on the local cytokine milieu. TGF-β1 alone could induce the differentiation of Treg from naive T cells in normal condition, while TGF-β1 could induce the
development of Th17 from naive T cells together with IL-6 in inflammatory state [27,28]. Veldhoen et al. [29] have also showed that the number of Th17 cells in TGF-β1-deficient mice was reduced, whereas increased in the presence of TGF-β1. In order to validate the suggestion that whether GE could revise the imbalance of Th17/Treg in AA rats, we determined the levels of IL-17, IL-6 and TGF-β1, which belong to Th17 and Treg cytokines, respectively. It was found that GE at low dose had no effect on the production of IL-17 and TGF-β1, while GE at moderate or high dose could remarkably decrease the production of IL-17 and IL-6, and increase the lowered level of TGF-β1 in AA rats. In all likelihood a low dose of GE was not sufficient to reduce the production of IL-17 and TGF-β1 and the effect could be dose-dependent. Therefore, we proposed an idea that GE in a dose-dependent manner could induce Th17 cell immune tolerance and give rise to a shift toward Treg cell-mediated antiinflammatory effects, which ultimately suppress the release of pro-
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Fig. 5. Effect of GE on expression of JNK, p-JNK and β-actin of MLNL and PBL in AA rats. JNK, p-JNK and β-actin were assessed by Western blot on day 24 after immunization, as described in Materials and methods. A: Western blot for JNK, p-JNK and β-actin of MLNL and PBL in AA rats. B: Quantitative evaluation of JNK and p-JNK of MLNL in AA rats by densitometry from triplicate independent experiments. C: Quantitative evaluation of JNK and p-JNK of PBL in AA rats. ⁎⁎P b 0.01 vs. AA, ## P b 0.01 vs. Normal. 1–3: GE (30, 60 and 120 mg/kg); 4: TGP (50 mg/kg); 5: AA; 6: Normal.
inflammatory cytokines IL-17 and IL-6, and play a lasting inhibitory effect on AA rats, so as to correct the imbalance of Th17/Treg. Previous reports have demonstrated the importance of JNK pathway in Th1/Th2 balance as well as in Th17/Treg balance [30,31]. One study reported that JNK1-deficient mice were resistant to experimental autoimmune encephalitis (EAE). Furthermore, fewer Th17 cells were observed in immunized JNK1-deficient mice [32]. Another paper showed that the JNK inhibitor (SP600125) not only strongly inhibited the Th17 cell-mediated in vivo disease model known as EAE, but also
completely abrogated in vitro Th17 cell induction from primary T cells in a dose-dependent manner, indicating that the JNK signal pathway was essential for Th17 cells development both in vitro and in vivo [20]. In our experiment, as shown in Fig. 5, we observed that the level of p-JNK was markedly decreased, while that of total JNK remained unchanged in MLNL and PBL in AA rats after administration of GE. The results showed that GE could down-regulate the protein expression of p-JNK, and retrieved the balance between JNK and p-JNK. As previously described, the JNK signal pathway had a significant impact on
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Fig. 6. Histopathologic examination of MLN in AA rats. A: In normal rats, fewer and smaller lymphatic follicles and no inflammatory cells were observed in MLN. B: In AA rats, the hyperplastic lymphatic follicle and the infiltration of inflammatory cells were observed. C: In AA rats treated with GE 120 mg/kg, only a small extent of hyperplastic lymphatic follicle was observed (original magnification 400×).
determining the developmental direction of Th17 and Treg. Consequently, it is possible that GE could induce immune balance of Th17/ Treg of MLNL and PBL via inhibiting excessive phosphorylation of JNK in AA rats. To further clarify the mechanisms by which GE induces immune balance of Th17/Treg of MLNL and PBL, we retrospected the effects of HSPs on cellular immunity. HSPs, also called stress proteins, were a highly conserved group of cytoprotective proteins which were typically upregulated in response to a range of stress stimuli, including heat shock, growth factors, inflammation and infection [33]. HSPs could interact with various signaling proteins of signal transduction pathways that regulate growth and development [34–36]. HSP70, an important member of HSPs, has vital immunoregulatory properties and has also been proved to display an anti-inflammatory effect on RA by downregulating levels of IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 in FLSs, which was mediated via the suppression of MAPKs and nuclear factor-κB (NF-κB) signal pathways induced by TNF-α [37]. Besides, recent investigations demonstrated that the HSP70 could restrain AA, which might partly be due to induction of self-HSP70 cross-reactive T cells that were capable of diminishing inflammation [38,39]. Furthermore, Kim et al. [40] demonstrated that arsenic trioxide induced HSP70 expression by JNK pathway in MDA231 cells and SP600125 suppressed HSP70 promoter-driven reporter gene expression, suggesting that JNK could be preferentially related to the protective heat shock response against arsenic trioxide stress. It has also been reported that in the control of apoptosis, HSP70 could not only inhibit directly or indirectly the activity of JNK, but also bind to JNK as a natural inhibitory protein in case of its phosphorylation, and thereby inhibit JNK-mediated apoptosis [41]. These results indicated that the activation of JNK signal pathway could enhance HSP70 expression; on the other hand, the high expression of HSP70 could suppress JNK activity. Works on experimental and human arthritis have presented that HSP70 could regulate autoimmunity indirectly by activating protective Treg cells that control pathogenic T cells specific for self-antigens, and HSP70 has also been identified as an immune target in RA [39]. Therefore, it's quite plausible that GE induces Th17 cell immune tolerance and enhances the activity of Treg cells through inducing the high expression of HSP70 and thereby inhibiting the phosphorylation of JNK. In conclusion, we have demonstrated the effects and mechanisms of GE on AA rats. GE induced Th17 cell immune tolerance and enhanced Treg cell-driving effects by decreasing pro-inflammatory cytokines IL-17 and IL-6 and increasing anti-inflammatory cytokine TGF-β1 . Furthermore, the fact that GE ameliorated the expression of p-JNK in MLNL and PBL indicated that GE could induce immune balance of Th17/Treg by suppressing over expression of p-JNK in AA rats. Therefore, this study provides more comprehensive evidences supporting GE as a potential alternative agent for the autoimmune disease, especially RA.
Further studies are needed to fully understand the details of the mechanism for the role of GE in RA.
Acknowledgments This project was financially supported by the National Natural Science Foundation of China (No. 81073122), the Anhui Provincial Natural Science Research Project in Colleges and Universities (KJ2009A045Z) and the youth of Natural Science Foundation of Anhui University of Chinese Medicine (qn201308).
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Effects and mechanisms of Geniposide on rats with adjuvant arthritis Miao-Miao Dai, Hong Wu ⁎, Hui Li, Jian Chen, Jin-Yun Chen, Shun-Li Hu, Chen Shen College of Pharmacy, Anhui University of Chinese Medicine, Key Laboratory of Modernized Chinese Medicine in Anhui Province, Hefei 230031, Anhui Province, China
a r t i c l e
i n f o
Article history: Received 23 December 2013 Received in revised form 18 January 2014 Accepted 11 February 2014 Available online 26 February 2014 Keywords: Adjuvant arthritis JNK signaling Mesenteric lymph node lymphocytes Peripheral blood lymphocytes Immune tolerance Geniposide
a b s t r a c t Geniposide (GE), an iridoid glycoside compound, is the major active ingredient of Gardenia jasminoides Ellis (GJ) fruit which has anti-inflammatory and other important therapeutic activities. The aim of this study was to investigate the effects of GE on adjuvant arthritis (AA) rats and its possible mechanisms. AA was induced by injecting with Freund's complete adjuvant (FCA). Male SD rats were subjected to treatment with GE at 30, 60 and 120 mg/kg from days 18 to 24 after immunization. Lymphocyte proliferation was assessed by MTT. Interleukin (IL)-6, IL-17, IL-4 and transforming growth factor-beta 1 (TGF-β1) were determined by ELISA. c-Jun N-terminal kinase (JNK) and phospho-JNK (p-JNK) were detected by Western blot. GE (60, 120 mg/kg) significantly relieved the secondary hind paw swelling and arthritis index, along with decreased Th17-cells cytokines and increased Treg-cell cytokines in mesenteric lymph node lymphocytes (MLNL) and peripheral blood lymphocytes (PBL) of AA rats. In addition, GE decreased the expression of p-JNK in MLNL and PBL of AA rats. In vivo study, it was also observed that GE attenuated histopathologic changes of MLN in AA rats. Collectively, GE might exert its antiinflammatory and immunoregulatory effects through inducing Th17 cell immune tolerance and enhancing Treg cell-mediated activities by down-regulating the expression of p-JNK. The mechanisms of GE on JNK signaling in MLNL and PBL may play critical roles in the pathogenesis of rheumatoid arthritis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease, which is characterized by synovial inflammation and joint destruction [1]. It is generally believed that the autoimmune response is associated with a variety of immune cells, especially CD4+ T helper (Th) cells [2]. CD4+ T cells were classically thought to differentiate into two subgroups: Th1 cells and Th2 cells. It has been reported that the imbalance of Th1/Th2 was closely related to RA [3]. However, recent studies have shown that CD4+ T cells could be divided into four subsets, such as pro-inflammatory Th1 cells, anti-inflammatory Th2 cells, and newly defined Th17 cells and regulatory T (Treg) cells. The imbalance of Th17/Treg played an even greater role in the development and pathogenesis of RA [4,5]. The recently identified Th17 cells were CD4+ T cells characterized by the secretion of IL-17. In addition to their key effector cytokine IL-17A (IL-17) which could participate in tissue inflammation and joint destruction of RA by inducing many pro-inflammatory cytokines and chemokines [6,7], they also produced other inflammatory cytokines, including IL-17F, IL-6 and tumor necrosis factor (TNF)-α [8,9]. Furthermore, Th17 cells were crucial for defending against extracellular bacteria and mediating chronic inflammation, autoimmune disease, malignant tumors, and so on [10]. Treg cells, characterized by the expression of the forkhead–winged-helix transcription factor (Foxp3), could suppress immune responses to exert anti-inflammatory ⁎ Corresponding author. Tel.: +86 551 6516 9230; fax: +86 551 6516 9222. E-mail address: [email protected] (H. Wu).
http://dx.doi.org/10.1016/j.intimp.2014.02.021 1567-5769/© 2014 Elsevier B.V. All rights reserved.
effect and maintain unresponsiveness to self-antigens by means of cell–cell contact inhibition and secreting anti-inflammatory cytokines such as IL-10 and TGF-β1 [11]. A previous study has shown that the imbalance of Th17/Treg was existed in the peripheral blood of patients with RA and related to pathogenetic condition [5]. However, how this imbalance is existed in patients with RA has not been reported. AA was chosen as an experimental model for RA, which shares some features with human RA in a number of pathological, histological and immunological aspects. AA was induced by injecting with FCA that containing heat-killed Mycobacterium tuberculosis (MT) by a mechanism involving heat shock proteins (HSPs). Geniposide (GE), an iridoid glycoside compound, is the main bioactive components (structure, see Fig. 1) of Gardenia jasminoides Ellis (GJ) fruit which commonly was used as a traditional medicine in many Asian countries for its antiphlogistic and antipyretic effects [12]. It has been shown that GE exhibited an anti-inflammatory property by downregulating the expression of Toll-like receptor 4 (TLR4) up-regulated by lipopolysaccharide (LPS) in the primary mouse macrophages and mouse models [13]. In addition, recent reports have demonstrated that GE exerted anti-inflammatory activity in the Carrageenan-induced rat paw edema model and displayed inhibitory effects on acetic acid-induced vascular permeability changes [14]. However, the anti-inflammatory effect and the mechanisms of GE on AA rats remain unclear. GE was extracted and purified from GJ by means of solvent extraction and column chromatography. The structure was identified by physicochemical properties and spectroscopic analysis, and the content was determined by Ultra-Performance Liquid Chromatography (UPLC).
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2.2. Materials Mouse anti-JNK monoclonal antibody (D-2, sc-7354) and mouse anti-p-JNK monoclonal antibody (G-7, sc-6254) were obtained from Santa Cruz Biotechnology, Inc. Mouse anti-beta actin monoclonal antibody (TA-09) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (2B-2305) were purchased from ZSGB-BIO. IL-6, IL-17, IL-4 and TGF-β1 ELISA kits were supplied from Elabscience Biotechnology Co., Ltd. The following reagents were offered commercially: Concanavalin A (ConA, stored at − 20 °C), lectin from Phaseolus vulgaris-phytohemagglutinin (PHA-P, stored at 2–8 °C) and 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) from Sigma Chemical Co. (St. Louis, MO, USA); Dulbecco's modified Eagle's medium (DMEM) from Thermo Scientific Co. (USA). Other chemicals used in these experiments were analytical grade from commercial sources. Fig. 1. Structure of Geniposide (formula C17H24O11, molecular weight 404.36).
2.3. Drugs
Total glucosides of paeony (TGP), as the positive control drug, were used for the treatment of RA. The c-Jun N-terminal kinases (JNKs), which belong to the mitogenactivated protein kinase (MAPK) family, play vital roles in the production of cytokines and the degradation of extracellular matrix by regulating matrix metalloproteinase (MMP) gene expression in fibroblast-like synoviocytes (FLSs) and mediating joint destruction in AA rats [15,16]. The JNK signal pathway could be activated by cytokines, growth factors, stresses, and so on. Many studies suggested that the JNK signal pathway plays a pivotal role in cell differentiation, apoptosis, initiation and progress of considerable human diseases as well as in animal models, including ischemia/reperfusion (I/R), RA, diabetes mellitus, and tumor [17,18]. Experience gathered has confirmed that IL-17 could synergize with local inflammatory mediator IL-6 to induce the expression of IL-6 by a transcriptional mechanism that involves JNK signal pathway, and blockage of JNK pathway with JNK inhibitor II could partially inhibit the synergistic effect of IL-17 and IL-6 on the expression of IL-6 [19]. Additionally, it has been shown that the transcription factor Eomesodermin (Eomes), whose expression could substantially suppress Th17 cell induction in primary T cells, was suppressed by TGF-β1 via the JNK-c-Jun signal pathway [20]. We therefore wondered whether the balance of Th17/Treg could be associated with the JNK signal pathway. In this study, we discussed the role of GE in rectifying Th17/Treg balance by inhibiting excessive phosphorylation of JNK. Here, we evaluated the effects and mechanisms of GE on immune balance of Th17/Treg at levels of organ, molecule, and protein, respectively. Our results showed that GE could inhibit the progressive inflammation by inducing Th17 cells tolerance and enhancing Treg-driving effects, suggesting that GE has a significant therapeutic potential in treating autoimmune and other chronic inflammatory disorders. This may provide useful information for both clinical therapy and basic scientific research in RA.
GE is a yellow powder with N94.6% purity (determined by UPLC, ACQuity H-CLASS, Waters Co., USA), which was provided by the Chemistry Lab of Anhui University of Chinese Medicine (Hefei, Anhui Province, China). Standard of TGP (purity N 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). GE and TGP were suspended in water before use. 2.4. Induction of AA AA was induced as previously described [21]. Briefly, rats were immunized on day 0 by a single intradermal injection into the left hind paw with 100 μL of FCA for each rat. 2.5. Treatment of AA Before the onset of arthritis, rats were divided into six groups randomly, in which the AA rats were given intragastrically GE (30, 60, 120 mg/kg) and TGP (50 mg/kg) from days 18 to 24 after immunizations. While in groups of normal and AA model, rats were given an equal volume of water at the same time. 2.6. Arthritis assessment Rats were assessed daily for signs of arthritis by two independent observers who were blinded to the experimental design. Non-injected hind paw volume was determined with YLS-7A volume meter (Shandong Academy of Medical Sciences Equipment Station, China). The severity of arthritis in each paw was graded on a scale of 0–4: 0, no swelling; 1, swelling of finger joints; 2, swelling of phalanx joint and digits; 3, severe swelling of the entire paws; 4, deformity or ankylosis. The maximum arthritis score was 12 including three secondary arthritis paws for each rat [22,23].
2. Materials and methods
2.7. Lymphocyte proliferation assay by MTT
2.1. Animals
MLNs and peripheral blood were removed in sterile condition. MLNL and PBL were isolated by routine method. Then, the cells were cultured in triplicate in a concentration of 1 × 1010 cell/L in 100 μL DMEM containing 10% FBS. First, the cells were stimulated with 5 mg/L ConA for 48 h at 37 °C and 5% of CO2. A 10 μL sample of MTT (5 g/L) was added before the end of stimulation for 4 h and then the cultures were stimulated for 4 h continuously. After incubation, the cultures were centrifuged (760 × g, 10 min) and the supernatants were discarded. A 150 μL of dimethylsulfoxide (DMSO) was added to each well and the absorbance (A) was examined at 490 nm using a MSS ELISA Microwell Reader (Thermo Scientific Co., USA).
Sprague–Dawley (SD) rats (♂, 180 ± 20 g. Grade II, Certificate No. 011) were purchased from the Animal Department of Anhui University of Chinese Medicine (Hefei, Anhui Province, China). All animals were housed under specific pathogen-free conditions with a 12-hour light/dark cycle in a temperature-controlled room at 23 °C (±1) and allowed food and water ad libitum in each group of no more than six. All experiments using rats were performed in accordance with protocols approved by the Ethics Review Committee for Animal Experimentation of Anhui University of Chinese Medicine.
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Levels of IL-6, IL-17, IL-4 and TGF-β1 in MLNL and PBL were evaluated using ELISA kits in accordance with the manufacturer's instructions. 2.9. Western blot Frozen cells were lysed in buffer and the lysates were separated by 10% sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat dry milk in Trisbuffered saline/Tween 20 (TBST) at room temperature for 3 h. Primary antibodies specific for JNK and p-JNK were incubated with the membranes overnight at 4 °C. Membranes were washed with phosphatebuffered saline (PBS) and incubated with the secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-mouse (1:1000) or goat anti-mouse (1:5000) IgG. Immunoreactive proteins were detected with 3, 3′-diaminobenzidine tetrahydrochloride (DAB). All the experiments were performed three times and the results were reproducible. 2.10. Histological analysis The MLNs of rats were removed under sterile conditions, fixed with 10% paraformaldehyde in PBS, and then embedded in paraffin for histological analysis. A series of paraffin sections (5 μm) were stained with hematoxylin and eosin (HE, 200×) and examined by two experienced pathologists in total darkness. 2.11. Statistical analysis Data were expressed as mean ± standard error of the mean (S.E.M.), in triplicate. One-way ANOVA test and Student's t -test (SPSS17.0 Software Products, Chicago, IL, USA) were used to determine significant differences between groups. The histopathologic analysis was analyzed by Ridit procedure. In all tests, P-value b 0.05 was considered statistically significant. 3. Results 3.1. Effect of GE on secondary arthritis in AA rats The onset and peak of inflammatory reaction occurred on day 14 and days 18–21 after immunization, respectively. There was a remarkable secondary inflammatory response in the model, which accompanied with paw swelling, pain, polyarthritis. The administration of GE (30, 60, 120 mg/kg) significantly suppressed the hind paw swelling (P b 0.01) (Fig. 2A). There was no significant reduction in polyarthritis index by treatment with GE at 30 mg/kg (P N 0.05), while a significant decrease in polyarthritis index was observed when the AA rats were treated with GE at 60 and 120 mg/kg (P b 0.01). The efficacy was similar to that of TGP (50 mg/kg) (Fig. 2B). 3.2. Effect of GE on lymphocyte proliferation of MLNL and PBL in AA rats As shown in Fig. 3A, the proliferation of MLNL in AA group decreased significantly compared with normal group. GE (60 and 120 mg/kg) could apparently increase the cell proliferation in AA. TGP (50 mg/kg) had similar effects as GE. However, the results of PBL were just the reverse of that of MLNL (Fig. 3B). 3.3. Effect of GE on production of IL-6, IL-17, IL-4 and TGF-β1 in AA rats The cytokines of AA rats were determined on day 24 after immunization. The production of IL-6 and IL-17 from MLNL and PBL was
A 0.9
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day Fig. 2. Effect of GE on secondary arthritis in AA rats. Rats were immunized on day 0 by a single intradermal injection into the left hind paw with 100 μL of FCA for each rat. GE (30, 60, 120 mg/kg) and TGP (50 mg/kg) were given intragastrically to AA rats from days 18 to 24 after immunization. The paw swelling and polyarthritis were evaluated as described in Materials and methods. A: paw swelling and B: arthritis index. Values are presented as means ± S.E.M. from 6 animals in each group. ##P b 0.01 vs. Normal group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. AA model.
increased, while that of IL-4 and TGF-β1 from MLNL and PBL was decreased when compared with normal group. GE at 30 mg/kg had no effect on the production of IL-17 and TGF-β1 (P N 0.05), while GE at 60, 120 mg/kg dramatically diminished the production of IL-17 and IL-6, and elevated the production of IL-4 and TGF-β1 from MLNL and PBL (P b 0.01), a result consistent with that from TGP (50 mg/kg) (Fig. 4).
3.4. Effect of GE on expression of JNK, p-JNK and β-actin of MLNL and PBL in AA rats The protein expression of JNK, p-JNK and β-actin of MLNL and PBL in AA rats were estimated on day 24 after immunization. It was found that the expression of p-JNK was heightened in AA rats when compared with normal group (P b 0.01). The administration of GE (30, 60, 120 mg/kg) markedly decreased that of p-JNK (P b 0.01), while the protein levels of total JNK had no change (Fig. 5).
3.5. Effect of GE on histopathology of MLNs in AA rats Rats were sacrificed on day 24 after immunization and subjected to histopathological examination. The hyperplasia of lymphatic follicle and the increased number of inflammatory cells were observed in MLN of AA rats. Treatment with GE at 120 mg/kg evidently reduced hyperplastic lymphatic follicle and the infiltration of inflammatory cells in AA rats (Fig. 6).
M.-M. Dai et al. / International Immunopharmacology 20 (2014) 46–53
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Group Fig. 3. Effect of GE on lymphocyte proliferation of MLNL and PBL in AA rats. MLNs and peripheral blood were removed in sterile condition. MLNL and PBL were isolated by routine method. Then, the cells were cultured in triplicate in a concentration of 1 × 1010 cell/L in 100 μL DMEM containing 10% FBS. The cells were stimulated with 5 mg/L ConA for 48 h at 37 °C and 5% of CO2. Lymphocyte proliferation was determined by MTT assay. A: the proliferation of MLNL and B: the proliferation of PBL. Values are presented as means ± S.E.M. from 6 animals in each group. ##P b 0.01 vs. Normal group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. AA model.
4. Discussion RA is a common autoimmune disease, which is characterized by joint swelling and pain, joint stiffness, deformity and serious functional damage. AA could act as an experimental model in our study to demonstrate the effects of GE on human RA because of its similarity to human RA in both clinical and histopathologic features. As shown in Fig. 2, the present study showed that GE significantly relieved the secondary hind paw swelling. GE at a moderate or high dose markedly reduced polyarthritis index, indicating a protective effect on FCA-induced injury in vivo, while GE at low dose did not show a significant inhibitory effect on polyarthritis index in AA rats. Several possibilities could account for this failure. For example, low dose of GE could not be high enough for attenuating polyarthritis index. Or, the treatment time was too short to see improvement in AA rats treated with GE at low dose. If we prolong the time, it could have a dose-dependent effect and time-dependent effect. Besides, it was found that GE improved histological status of
MLNs in AA rats. These findings indicated that GE could alleviate the extent of arthritis in AA rats with FCA-induced. AA was closely related to the hyperfunction of immune function. GE could reduce the proliferation of PBL in AA rats, suggesting that GE could get the immune function back to normal levels by acting on immune cells, so as to relieve AA. Interestingly, in the intestinal mucosal immune system, the proliferation of MLNL was inhibited by AA, while GE could restore and even enhance the proliferation activity of MLNL. This hinted that the effect of GE on AA may also be played by the mucosal immune tolerance mechanisms. Scilicet, in the intestinal immune system, GE stimulated the mucosal immune system and then the intestinal mucosal immunity was enhanced, but the immune response of lesions was suppressed. The balance was skewed toward Th17, resulting in excessive production of IL-17, TNF-α and IL-6, whereas Treg activity and the level of TGF-β1 were deficient in RA patients compared to healthy individuals [24]. One study supported the concept that there was a reciprocal
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Fig. 4. Effect of GE on production of IL-6, IL-17, IL-4 and TGF-β1 in AA rats. MLNL and PBL were collected on day 24 after immunization. Levels of IL-6, IL-17, IL-4 and TGF-β1 were measured by ELISA. A: Levels of IL-6, IL-17, IL-4 and TGF-β1 in MLNL, B: Levels of IL-6, IL-17, IL-4 and TGF-β1 in PBL. Values are presented as means ± S.E.M. from 6 animals in each group. ##P b 0.01 vs. Normal group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. AA model.
relationship between pathogenic Th17 and protective Treg in both developmental pathways and function in the immune system depending on the state of the innate immune system as well as the production of IL-6 [25]. Recent researches have demonstrated that IL-6 was a key factor in determining the balance of Th17/Treg. IL-6 could induce the differentiation of Th17 cells from naive T cells in the presence of TGF-β1; on the contrary, IL-6 could inhibit TGF-β1-induced Treg cell differentiation. Dysregulation or overproduction of IL-6 could result in autoimmune diseases such as systemic lupus erythematous (SLE) and RA, in which Th17 cells were considered to be the leading cause of pathology [26]. Besides, evidence indicated that TGF-β1 was required for the generation of Th17 and Treg. TGF-β1 could induce both anti-inflammatory Treg and pro-inflammatory Th17 depending on the local cytokine milieu. TGF-β1 alone could induce the differentiation of Treg from naive T cells in normal condition, while TGF-β1 could induce the
development of Th17 from naive T cells together with IL-6 in inflammatory state [27,28]. Veldhoen et al. [29] have also showed that the number of Th17 cells in TGF-β1-deficient mice was reduced, whereas increased in the presence of TGF-β1. In order to validate the suggestion that whether GE could revise the imbalance of Th17/Treg in AA rats, we determined the levels of IL-17, IL-6 and TGF-β1, which belong to Th17 and Treg cytokines, respectively. It was found that GE at low dose had no effect on the production of IL-17 and TGF-β1, while GE at moderate or high dose could remarkably decrease the production of IL-17 and IL-6, and increase the lowered level of TGF-β1 in AA rats. In all likelihood a low dose of GE was not sufficient to reduce the production of IL-17 and TGF-β1 and the effect could be dose-dependent. Therefore, we proposed an idea that GE in a dose-dependent manner could induce Th17 cell immune tolerance and give rise to a shift toward Treg cell-mediated antiinflammatory effects, which ultimately suppress the release of pro-
M.-M. Dai et al. / International Immunopharmacology 20 (2014) 46–53
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Fig. 5. Effect of GE on expression of JNK, p-JNK and β-actin of MLNL and PBL in AA rats. JNK, p-JNK and β-actin were assessed by Western blot on day 24 after immunization, as described in Materials and methods. A: Western blot for JNK, p-JNK and β-actin of MLNL and PBL in AA rats. B: Quantitative evaluation of JNK and p-JNK of MLNL in AA rats by densitometry from triplicate independent experiments. C: Quantitative evaluation of JNK and p-JNK of PBL in AA rats. ⁎⁎P b 0.01 vs. AA, ## P b 0.01 vs. Normal. 1–3: GE (30, 60 and 120 mg/kg); 4: TGP (50 mg/kg); 5: AA; 6: Normal.
inflammatory cytokines IL-17 and IL-6, and play a lasting inhibitory effect on AA rats, so as to correct the imbalance of Th17/Treg. Previous reports have demonstrated the importance of JNK pathway in Th1/Th2 balance as well as in Th17/Treg balance [30,31]. One study reported that JNK1-deficient mice were resistant to experimental autoimmune encephalitis (EAE). Furthermore, fewer Th17 cells were observed in immunized JNK1-deficient mice [32]. Another paper showed that the JNK inhibitor (SP600125) not only strongly inhibited the Th17 cell-mediated in vivo disease model known as EAE, but also
completely abrogated in vitro Th17 cell induction from primary T cells in a dose-dependent manner, indicating that the JNK signal pathway was essential for Th17 cells development both in vitro and in vivo [20]. In our experiment, as shown in Fig. 5, we observed that the level of p-JNK was markedly decreased, while that of total JNK remained unchanged in MLNL and PBL in AA rats after administration of GE. The results showed that GE could down-regulate the protein expression of p-JNK, and retrieved the balance between JNK and p-JNK. As previously described, the JNK signal pathway had a significant impact on
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M.-M. Dai et al. / International Immunopharmacology 20 (2014) 46–53
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Fig. 6. Histopathologic examination of MLN in AA rats. A: In normal rats, fewer and smaller lymphatic follicles and no inflammatory cells were observed in MLN. B: In AA rats, the hyperplastic lymphatic follicle and the infiltration of inflammatory cells were observed. C: In AA rats treated with GE 120 mg/kg, only a small extent of hyperplastic lymphatic follicle was observed (original magnification 400×).
determining the developmental direction of Th17 and Treg. Consequently, it is possible that GE could induce immune balance of Th17/ Treg of MLNL and PBL via inhibiting excessive phosphorylation of JNK in AA rats. To further clarify the mechanisms by which GE induces immune balance of Th17/Treg of MLNL and PBL, we retrospected the effects of HSPs on cellular immunity. HSPs, also called stress proteins, were a highly conserved group of cytoprotective proteins which were typically upregulated in response to a range of stress stimuli, including heat shock, growth factors, inflammation and infection [33]. HSPs could interact with various signaling proteins of signal transduction pathways that regulate growth and development [34–36]. HSP70, an important member of HSPs, has vital immunoregulatory properties and has also been proved to display an anti-inflammatory effect on RA by downregulating levels of IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 in FLSs, which was mediated via the suppression of MAPKs and nuclear factor-κB (NF-κB) signal pathways induced by TNF-α [37]. Besides, recent investigations demonstrated that the HSP70 could restrain AA, which might partly be due to induction of self-HSP70 cross-reactive T cells that were capable of diminishing inflammation [38,39]. Furthermore, Kim et al. [40] demonstrated that arsenic trioxide induced HSP70 expression by JNK pathway in MDA231 cells and SP600125 suppressed HSP70 promoter-driven reporter gene expression, suggesting that JNK could be preferentially related to the protective heat shock response against arsenic trioxide stress. It has also been reported that in the control of apoptosis, HSP70 could not only inhibit directly or indirectly the activity of JNK, but also bind to JNK as a natural inhibitory protein in case of its phosphorylation, and thereby inhibit JNK-mediated apoptosis [41]. These results indicated that the activation of JNK signal pathway could enhance HSP70 expression; on the other hand, the high expression of HSP70 could suppress JNK activity. Works on experimental and human arthritis have presented that HSP70 could regulate autoimmunity indirectly by activating protective Treg cells that control pathogenic T cells specific for self-antigens, and HSP70 has also been identified as an immune target in RA [39]. Therefore, it's quite plausible that GE induces Th17 cell immune tolerance and enhances the activity of Treg cells through inducing the high expression of HSP70 and thereby inhibiting the phosphorylation of JNK. In conclusion, we have demonstrated the effects and mechanisms of GE on AA rats. GE induced Th17 cell immune tolerance and enhanced Treg cell-driving effects by decreasing pro-inflammatory cytokines IL-17 and IL-6 and increasing anti-inflammatory cytokine TGF-β1 . Furthermore, the fact that GE ameliorated the expression of p-JNK in MLNL and PBL indicated that GE could induce immune balance of Th17/Treg by suppressing over expression of p-JNK in AA rats. Therefore, this study provides more comprehensive evidences supporting GE as a potential alternative agent for the autoimmune disease, especially RA.
Further studies are needed to fully understand the details of the mechanism for the role of GE in RA.
Acknowledgments This project was financially supported by the National Natural Science Foundation of China (No. 81073122), the Anhui Provincial Natural Science Research Project in Colleges and Universities (KJ2009A045Z) and the youth of Natural Science Foundation of Anhui University of Chinese Medicine (qn201308).
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