- Email: [email protected]
GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling pathway through the α7 nicotinic acetylcholine receptor
INTIMP-03909; No of Pages 9 International Immunopharmacology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling pathway through the α7 nicotinic acetylcholine receptor Ye Yue a,1, Ruoxi Liu a,b,1, Wenxiang Cheng a, Yiping Hu a, Jinchao Li a, Xiaohua Pan c, Jiang Peng b,⁎, Peng Zhang a,⁎ a Shenzhen Bioactive Materials Engineering Key Lab for Medicine, Translational Medicine R&D Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China b Institute of Orthopedics, Chinese PLA General Hospital, Fuxing 28# Road, Beijing, China c The Department of Orthopedics, The Second Clinical Medical College, Jinan University, Shenzhen, Guangdong, China
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 16 September 2015 Accepted 5 October 2015 Available online xxxx Keywords: GTS-21 RAW264.7 cells Inflammation NF-κB Akt α7 nicotinic acetylcholine receptor
a b s t r a c t Objective: GTS-21, a selective α7 nicotinic acetylcholine receptor agonist, has recently been established as a promising treatment for inflammation. However, the detailed molecular mechanism of GTS-21 in suppressing pro-inflammatory cytokine production is only partially explored. The study aimed to analyze cytokine expression suppressed by GTS-21 with lipopolysaccharide (LPS)-induced inflammation in vitro and to gain insights into the role of Akt/NF-κB signaling pathway in this process. Materials and methods: Cell Counting Kit-8 (CCK-8) assay was performed to detect drug cytotoxicity. RAW 264.7 cells were stimulated with LPS and treated with GTS-21. Interleukin (IL)-1β, IL-6, or tumor necrosis factor (TNF)α production was detected using enzyme-linked immunosorbent assay. Western blot was used to assess the expression patterns of signal transduction protein. Nuclear translocation of nuclear factor (NF)-κB was analyzed by confocal fluorescence microscopy. In addition, α7 nicotinic acetylcholine receptors (α7 nAChR) were detected on RAW264.7, and the α7 nAChR-specific antagonist was adopted to verify whether the effect of GTS-21 was mediated by α7 nAChR. Results: The CCK-8 assay showed that GTS-21 did not significantly affect cell proliferation. The production of IL1β, IL-6, and TNF-α decreased after being treated with GTS-21 in LPS-stimulated RAW 264.7 cells. GTS-21 also suppressed LPS-induced phosphorylation of NF-κBp65, IKKα/β, IκBα, and Akt, as well as NF-κB p65 nuclear translocation. Moreover, α7 nAChR was expressed on the surfaces of RAW264.7 cells, and the α7 nAChRspecific antagonist almost completely prohibited the inhibitory effect of GTS-21 on NF-κB activation. Conclusion: These findings indicate that GTS-21 suppresses LPS-induced inflammation by inhibiting the Akt/NFκB signal pathway through α7 nAChR. GTS-21 has a potential application in inflammatory disease therapy. © 2015 Published by Elsevier B.V.
1. Introduction A novel link between the vagus nerve and inflammatory responses was determined in past decades [1,2]. Recently, the efferent vagus nerve has been demonstrated to inhibit inflammatory responses and to attenuate the development of endotoxin-induced shock in rodents; these actions are called “the cholinergic anti-inflammatory pathway” [3]. α7 nAChR, expressed on the surfaces of multiple inflammatory cells, including mononuclear macrophages, T lymphocytes, B lymphocytes, and dendritic cells, and fibroblast-like synoviocytes [4–6] was reported to significantly influence the anti-inflammatory effect of the ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Peng), [email protected] (P. Zhang). 1 These two authors contribute equally as the first authors.
vagus nerve [7]. This evidence implies that employing cholinergic agonists could be a potential approach in dealing with inflammation. GTS-21 (3-(2,4-dimethoxybenzylidene)-anabaseine), one of the most potent α7nAChR agonists, has been reported to attenuate proinflammatory cytokine production [8], improve outcomes in sepsis models [9], pancreatitis [10], and ischemia–reperfusion injury [11], and inhibit the production of endotoxin-induced TNF in lung tissue [12]. In addition, recent studies have demonstrated that GTS-21 inhibits the activities of endothelial cells and monocyte macrophages, as well as the secretion of pro-inflammatory cytokines in peripheral blood samples, by regulating the JAK2-STAT3 pathway [13–15]. Moreover, cholinergic receptor agonists such as AR-R17779 and nicotine have positive therapeutic effects on rheumatoid arthritis (RA) [16,17]. GTS-21, which is being investigated for the treatment of Alzheimer's disease [18] and schizophrenia [19], has been proven safe for humans in clinical
http://dx.doi.org/10.1016/j.intimp.2015.10.005 1567-5769/© 2015 Published by Elsevier B.V.
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
2
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
trials. At present, GTS-21 has gained increased popularity and is considered a potential therapeutic agent for inflammatory disorders. However, the detailed molecular mechanisms of the anti-inflammatory effects of GTS-21 are still limited. RA, a systemic autoimmune disease pathologically characterized by chronic inflammation and joint destruction, approximately affects 1% of the global population [20,21]. Although several treatment protocols with certain alleviatory effects are clinically available, RA treatments still need to be improved. Our previous in-vivo study was performed to observe the effect of GTS-21 on collagen-induced arthritis (CIA) in rats [22]. The results showed that the levels of TNF-α, IL-1β, and IL-6 in the serum were significantly reduced after GTS-21 management, and bone degradation was inhibited as well. The pathogenesis of RA is a complex process involving many cell types, including T cells, B cells, and macrophages. Considering the continuous exploration of the antiinflammatory molecular mechanism of GTS-21 at the cell level in RA, we chose macrophage RAW264.7 cell line as our research object. In this study, we explored whether GTS-21 attenuated lipopolysaccharide (LPS)-induced inflammatory cytokine production in RAW264.7 cells and to gain insights into the role of Akt/NF-κB in inflammation to assess its application prospect in RA. 2. Materials and methods 2.1. Cell culture RAW264.7 cells were obtained from the American Type Culture Collection (ATCC TIB-71). The cells were maintained in Dulbecco's modified Eagle's medium (Thermo Fisher, USA) and supplemented with 10% fetal bovine serum (Gibco, USA) and penicillin/streptomycin (1:100, Sigma, St. Louis, MO) in a humidified atmosphere of 95% air and 5% CO2 incubator at 37 °C.
(Cambridge, UK). The antibody of β-actin was purchased from EarthOx (San Francisco, CA, USA). Phosphoinositide-3 kinase (PI3K)/Akt inhibitor LY294002 was obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.5. Immunocytochemistry The nuclei translocation of NF-κB p65 was assessed using confocal fluorescence microscopy. RAW 264.7 cells, which were grown on glass coverslips, were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS), stained with anti-NF-κB p65 antibody (diluted 1:100) for 1 h at room temperature, and incubated with TRITC-conjugated second antibody (Santa Cruz, USA) for 1 h at room temperature. After washing with PBS, the nuclei were stained for 3 min with the fluorescent dye 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). The stained cells were analyzed using confocal fluorescence microscopy (Leica TCS SP5-II). Immunofluorescence assay was used to detect the expression of α7nAChR on the surfaces of RAW264.7 cells using an anti-α7 nAChR antibody or a specific α7 nAChR antagonist. Cells grown on chambered coverglass were fixed with freshly prepared 40 g/L paraformaldehyde in PBS for 30 min at room temperature, washed twice with cold PBS, and blocked with 1% BSA for 30 min at room temperature. The cells were incubated first in the diluted anti-α7-nAChR antibody (Abcam, UK) overnight at 4 °C and with the TRITC-conjugated secondary antibodies (BOSTER, China) for 1 h at room temperature with three washes after each incubation period. The stained cells were analyzed using fluorescence microscopy (Olympus, IX71). To confirm the antibody test results, the α7-nAChR-specific antagonist α-bungarotoxin (Bgt) was used for further identification. After fixing, RAW264.7 cells were incubated with tetramethylrhodamine-α-Bgt (Sigma, USA) for 1 h at room temperature and observed using fluorescence microscopy. 2.6. Statistical analysis
2.2. Cell viability assay (CCK-8 assay) The viability of RAW 264.7 cells was determined by the CCK-8 assay (Dojindo, Japan). Cells were treated with specific concentrations of GTS21 (5 μM to 100 μM; the compound was purchased from Abcam (Cambridge, UK)) for 72 h. CCK-8 reagent was added and incubated at 37 °C for 2.5 h. The absorbance of CCK-8 was detected at 450 nm by a microplate reader (BioTek Synergy 4, USA). The viability of the cells was expressed as the fraction of surviving cells relative to untreated controls.
All quantitative data were expressed as means ± standard deviation from at least three independent experiments. Statistical analysis was conducted by one-way ANOVA, followed by Dunnett's t-test. All groups were tested against a control group as reference using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). A difference is considered significant when P b 0.05. 3. Results
2.3. Enzyme-linked immunosorbent assay (ELISA)
3.1. Cytotoxicity of GTS-21 in RAW 264.7 cells
RAW 264.7 cells were pretreated with various concentrations of GTS-21 for 2 h and incubated for another 22 h with or without stimulation by 100 ng/mL of LPS (Sigma-Aldrich, USA). The culture medium was collected, and the cells were lysed by lysis buffer (Thermo Scientific, USA). Protein concentration was measured by BCA Protein Assay Kit (Thermo Scientific, USA). Concentrations of the cytokines were determined by ELISA (eBioscience, USA).
CCK-8 assay was performed to investigate the cytotoxicity of GTS21. No cytotoxicity was observed when RAW264.7 cells were exposed to GTS-21 (5 μM to 100 μM) for 72 h (Fig. 1). We decided to set the highest concentration of GTS-21 to 100 μM for the following study.
2.4. Western blot Cells were lysed by lysis buffer (Thermo Scientific, USA), and the protein concentration was measured using BCA Protein Assay Kit (Thermo Scientific, USA). A total of 40 μg of protein from each sample was separated by 10% SDS-PAGE and transferred to a PVDF membrane. After incubating the protein with specific antibodies overnight at 4 °C and with the secondary antibody, antibody binding was detected with the enhanced ECL detection system (Millipore, USA). The antibodies against phospho-Akt (Ser473), phospho-IKKα/β (Ser176/180), phospho-IκBα (Ser32), NF-κB p65, and phospho-NF-κB p65 (Ser536) were purchased from Cell Signaling Technology (Beverly, MA, USA), and the antibody for Akt1 (Y89) was purchased from Abcam
3.2. Effects of GTS-21 on LPS-induced pro-inflammatory cytokine production To determine the inhibitory action of GTS-21 on the proinflammatory cytokines in LPS-stimulated RAW264.7 cells, the levels of IL-1β, IL-6, or TNF-α expression were analyzed by ELISA. As shown in Fig. 2, the production of IL-1β, IL-6, and TNF-α was significantly increased by the stimulation with LPS. Pretreatment with GTS-21 reduced the release of IL-6 in LPS-stimulated cells in a concentration-dependent manner. However, the dose-dependent attenuation of IL-1β and TNF-α secretion was relatively weaker. 3.3. GTS-21 suppressed NF-κB activation in LPS-stimulated RAW 264.7 cells NF-κB is a transcriptional regulator that significantly affects inflammatory response. Cells were pretreated with or without GTS-21 for
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
3
Fig. 1. Cytotoxicity of GTS-21 in RAW 264.7 cells. RAW 264.7 cells were pretreated with different concentrations of GTS-21 (5, 10, 20, and 100 μM) for 72 h. The viability of the cells was determined using CCK-8 assay, and no cytotoxicity was observed. The data are presented as the means ± standard deviation from three independent experiments.
2 h and stimulated with LPS (100 ng/mL) in the indicated times. As shown in Fig. 3A, GTS-21 substantially inhibited the enhanced phosphorylation of Ser536 of NF-κB p65 stimulated by LPS in different times. Furthermore, GTS-21 inhibited the phosphorylation of Ser536 of NF-κB p65 dose-dependently at 30 min (Fig. 3B). We used immunocytochemistry to examine the effect of GTS-21 on the nuclear translocation of phospho(p)-NF-κB p65 stimulated by LPS. Treatment with GTS21 (100 μM) evidently inhibited the LPS-induced NF-κB p65 nuclear translocation (Fig. 3C). NF-κB is associated with IκB protein, which inhibits NF-κB activity, in the cytoplasm as an inactive NF-κB–IκB complex. IKK is an important upstream kinase for the phosphorylation of IκB. Therefore, we examined p-IKKα/β and p-IκBα by Western blot analysis. As expected, LPS (100 ng/mL) induced IKKα/β and IκBα phosphorylation. GTS-21 dramatically blocked these effects at different time points and in a dose-dependent manner at 30 min (Fig. 3A and B). 3.4. GTS-21 inhibited Akt activation in LPS-stimulated RAW 264.7 cells PI3K/Akt pathway was determined to be involved in the LPS activation of signaling pathways and the expression of inflammatory cytokines in RAW 264.7 cells [23]. We examined the effects of GTS-21 on the LPS-induced phosphorylation of Akt in the cells. As shown in Fig. 4A–B, GTS-21 significantly inhibited the LPS-induced-phosphorylation of Akt in Ser473 at different time points and in a dose-dependent manner at 30 min. 3.5. Akt pathway was involved in NF-κB activation stimulated by LPS in RAW 264.7 cells Evidence shows that multiple signaling pathways are involved in the regulation of NF-κB activation and a complex crosstalk exists among them, including PI3K/Akt, PKC/MAPK, STAT5 [24–26]. In our study, in order to investigate the relationship between PI3K/Akt pathway and NF-κB, the cells were pretreated with 20 μM PI3K/Akt inhibitor LY294002 for 1 h and exposed to LPS (100 ng/mL). LY294002 pretreatment blocked the increased p-Akt, p-IKK, and p-NF-κB p65 levels by LPS stimulation (100 ng/mL) (Fig. 5A). In addition, LY294002 significantly suppressed the pro-inflammatory cytokine production in LPSstimulated RAW 264.7 cells (Fig. 5B).
Fig. 2. Effects of GTS-21 on LPS-induced pro-inflammatory cytokine production. RAW264.7 cells were pretreated with and without GTS-21 (5, 10, 20, and 100 μM) for 2 h and stimulated with LPS (100 ng/mL). The control group was not subject to any stimulus or GTS-21. After 22 h, the levels of interleukin (IL)-1β, IL-6, or tumor necrosis factor (TNF)-α were measured in the culture medium by ELISA. *P b 0.05 and **P b 0.01 versus the group with LPS treatment alone. The data are presented as the means ± standard deviation from three independent experiments.
pheochromocytoma cells were provided by Prof. Liping-Wang from the Neural Engineering Research Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences) are known to express α7 nAChR at high levels [28,29], and the cells were used as the positive control in the test. RAW264.7 cells and human rheumatoid fibroblast-like synoviocyte line MH7A cells were detected by immunofluorescence assay using the antibody specific for α7 nAChR. The results showed that RAW264.7 cells were covered with the red fluorescence of α7nAChR (Fig. 6A). Subsequently, we chose the a7 nAChR selective antagonist a-Bgt labeled with tetramethylrhodamine to further confirm that the positive signals in RAW264.7 cells represented the α7 subunit. Consistent with the previous result, the immunofluorescence images showed that RAW264.7 cells were similarly covered with the red fluorescence (Fig. 6B). However, no significant positive results were observed in both experiments in MH7A cells (Fig. 6A and B). 3.7. α7 nAChR selective antagonist α-Bgt inhibited the negative regulation of GTS-21 on NF-κB activation in RAW264.7 cells
3.6. α7 nAChR is expressed on RAW264.7 In recent years, α7 nAChR has been considered a potential therapeutic target for inflammatory diseases [5,27]. GTS-21 acts as an agonist at neural nicotinic acetylcholine receptors. To assess whether the antiinflammation effect of GTS-21 was caused by α7 nAChR, we first examined α7 nAChR expression on RAW264.7. PC12 cells (rat
To determine whether α7 nAChR is required for the GTS-21 inhibition of NF-κB activation in RAW264.7, we chose α-Bgt, a strong and irreversible antagonist of nAChR with partial selectivity to α7 nAChR [30–32], to block α7 nAChR on RAW264.7 cells. These cells were pretreated for 30 min in the absence or presence of α-Bgt (10 nM or 100 nM) before adding 100 μM GTS-21. About 2 h later, cells were
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
4
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
5
Fig. 4. Effects of GTS-21 on Akt activation in RAW 264.7 cells. (A) RAW 264.7 cells were pretreated with GTS-21 (100 μM) for 2 h and incubated with LPS (100 ng/mL) for 15 min to 60 min (A) or with different concentrations of GTS-21 for 30 min (B). Akt and p-Akt levels were determined by Western blot analysis. Results are presented as means ± standard deviation of three independent experiments. *P b 0.05 and **P b 0.01 versus the group with LPS treatment alone.
Fig. 5. Involvement of Akt pathway in NF-κB activation and effects of inhibition of PI3k/Akt on pro-inflammatory cytokine production. (A) RAW 264.7 cells were pretreated with LY294002 (20 μM) or GTS-21 for 2 h and incubated with LPS (100 ng/mL) for 30 min. p-Akt, p-IKKα/β, p-IκBα, and p-NF-κB p65 levels were determined by Western blot analysis. (B) IL-1β, IL-6, and TNF-α production was determined in RAW 264.7 cells treated with LPS (100 ng/mL) with and without LY294002 (20 μM) for 24 h by ELISA. The data are presented as the means ± standard deviation from three independent experiments. **P b 0.01 versus the group with LPS treatment alone.
induced with 100 ng/mL LPS for 30 min. NF-κB activation in RAW264.7 cells was determined by Western blot assay and laser confocal imaging. As shown in Fig. 7A, the level of p-NF-κB p65 significantly increased in
the RAW264.7 cells exposed to α-Bgt (10 nM or 100 nM). Moreover, α-Bgt almost reversed the inhibitory effect of GTS-21 on the NF-κB p65 nuclear translocation (Fig. 7B).
Fig. 3. Effects of GTS-21 on NF-κB activation in RAW 264.7 cells. (A–B) RAW 264.7 cells were pretreated with and without GTS-21 (100 μM) for 2 h and incubated with LPS (100 ng/mL) for 15 min to 60 min (A) or with different concentrations of GTS-21 for 30 min (B). Phospho-IKKα/β (p-IKK-α/β), p-IκBα, and p-NF-κB p65 levels were determined by Western blot analysis. Results are presented as means ± standard deviation of three independent experiments. *P b 0.05 and **P b 0.01 versus the group with LPS treatment only. (C) After pretreatment with and without GTS-21 (100 μM) for 2 h, RAW 264.7 cells were incubated with LPS (100 ng/mL) for 30 min. The control group was not subject to any stimulus or GTS-21. The nuclei translocation of NF-κB p65 was assessed by confocal fluorescence microscopy. Cells were immunostained with NF-κB p65 antibody labeled with TRITC in red, and nuclei were stained with DAPI in blue. Bar, 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
6
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
Fig. 6. Analysis of α7 nAChR expressed in RAW264.7 cells. Expression of α7 nAChR on the cell surfaces of RAW264.7 cells. (A). Immunofluorescence analysis of RAW264.7 cells using an anti-α7 nAChR antibody in red. (B) Immunofluorescence analysis of RAW264.7 cells using tetramethylrhodamine-α-Bgt in red. PC12 cells and MH7A cells were used as positive and negative control respectively. Each middle image was a large version of the corresponding parts marked in red in top images. The nuclei were stained with DAPI in blue in bottom images. Bar, 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Discussion The discovery of the cholinergic anti-inflammatory pathway suggests a novel treatment method for RA therapy. GTS-21 is a selective α7nAChR agonist, and both in vitro and in vivo investigations suggest
that GTS-21 has immunomodulatory and anti-inflammatory effects. Although the health-promoting properties are not fully understood, these benefits have been mainly related to the interactions of GTS-21 with several cytokines and regulatory transcription factors. The inhibitory properties in these pathways are likely to significantly affect cellular
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
7
Fig. 7. α7 nAChR selective antagonist α-Bgt inhibited the effect of GTS-21 on NF-κB activation in RAW264.7 cells. NF-κB activation in RAW264.7 cells was determined by Western blot assay and laser confocal imaging. RAW264.7 cells were pre-treated for 30 min with and without α-Bgt (10 nM or 100 nM) before adding 100 μM GTS-21. After 2 h, these cells were induced in 100 ng/mL LPS for 30 min. (A) p-NF-κB p65 levels were determined by Western blot analysis. Results are presented as means ± standard deviation of three independent experiments. **P b 0.01 versus the group with GTS-21 and LPS treatment. (B) The nuclei translocation of NF-κB p65 was assessed by confocal fluorescence microscopy. Cells were immunostained with NF-κB p65 antibody labeled with TRITC in red. The nuclei were stained with DAPI in blue. Bar, 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
function by changing the phosphorylation state of target molecules and/ or modulating gene expressions. GTS-21 is a drug that has been used safely in humans for the treatment of Alzheimer's disease, nicotine dependence, and schizophrenia in clinical trials. This drug also has a rapid transformational application in anti-inflammation treatment. The mature manufacturing process for GTS-21 may greatly reduce the research cost of novel RA therapies. In our study, we explored the molecular mechanism of GTS-21 action against LPS-induced inflammation in RAW 264.7 cells. The results showed that GTS-21(100 μM) did not exhibit cytoxicity effects and significantly reduced the inflammatory mediators IL-1 β, IL-6, and TNF-α in LPS-activated RAW 264.7 cells. These findings suggest the marked anti-inflammatory effects of GTS-21 in LPS-stimulated RAW 264.7 cells, consistent with the reports from earlier studies. The present study demonstrated that GTS-21 significantly suppressed the NF-κB transcriptional activity for the first time by attenuating the LPS-induced IKKα/β, IκBα, p65 phosphorylation, and subsequent p65 nuclear translocation. NF-κB is known to significantly affect the regulation of the expressions of pro-inflammatory enzymes and cytokines, such as TNF-α and IL-6 [33]. NF-κB is represented mainly by the p65/p50 heterodimeric complex in most cell types. In unstimulated cells, NF-κB resides in the cytoplasm as an inactive NF-κB– IκBα complex. In the activation process, the inhibitory NF-κB kinase (IKK) is activated. IκBα, the inhibitory subunit, is rapidly phosphorylated in Ser32 and Ser36 residues by IKKα/β, subsequently ubiquitinated, and degraded by 26S proteasome complex; thus NF-κB is translocated to the nucleus and activates the transcription of various inflammatory gene products [34]. Therefore, GTS-21 significantly inhibited NF-κB transcriptional activity in RAW 264.7 cells by attenuating the LPS-induced IKK α/β, IκBα, and p65 phosphorylation and subsequent p65 nuclear translocation. Other studies have proved the involvement of the PI3K/Akt pathway in NF-κB transcriptional activation [35]. The PI3K/Akt pathway was
reported to significantly influence inflammation-mediated diseases, such as rhinosinusitis [36], RA [37], lung injury [38], and psoriasis [39]. Akt, a downstream protein of PI3K, is a serine kinase that is recruited to the plasma membrane in cells phosphorylated and activated with a variety of stimulants, including growth factors and cytokines [40–42]. Ojaniemi et al. found that the inhibition of PI3k/Akt decreased the LPS-induced transcriptional activity of NF-κB in RAW 264.7 cells [23]. Our results showed that the inhibition of PI3K/Akt by LY294002 could suppress the phosphorylation of IKKα/β, IκBα, and NF-κB-p65. LY294002 also inhibited the secretion of LPS-induced TNF-α, IL-1β, and IL-6 in RAW264.7. These results indicate a probable antiinflammation mechanism of GTS-21 underlying the suppression of the inducible activation of NF-κB by inhibiting the PI3K/Akt pathway. Previous studies have suggested that the α7 subunit of nAChR is essential for the so-called “cholinergic anti-inflammatory pathway” [43]. GTS-21 is a partial agonist at α7 nAChR, and our study showed that GTS-21exhibited good anti-inflammatory properties. To confirm the effectiveness of GTS-21 on RAW264.7 cells through α7 nAChR for GTS-21, we detected the expression of α7 nAChR and NF-κB activation after blocking this receptor in RAW264.7 cells. We found that α7 nAChR did express on the surface of RAW264.7 cells. α-bungarotoxin (α-Bgt) is an α7 nAChR-specific antagonist that can be used in distinguishing acetylcholine receptor types. Our study indicated that α-Bgt pretreatment almost prevented the inhibition of NF-κB activation and the nuclear translocation by GTS-21. This finding establishes that the GTS-21 function is a result of its combination with α7 nAChR. The main symptoms of RA are persistent inflammation and abnormal synovial hyperplasia with marked pannus formation, as well as the subsequent invasion and destruction of cartilage and bone with eventual bone tissue necrosis. As an important component of articular synovial tissues, fibroblast-like synoviocytes (FLS) are also crucial in perpetuating inflammation and promoting cartilage destruction [44]. Therefore, FLS has consequently gained significant attention in RA. FLS
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
8
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
and macrophage-like synoviocytes are two main effector cells in the joint synovial of RA patients. On the basis of these theories, we selected both the murine macrophage cell line RAW264.7 cells and the human rheumatoid fibroblast-like synoviocyte line MH7A cells to assess the anti-inflammatory prospect of GTS-21 in RA. However, our results did not indicate any significant anti-inflammatory effects of GTS-21 on MH7A cells or any significant changes in the anti-inflammatory signaling pathway mediators (data not shown). With uncertainties, we found that α7nAChR was not expressed in MH7A cells (Fig. 6A and B). Maybe that is the reason for the difference in the effect of GTS-21 between MH7A cells and RAW264.7 cells. 5. Conclusion Our previous in vivo study suggested that GTS-21 significantly reduced serum pro-inflammatory cytokine titers and bone degradation in rats with CIA [22]. In this article, our in vitro study provided new insight into the anti-inflammatory mechanisms of GTS-21 and demonstrated that GTS-21 downregulated the inflammatory cytokines by inhibiting the Akt activation. The subsequent IKKα/β mediated the IκBα phosphorylation and inhibited the NF-κB activation and gene expression in LPS-induced RAW 264.7 cells through α7nAChR. Our in vivo and in vitro studies may provide a basis for the use of GTS-21 for inflammatory disorders such as RA. Other effector cells such as endotheliocytes and other kinds of pro-inflammatory mediator production, such as nitric oxide and vascular endothelial growth factor, will be detected in further studies. Furthermore, we intend to explore the underlying mechanisms and other signaling pathways in the antiinflammatory effect of GTS-21 and evaluate its therapeutic potency in inflammatory disease. Conflict of interest The authors have no potential conflicts of interest to disclose. Acknowledgments This work was supported by the National Natural Science Foundation of China (81271952), the Science and Technology Project of Guangdong Province (2013B050800005), the Science and Technology Project of Shenzhen City (JCYJ20140610151856738), and the Shanghai Key Laboratory of Orthopedic Implants. References [1] M. Fleshner, L.E. Goehler, B.A. Schwartz, M. McGorry, D. Martin, S.F. Maier, et al., Thermogenic and corticosterone responses to intravenous cytokines (IL-1beta and TNF-alpha) are attenuated by subdiaphragmatic vagotomy, J. Neuroimmunol. 86 (1998) 134–141. [2] L.E. Goehler, C.R. Busch, N. Tartaglia, J. Relton, D. Sisk, S.F. Maier, et al., Blockade of cytokine induced conditioned taste aversion by subdiaphragmatic vagotomy: further evidence for vagal mediation of immune-brain communication, Neurosci. Lett. 185 (1995) 163–166. [3] L.V. Borovikova, S. Ivanova, M. Zhang, H. Yang, G.I. Botchkina, L.R. Watkins, et al., Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin, Nature 405 (2000) 458–462. [4] M.A. van Maanen, S.P. Stoof, E.P. van der Zanden, W.J. de Jonge, R.A. Janssen, D.F. Fischer, et al., The alpha7 nicotinic acetylcholine receptor on fibroblast-like synoviocytes and in synovial tissue from rheumatoid arthritis patients: a possible role for a key neurotransmitter in synovial inflammation, Arthritis Rheum. 60 (2009) 1272–1281. [5] J.M. Waldburger, D.L. Boyle, V.A. Pavlov, K.J. Tracey, G.S. Firestein, Acetylcholine regulation of synoviocyte cytokine expression by the alpha7 nicotinic receptor, Arthritis Rheum. 58 (2008) 3439–3449. [6] K.Z. Sato, T. Fujii, Y. Watanabe, S. Yamada, T. Ando, F. Kazuko, et al., Diversity of mRNA expression for muscarinic acetylcholine receptor subtypes and neuronal nicotinic acetylcholine receptor subunits in human mononuclear leukocytes and leukemic cell lines, Neurosci. Lett. 266 (1999) 17–20. [7] H. Wang, M. Yu, M. Ochani, C.A. Amella, M. Tanovic, S. Susarla, et al., Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation, Nature 421 (2003) 384–388.
[8] I.A. Giebelen, D.J. van Westerloo, G.J. LaRosa, A.F. de Vos, T. van der Poll, Stimulation of alpha 7 cholinergic receptors inhibits lipopolysaccharide-induced neutrophil recruitment by a tumor necrosis factor alpha-independent mechanism, Shock 27 (2007) 443–447. [9] V.A. Pavlov, M. Ochani, L.H. Yang, M. Gallowitsch-Puerta, K. Ochani, X. Lin, et al., Selective alpha7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis, Crit. Care Med. 35 (2007) 1139–1144. [10] D.J. van Westerloo, I.A. Giebelen, S. Florquin, M.J. Bruno, G.J. Larosa, L. Ulloa, et al., The vagus nerve and nicotinic receptors modulate experimental pancreatitis severity in mice, Gastroenterology 130 (2006) 1822–1830. [11] M.M. Yeboah, X. Xue, B. Duan, M. Ochani, K.J. Tracey, M. Susin, et al., Cholinergic agonists attenuate renal ischemia–reperfusion injury in rats, Kidney Int. 74 (2008) 62–69. [12] I.A. Giebelen, D.J. van Westerloo, G.J. LaRosa, A.F. de Vos, T. van der Poll, Local stimulation of alpha7 cholinergic receptors inhibits LPS-induced TNF-alpha release in the mouse lung, Shock 28 (2007) 700–703. [13] P.K. Chatterjee, Y. Al-Abed, B. Sherry, C.N. Metz, Cholinergic agonists regulate JAK2/ STAT3 signaling to suppress endothelial cell activation, Am. J. Physiol. Cell Physiol. 297 (2009) C1294–C1306. [14] M. Rosas-Ballina, R.S. Goldstein, M. Gallowitsch-Puerta, L. Yang, S.I. Valdes-Ferrer, N.B. Patel, et al., The selective alpha7 agonist GTS-21 attenuates cytokine production in human whole blood and human monocytes activated by ligands for TLR2, TLR3, TLR4, TLR9, and RAGE, Mol. Med. 15 (2009) 195–202. [15] M. Kox, J.F. van Velzen, J.C. Pompe, C.W. Hoedemaekers, J.G. van der Hoeven, P. Pickkers, GTS-21 inhibits pro-inflammatory cytokine release independent of the Toll-like receptor stimulated via a transcriptional mechanism involving JAK2 activation, Biochem. Pharmacol. 78 (2009) 863–872. [16] M.A. van Maanen, M.C. Lebre, T. van der Poll, G.J. LaRosa, D. Elbaum, M.J. Vervoordeldonk, et al., Stimulation of nicotinic acetylcholine receptors attenuates collagen-induced arthritis in mice, Arthritis Rheum. 60 (2009) 114–122. [17] T. Li, X. Zuo, Y. Zhou, Y. Wang, H. Zhuang, L. Zhang, et al., The vagus nerve and nicotinic receptors involve inhibition of HMGB1 release and early pro-inflammatory cytokines function in collagen-induced arthritis, J. Clin. Immunol. 30 (2010) 213–220. [18] W.R. Kem, The brain alpha 7 nicotinic receptor may be an important therapeutic target for the treatment of Alzheimer's disease: studies with DMXBA (GTS-21), Behav. Brain Res. 113 (2000) 169–181. [19] R. Freedman, A. Olincy, R.W. Buchanan, J.G. Harris, J.M. Gold, L. Johnson, et al., Initial phase 2 trial of a nicotinic agonist in schizophrenia, Am. J. Psychiatry 165 (2008) 1040–1047. [20] B. Jonsson, Patient access to rheumatoid arthritis treatments, Eur. J. Health Econ. 8 (Suppl. 2) (2008) S35–S38. [21] A. Morse, Services For People With Rheumatoid Arthritis International Comparisons, The National Audit Office, London, 2009. [22] Y. Hu, R. Liu, J. Li, Y. Yue, W. Cheng, P. Zhang, Attenuation of collagen-induced arthritis in rat by nicotinic alpha7 receptor partial agonist GTS-21, Biomed. Res. Int. 2014 (2014) 325875. [23] M. Ojaniemi, V. Glumoff, K. Harju, M. Liljeroos, K. Vuori, M. Hallman, Phosphatidylinositol 3-kinase is involved in Toll-like receptor 4-mediated cytokine expression in mouse macrophages, Eur. J. Immunol. 33 (2003) 597–605. [24] O.N. Ozes, L.D. Mayo, J.A. Gustin, S.R. Pfeffer, L.M. Pfeffer, D.B. Donner, NF-kappaB activation by tumour necrosis factor requires the Akt serine–threonine kinase, Nature 401 (1999) 82–85. [25] Z. Wei, F. Wang, J. Song, Q. Lu, P. Zhao, Y. Xia, et al., Norisoboldine inhibits the production of interleukin-6 in fibroblast-like synoviocytes from adjuvant arthritis rats through PKC/MAPK/NF-kappaB-p65/CREB pathways, J. Cell. Biochem. 113 (2012) 2785–2795. [26] Z.S. Nagy, M.J. LeBaron, J.A. Ross, A. Mitra, H. Rui, R.A. Kirken, STAT5 regulation of BCL10 parallels constitutive NFkappaB activation in lymphoid tumor cells, Mol. Cancer 8 (2009) 67. [27] M. Kox, J.C. Pompe, E. Peters, M. Vaneker, J.W. van der Laak, J.G. van der Hoeven, et al., alpha7 nicotinic acetylcholine receptor agonist GTS-21 attenuates ventilatorinduced tumour necrosis factor-alpha production and lung injury, Br. J. Anaesth. 107 (2011) 559–566. [28] H. Kawai, W. Zago, D.K. Berg, Nicotinic alpha 7 receptor clusters on hippocampal GABAergic neurons: regulation by synaptic activity and neurotrophins, J. Neurosci. 22 (2002) 7903–7912. [29] R.R. Jonnala, J.J. Buccafusco, Relationship between the increased cell surface alpha7 nicotinic receptor expression and neuroprotection induced by several nicotinic receptor agonists, J. Neurosci. Res. 66 (2001) 565–572. [30] M. Pohanka, Alpha7 nicotinic acetylcholine receptor is a target in pharmacology and toxicology, Int. J. Mol. Sci. 13 (2012) 2219–2238. [31] R. Doley, R.M. Kini, Protein complexes in snake venom, Cell. Mol. Life Sci. 66 (2009) 2851–2871. [32] A. Orr-Urtreger, F.M. Goldner, M. Saeki, I. Lorenzo, L. Goldberg, M. De Biasi, et al., Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alphabungarotoxin binding sites and hippocampal fast nicotinic currents, J. Neurosci. 17 (1997) 9165–9171. [33] P.A. Baeuerle, D. Baltimore, NF-kappa B: ten years after, Cell 87 (1996) 13–20. [34] S. Akira, K. Takeda, Toll-like receptor signalling, Nat. Rev. Immunol. 4 (2004) 499–511. [35] H.C. Dan, M.J. Cooper, P.C. Cogswell, J.A. Duncan, J.P. Ting, A.S. Baldwin, Aktdependent regulation of NF-{kappa}B is controlled by mTOR and Raptor in association with IKK, Genes Dev. 22 (2008) 1490–1500. [36] W. Chen, Z.J. Liu, J. Ye, Relationship among the expression of GSK3beta, PI3K/Akt, and IL-6 in chronic rhinosinusitis, Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 48 (2013) 128–134.
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx [37] M. Camps, T. Ruckle, H. Ji, V. Ardissone, F. Rintelen, J. Shaw, et al., Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis, Nat. Med. 11 (2005) 936–943. [38] Yang Y, Cheng Y, Lian QQ, Yang L, Qi W, Wu DR, et al. Contribution of CFTR to alveolar fluid clearance by lipoxin A4 via PI3K/Akt pathway in LPS-induced acute lung injury. Mediat. Inflamm. 2013;2013:862628. [39] M.P. Schon, W.H. Boehncke, Psoriasis, N. Engl. J. Med. 352 (2005) 1899–1912. [40] D. Stokoe, L.R. Stephens, T. Copeland, P.R. Gaffney, C.B. Reese, G.F. Painter, et al., Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B, Science 277 (1997) 567–570.
9
[41] A. Bellacosa, T.O. Chan, N.N. Ahmed, K. Datta, S. Malstrom, D. Stokoe, et al., Akt activation by growth factors is a multiple-step process: the role of the PH domain, Oncogene 17 (1998) 313–325. [42] A. Toker, L.C. Cantley, Signalling through the lipid products of phosphoinositide-3OH kinase, Nature 387 (1997) 673–676. [43] De Simone R, Ajmone-Cat MA, Carnevale D, Minghetti L. Activation of alpha7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures. J. Neuroinflammation 2005;2:4. [44] Bartok B, Firestein GS. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol. Rev. 2010;233:233–55.
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling pathway through the α7 nicotinic acetylcholine receptor Ye Yue a,1, Ruoxi Liu a,b,1, Wenxiang Cheng a, Yiping Hu a, Jinchao Li a, Xiaohua Pan c, Jiang Peng b,⁎, Peng Zhang a,⁎ a Shenzhen Bioactive Materials Engineering Key Lab for Medicine, Translational Medicine R&D Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China b Institute of Orthopedics, Chinese PLA General Hospital, Fuxing 28# Road, Beijing, China c The Department of Orthopedics, The Second Clinical Medical College, Jinan University, Shenzhen, Guangdong, China
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 16 September 2015 Accepted 5 October 2015 Available online xxxx Keywords: GTS-21 RAW264.7 cells Inflammation NF-κB Akt α7 nicotinic acetylcholine receptor
a b s t r a c t Objective: GTS-21, a selective α7 nicotinic acetylcholine receptor agonist, has recently been established as a promising treatment for inflammation. However, the detailed molecular mechanism of GTS-21 in suppressing pro-inflammatory cytokine production is only partially explored. The study aimed to analyze cytokine expression suppressed by GTS-21 with lipopolysaccharide (LPS)-induced inflammation in vitro and to gain insights into the role of Akt/NF-κB signaling pathway in this process. Materials and methods: Cell Counting Kit-8 (CCK-8) assay was performed to detect drug cytotoxicity. RAW 264.7 cells were stimulated with LPS and treated with GTS-21. Interleukin (IL)-1β, IL-6, or tumor necrosis factor (TNF)α production was detected using enzyme-linked immunosorbent assay. Western blot was used to assess the expression patterns of signal transduction protein. Nuclear translocation of nuclear factor (NF)-κB was analyzed by confocal fluorescence microscopy. In addition, α7 nicotinic acetylcholine receptors (α7 nAChR) were detected on RAW264.7, and the α7 nAChR-specific antagonist was adopted to verify whether the effect of GTS-21 was mediated by α7 nAChR. Results: The CCK-8 assay showed that GTS-21 did not significantly affect cell proliferation. The production of IL1β, IL-6, and TNF-α decreased after being treated with GTS-21 in LPS-stimulated RAW 264.7 cells. GTS-21 also suppressed LPS-induced phosphorylation of NF-κBp65, IKKα/β, IκBα, and Akt, as well as NF-κB p65 nuclear translocation. Moreover, α7 nAChR was expressed on the surfaces of RAW264.7 cells, and the α7 nAChRspecific antagonist almost completely prohibited the inhibitory effect of GTS-21 on NF-κB activation. Conclusion: These findings indicate that GTS-21 suppresses LPS-induced inflammation by inhibiting the Akt/NFκB signal pathway through α7 nAChR. GTS-21 has a potential application in inflammatory disease therapy. © 2015 Published by Elsevier B.V.
1. Introduction A novel link between the vagus nerve and inflammatory responses was determined in past decades [1,2]. Recently, the efferent vagus nerve has been demonstrated to inhibit inflammatory responses and to attenuate the development of endotoxin-induced shock in rodents; these actions are called “the cholinergic anti-inflammatory pathway” [3]. α7 nAChR, expressed on the surfaces of multiple inflammatory cells, including mononuclear macrophages, T lymphocytes, B lymphocytes, and dendritic cells, and fibroblast-like synoviocytes [4–6] was reported to significantly influence the anti-inflammatory effect of the ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Peng), [email protected] (P. Zhang). 1 These two authors contribute equally as the first authors.
vagus nerve [7]. This evidence implies that employing cholinergic agonists could be a potential approach in dealing with inflammation. GTS-21 (3-(2,4-dimethoxybenzylidene)-anabaseine), one of the most potent α7nAChR agonists, has been reported to attenuate proinflammatory cytokine production [8], improve outcomes in sepsis models [9], pancreatitis [10], and ischemia–reperfusion injury [11], and inhibit the production of endotoxin-induced TNF in lung tissue [12]. In addition, recent studies have demonstrated that GTS-21 inhibits the activities of endothelial cells and monocyte macrophages, as well as the secretion of pro-inflammatory cytokines in peripheral blood samples, by regulating the JAK2-STAT3 pathway [13–15]. Moreover, cholinergic receptor agonists such as AR-R17779 and nicotine have positive therapeutic effects on rheumatoid arthritis (RA) [16,17]. GTS-21, which is being investigated for the treatment of Alzheimer's disease [18] and schizophrenia [19], has been proven safe for humans in clinical
http://dx.doi.org/10.1016/j.intimp.2015.10.005 1567-5769/© 2015 Published by Elsevier B.V.
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
2
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
trials. At present, GTS-21 has gained increased popularity and is considered a potential therapeutic agent for inflammatory disorders. However, the detailed molecular mechanisms of the anti-inflammatory effects of GTS-21 are still limited. RA, a systemic autoimmune disease pathologically characterized by chronic inflammation and joint destruction, approximately affects 1% of the global population [20,21]. Although several treatment protocols with certain alleviatory effects are clinically available, RA treatments still need to be improved. Our previous in-vivo study was performed to observe the effect of GTS-21 on collagen-induced arthritis (CIA) in rats [22]. The results showed that the levels of TNF-α, IL-1β, and IL-6 in the serum were significantly reduced after GTS-21 management, and bone degradation was inhibited as well. The pathogenesis of RA is a complex process involving many cell types, including T cells, B cells, and macrophages. Considering the continuous exploration of the antiinflammatory molecular mechanism of GTS-21 at the cell level in RA, we chose macrophage RAW264.7 cell line as our research object. In this study, we explored whether GTS-21 attenuated lipopolysaccharide (LPS)-induced inflammatory cytokine production in RAW264.7 cells and to gain insights into the role of Akt/NF-κB in inflammation to assess its application prospect in RA. 2. Materials and methods 2.1. Cell culture RAW264.7 cells were obtained from the American Type Culture Collection (ATCC TIB-71). The cells were maintained in Dulbecco's modified Eagle's medium (Thermo Fisher, USA) and supplemented with 10% fetal bovine serum (Gibco, USA) and penicillin/streptomycin (1:100, Sigma, St. Louis, MO) in a humidified atmosphere of 95% air and 5% CO2 incubator at 37 °C.
(Cambridge, UK). The antibody of β-actin was purchased from EarthOx (San Francisco, CA, USA). Phosphoinositide-3 kinase (PI3K)/Akt inhibitor LY294002 was obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.5. Immunocytochemistry The nuclei translocation of NF-κB p65 was assessed using confocal fluorescence microscopy. RAW 264.7 cells, which were grown on glass coverslips, were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS), stained with anti-NF-κB p65 antibody (diluted 1:100) for 1 h at room temperature, and incubated with TRITC-conjugated second antibody (Santa Cruz, USA) for 1 h at room temperature. After washing with PBS, the nuclei were stained for 3 min with the fluorescent dye 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). The stained cells were analyzed using confocal fluorescence microscopy (Leica TCS SP5-II). Immunofluorescence assay was used to detect the expression of α7nAChR on the surfaces of RAW264.7 cells using an anti-α7 nAChR antibody or a specific α7 nAChR antagonist. Cells grown on chambered coverglass were fixed with freshly prepared 40 g/L paraformaldehyde in PBS for 30 min at room temperature, washed twice with cold PBS, and blocked with 1% BSA for 30 min at room temperature. The cells were incubated first in the diluted anti-α7-nAChR antibody (Abcam, UK) overnight at 4 °C and with the TRITC-conjugated secondary antibodies (BOSTER, China) for 1 h at room temperature with three washes after each incubation period. The stained cells were analyzed using fluorescence microscopy (Olympus, IX71). To confirm the antibody test results, the α7-nAChR-specific antagonist α-bungarotoxin (Bgt) was used for further identification. After fixing, RAW264.7 cells were incubated with tetramethylrhodamine-α-Bgt (Sigma, USA) for 1 h at room temperature and observed using fluorescence microscopy. 2.6. Statistical analysis
2.2. Cell viability assay (CCK-8 assay) The viability of RAW 264.7 cells was determined by the CCK-8 assay (Dojindo, Japan). Cells were treated with specific concentrations of GTS21 (5 μM to 100 μM; the compound was purchased from Abcam (Cambridge, UK)) for 72 h. CCK-8 reagent was added and incubated at 37 °C for 2.5 h. The absorbance of CCK-8 was detected at 450 nm by a microplate reader (BioTek Synergy 4, USA). The viability of the cells was expressed as the fraction of surviving cells relative to untreated controls.
All quantitative data were expressed as means ± standard deviation from at least three independent experiments. Statistical analysis was conducted by one-way ANOVA, followed by Dunnett's t-test. All groups were tested against a control group as reference using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). A difference is considered significant when P b 0.05. 3. Results
2.3. Enzyme-linked immunosorbent assay (ELISA)
3.1. Cytotoxicity of GTS-21 in RAW 264.7 cells
RAW 264.7 cells were pretreated with various concentrations of GTS-21 for 2 h and incubated for another 22 h with or without stimulation by 100 ng/mL of LPS (Sigma-Aldrich, USA). The culture medium was collected, and the cells were lysed by lysis buffer (Thermo Scientific, USA). Protein concentration was measured by BCA Protein Assay Kit (Thermo Scientific, USA). Concentrations of the cytokines were determined by ELISA (eBioscience, USA).
CCK-8 assay was performed to investigate the cytotoxicity of GTS21. No cytotoxicity was observed when RAW264.7 cells were exposed to GTS-21 (5 μM to 100 μM) for 72 h (Fig. 1). We decided to set the highest concentration of GTS-21 to 100 μM for the following study.
2.4. Western blot Cells were lysed by lysis buffer (Thermo Scientific, USA), and the protein concentration was measured using BCA Protein Assay Kit (Thermo Scientific, USA). A total of 40 μg of protein from each sample was separated by 10% SDS-PAGE and transferred to a PVDF membrane. After incubating the protein with specific antibodies overnight at 4 °C and with the secondary antibody, antibody binding was detected with the enhanced ECL detection system (Millipore, USA). The antibodies against phospho-Akt (Ser473), phospho-IKKα/β (Ser176/180), phospho-IκBα (Ser32), NF-κB p65, and phospho-NF-κB p65 (Ser536) were purchased from Cell Signaling Technology (Beverly, MA, USA), and the antibody for Akt1 (Y89) was purchased from Abcam
3.2. Effects of GTS-21 on LPS-induced pro-inflammatory cytokine production To determine the inhibitory action of GTS-21 on the proinflammatory cytokines in LPS-stimulated RAW264.7 cells, the levels of IL-1β, IL-6, or TNF-α expression were analyzed by ELISA. As shown in Fig. 2, the production of IL-1β, IL-6, and TNF-α was significantly increased by the stimulation with LPS. Pretreatment with GTS-21 reduced the release of IL-6 in LPS-stimulated cells in a concentration-dependent manner. However, the dose-dependent attenuation of IL-1β and TNF-α secretion was relatively weaker. 3.3. GTS-21 suppressed NF-κB activation in LPS-stimulated RAW 264.7 cells NF-κB is a transcriptional regulator that significantly affects inflammatory response. Cells were pretreated with or without GTS-21 for
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
3
Fig. 1. Cytotoxicity of GTS-21 in RAW 264.7 cells. RAW 264.7 cells were pretreated with different concentrations of GTS-21 (5, 10, 20, and 100 μM) for 72 h. The viability of the cells was determined using CCK-8 assay, and no cytotoxicity was observed. The data are presented as the means ± standard deviation from three independent experiments.
2 h and stimulated with LPS (100 ng/mL) in the indicated times. As shown in Fig. 3A, GTS-21 substantially inhibited the enhanced phosphorylation of Ser536 of NF-κB p65 stimulated by LPS in different times. Furthermore, GTS-21 inhibited the phosphorylation of Ser536 of NF-κB p65 dose-dependently at 30 min (Fig. 3B). We used immunocytochemistry to examine the effect of GTS-21 on the nuclear translocation of phospho(p)-NF-κB p65 stimulated by LPS. Treatment with GTS21 (100 μM) evidently inhibited the LPS-induced NF-κB p65 nuclear translocation (Fig. 3C). NF-κB is associated with IκB protein, which inhibits NF-κB activity, in the cytoplasm as an inactive NF-κB–IκB complex. IKK is an important upstream kinase for the phosphorylation of IκB. Therefore, we examined p-IKKα/β and p-IκBα by Western blot analysis. As expected, LPS (100 ng/mL) induced IKKα/β and IκBα phosphorylation. GTS-21 dramatically blocked these effects at different time points and in a dose-dependent manner at 30 min (Fig. 3A and B). 3.4. GTS-21 inhibited Akt activation in LPS-stimulated RAW 264.7 cells PI3K/Akt pathway was determined to be involved in the LPS activation of signaling pathways and the expression of inflammatory cytokines in RAW 264.7 cells [23]. We examined the effects of GTS-21 on the LPS-induced phosphorylation of Akt in the cells. As shown in Fig. 4A–B, GTS-21 significantly inhibited the LPS-induced-phosphorylation of Akt in Ser473 at different time points and in a dose-dependent manner at 30 min. 3.5. Akt pathway was involved in NF-κB activation stimulated by LPS in RAW 264.7 cells Evidence shows that multiple signaling pathways are involved in the regulation of NF-κB activation and a complex crosstalk exists among them, including PI3K/Akt, PKC/MAPK, STAT5 [24–26]. In our study, in order to investigate the relationship between PI3K/Akt pathway and NF-κB, the cells were pretreated with 20 μM PI3K/Akt inhibitor LY294002 for 1 h and exposed to LPS (100 ng/mL). LY294002 pretreatment blocked the increased p-Akt, p-IKK, and p-NF-κB p65 levels by LPS stimulation (100 ng/mL) (Fig. 5A). In addition, LY294002 significantly suppressed the pro-inflammatory cytokine production in LPSstimulated RAW 264.7 cells (Fig. 5B).
Fig. 2. Effects of GTS-21 on LPS-induced pro-inflammatory cytokine production. RAW264.7 cells were pretreated with and without GTS-21 (5, 10, 20, and 100 μM) for 2 h and stimulated with LPS (100 ng/mL). The control group was not subject to any stimulus or GTS-21. After 22 h, the levels of interleukin (IL)-1β, IL-6, or tumor necrosis factor (TNF)-α were measured in the culture medium by ELISA. *P b 0.05 and **P b 0.01 versus the group with LPS treatment alone. The data are presented as the means ± standard deviation from three independent experiments.
pheochromocytoma cells were provided by Prof. Liping-Wang from the Neural Engineering Research Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences) are known to express α7 nAChR at high levels [28,29], and the cells were used as the positive control in the test. RAW264.7 cells and human rheumatoid fibroblast-like synoviocyte line MH7A cells were detected by immunofluorescence assay using the antibody specific for α7 nAChR. The results showed that RAW264.7 cells were covered with the red fluorescence of α7nAChR (Fig. 6A). Subsequently, we chose the a7 nAChR selective antagonist a-Bgt labeled with tetramethylrhodamine to further confirm that the positive signals in RAW264.7 cells represented the α7 subunit. Consistent with the previous result, the immunofluorescence images showed that RAW264.7 cells were similarly covered with the red fluorescence (Fig. 6B). However, no significant positive results were observed in both experiments in MH7A cells (Fig. 6A and B). 3.7. α7 nAChR selective antagonist α-Bgt inhibited the negative regulation of GTS-21 on NF-κB activation in RAW264.7 cells
3.6. α7 nAChR is expressed on RAW264.7 In recent years, α7 nAChR has been considered a potential therapeutic target for inflammatory diseases [5,27]. GTS-21 acts as an agonist at neural nicotinic acetylcholine receptors. To assess whether the antiinflammation effect of GTS-21 was caused by α7 nAChR, we first examined α7 nAChR expression on RAW264.7. PC12 cells (rat
To determine whether α7 nAChR is required for the GTS-21 inhibition of NF-κB activation in RAW264.7, we chose α-Bgt, a strong and irreversible antagonist of nAChR with partial selectivity to α7 nAChR [30–32], to block α7 nAChR on RAW264.7 cells. These cells were pretreated for 30 min in the absence or presence of α-Bgt (10 nM or 100 nM) before adding 100 μM GTS-21. About 2 h later, cells were
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
4
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
5
Fig. 4. Effects of GTS-21 on Akt activation in RAW 264.7 cells. (A) RAW 264.7 cells were pretreated with GTS-21 (100 μM) for 2 h and incubated with LPS (100 ng/mL) for 15 min to 60 min (A) or with different concentrations of GTS-21 for 30 min (B). Akt and p-Akt levels were determined by Western blot analysis. Results are presented as means ± standard deviation of three independent experiments. *P b 0.05 and **P b 0.01 versus the group with LPS treatment alone.
Fig. 5. Involvement of Akt pathway in NF-κB activation and effects of inhibition of PI3k/Akt on pro-inflammatory cytokine production. (A) RAW 264.7 cells were pretreated with LY294002 (20 μM) or GTS-21 for 2 h and incubated with LPS (100 ng/mL) for 30 min. p-Akt, p-IKKα/β, p-IκBα, and p-NF-κB p65 levels were determined by Western blot analysis. (B) IL-1β, IL-6, and TNF-α production was determined in RAW 264.7 cells treated with LPS (100 ng/mL) with and without LY294002 (20 μM) for 24 h by ELISA. The data are presented as the means ± standard deviation from three independent experiments. **P b 0.01 versus the group with LPS treatment alone.
induced with 100 ng/mL LPS for 30 min. NF-κB activation in RAW264.7 cells was determined by Western blot assay and laser confocal imaging. As shown in Fig. 7A, the level of p-NF-κB p65 significantly increased in
the RAW264.7 cells exposed to α-Bgt (10 nM or 100 nM). Moreover, α-Bgt almost reversed the inhibitory effect of GTS-21 on the NF-κB p65 nuclear translocation (Fig. 7B).
Fig. 3. Effects of GTS-21 on NF-κB activation in RAW 264.7 cells. (A–B) RAW 264.7 cells were pretreated with and without GTS-21 (100 μM) for 2 h and incubated with LPS (100 ng/mL) for 15 min to 60 min (A) or with different concentrations of GTS-21 for 30 min (B). Phospho-IKKα/β (p-IKK-α/β), p-IκBα, and p-NF-κB p65 levels were determined by Western blot analysis. Results are presented as means ± standard deviation of three independent experiments. *P b 0.05 and **P b 0.01 versus the group with LPS treatment only. (C) After pretreatment with and without GTS-21 (100 μM) for 2 h, RAW 264.7 cells were incubated with LPS (100 ng/mL) for 30 min. The control group was not subject to any stimulus or GTS-21. The nuclei translocation of NF-κB p65 was assessed by confocal fluorescence microscopy. Cells were immunostained with NF-κB p65 antibody labeled with TRITC in red, and nuclei were stained with DAPI in blue. Bar, 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
6
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
Fig. 6. Analysis of α7 nAChR expressed in RAW264.7 cells. Expression of α7 nAChR on the cell surfaces of RAW264.7 cells. (A). Immunofluorescence analysis of RAW264.7 cells using an anti-α7 nAChR antibody in red. (B) Immunofluorescence analysis of RAW264.7 cells using tetramethylrhodamine-α-Bgt in red. PC12 cells and MH7A cells were used as positive and negative control respectively. Each middle image was a large version of the corresponding parts marked in red in top images. The nuclei were stained with DAPI in blue in bottom images. Bar, 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Discussion The discovery of the cholinergic anti-inflammatory pathway suggests a novel treatment method for RA therapy. GTS-21 is a selective α7nAChR agonist, and both in vitro and in vivo investigations suggest
that GTS-21 has immunomodulatory and anti-inflammatory effects. Although the health-promoting properties are not fully understood, these benefits have been mainly related to the interactions of GTS-21 with several cytokines and regulatory transcription factors. The inhibitory properties in these pathways are likely to significantly affect cellular
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
7
Fig. 7. α7 nAChR selective antagonist α-Bgt inhibited the effect of GTS-21 on NF-κB activation in RAW264.7 cells. NF-κB activation in RAW264.7 cells was determined by Western blot assay and laser confocal imaging. RAW264.7 cells were pre-treated for 30 min with and without α-Bgt (10 nM or 100 nM) before adding 100 μM GTS-21. After 2 h, these cells were induced in 100 ng/mL LPS for 30 min. (A) p-NF-κB p65 levels were determined by Western blot analysis. Results are presented as means ± standard deviation of three independent experiments. **P b 0.01 versus the group with GTS-21 and LPS treatment. (B) The nuclei translocation of NF-κB p65 was assessed by confocal fluorescence microscopy. Cells were immunostained with NF-κB p65 antibody labeled with TRITC in red. The nuclei were stained with DAPI in blue. Bar, 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
function by changing the phosphorylation state of target molecules and/ or modulating gene expressions. GTS-21 is a drug that has been used safely in humans for the treatment of Alzheimer's disease, nicotine dependence, and schizophrenia in clinical trials. This drug also has a rapid transformational application in anti-inflammation treatment. The mature manufacturing process for GTS-21 may greatly reduce the research cost of novel RA therapies. In our study, we explored the molecular mechanism of GTS-21 action against LPS-induced inflammation in RAW 264.7 cells. The results showed that GTS-21(100 μM) did not exhibit cytoxicity effects and significantly reduced the inflammatory mediators IL-1 β, IL-6, and TNF-α in LPS-activated RAW 264.7 cells. These findings suggest the marked anti-inflammatory effects of GTS-21 in LPS-stimulated RAW 264.7 cells, consistent with the reports from earlier studies. The present study demonstrated that GTS-21 significantly suppressed the NF-κB transcriptional activity for the first time by attenuating the LPS-induced IKKα/β, IκBα, p65 phosphorylation, and subsequent p65 nuclear translocation. NF-κB is known to significantly affect the regulation of the expressions of pro-inflammatory enzymes and cytokines, such as TNF-α and IL-6 [33]. NF-κB is represented mainly by the p65/p50 heterodimeric complex in most cell types. In unstimulated cells, NF-κB resides in the cytoplasm as an inactive NF-κB– IκBα complex. In the activation process, the inhibitory NF-κB kinase (IKK) is activated. IκBα, the inhibitory subunit, is rapidly phosphorylated in Ser32 and Ser36 residues by IKKα/β, subsequently ubiquitinated, and degraded by 26S proteasome complex; thus NF-κB is translocated to the nucleus and activates the transcription of various inflammatory gene products [34]. Therefore, GTS-21 significantly inhibited NF-κB transcriptional activity in RAW 264.7 cells by attenuating the LPS-induced IKK α/β, IκBα, and p65 phosphorylation and subsequent p65 nuclear translocation. Other studies have proved the involvement of the PI3K/Akt pathway in NF-κB transcriptional activation [35]. The PI3K/Akt pathway was
reported to significantly influence inflammation-mediated diseases, such as rhinosinusitis [36], RA [37], lung injury [38], and psoriasis [39]. Akt, a downstream protein of PI3K, is a serine kinase that is recruited to the plasma membrane in cells phosphorylated and activated with a variety of stimulants, including growth factors and cytokines [40–42]. Ojaniemi et al. found that the inhibition of PI3k/Akt decreased the LPS-induced transcriptional activity of NF-κB in RAW 264.7 cells [23]. Our results showed that the inhibition of PI3K/Akt by LY294002 could suppress the phosphorylation of IKKα/β, IκBα, and NF-κB-p65. LY294002 also inhibited the secretion of LPS-induced TNF-α, IL-1β, and IL-6 in RAW264.7. These results indicate a probable antiinflammation mechanism of GTS-21 underlying the suppression of the inducible activation of NF-κB by inhibiting the PI3K/Akt pathway. Previous studies have suggested that the α7 subunit of nAChR is essential for the so-called “cholinergic anti-inflammatory pathway” [43]. GTS-21 is a partial agonist at α7 nAChR, and our study showed that GTS-21exhibited good anti-inflammatory properties. To confirm the effectiveness of GTS-21 on RAW264.7 cells through α7 nAChR for GTS-21, we detected the expression of α7 nAChR and NF-κB activation after blocking this receptor in RAW264.7 cells. We found that α7 nAChR did express on the surface of RAW264.7 cells. α-bungarotoxin (α-Bgt) is an α7 nAChR-specific antagonist that can be used in distinguishing acetylcholine receptor types. Our study indicated that α-Bgt pretreatment almost prevented the inhibition of NF-κB activation and the nuclear translocation by GTS-21. This finding establishes that the GTS-21 function is a result of its combination with α7 nAChR. The main symptoms of RA are persistent inflammation and abnormal synovial hyperplasia with marked pannus formation, as well as the subsequent invasion and destruction of cartilage and bone with eventual bone tissue necrosis. As an important component of articular synovial tissues, fibroblast-like synoviocytes (FLS) are also crucial in perpetuating inflammation and promoting cartilage destruction [44]. Therefore, FLS has consequently gained significant attention in RA. FLS
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
8
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx
and macrophage-like synoviocytes are two main effector cells in the joint synovial of RA patients. On the basis of these theories, we selected both the murine macrophage cell line RAW264.7 cells and the human rheumatoid fibroblast-like synoviocyte line MH7A cells to assess the anti-inflammatory prospect of GTS-21 in RA. However, our results did not indicate any significant anti-inflammatory effects of GTS-21 on MH7A cells or any significant changes in the anti-inflammatory signaling pathway mediators (data not shown). With uncertainties, we found that α7nAChR was not expressed in MH7A cells (Fig. 6A and B). Maybe that is the reason for the difference in the effect of GTS-21 between MH7A cells and RAW264.7 cells. 5. Conclusion Our previous in vivo study suggested that GTS-21 significantly reduced serum pro-inflammatory cytokine titers and bone degradation in rats with CIA [22]. In this article, our in vitro study provided new insight into the anti-inflammatory mechanisms of GTS-21 and demonstrated that GTS-21 downregulated the inflammatory cytokines by inhibiting the Akt activation. The subsequent IKKα/β mediated the IκBα phosphorylation and inhibited the NF-κB activation and gene expression in LPS-induced RAW 264.7 cells through α7nAChR. Our in vivo and in vitro studies may provide a basis for the use of GTS-21 for inflammatory disorders such as RA. Other effector cells such as endotheliocytes and other kinds of pro-inflammatory mediator production, such as nitric oxide and vascular endothelial growth factor, will be detected in further studies. Furthermore, we intend to explore the underlying mechanisms and other signaling pathways in the antiinflammatory effect of GTS-21 and evaluate its therapeutic potency in inflammatory disease. Conflict of interest The authors have no potential conflicts of interest to disclose. Acknowledgments This work was supported by the National Natural Science Foundation of China (81271952), the Science and Technology Project of Guangdong Province (2013B050800005), the Science and Technology Project of Shenzhen City (JCYJ20140610151856738), and the Shanghai Key Laboratory of Orthopedic Implants. References [1] M. Fleshner, L.E. Goehler, B.A. Schwartz, M. McGorry, D. Martin, S.F. Maier, et al., Thermogenic and corticosterone responses to intravenous cytokines (IL-1beta and TNF-alpha) are attenuated by subdiaphragmatic vagotomy, J. Neuroimmunol. 86 (1998) 134–141. [2] L.E. Goehler, C.R. Busch, N. Tartaglia, J. Relton, D. Sisk, S.F. Maier, et al., Blockade of cytokine induced conditioned taste aversion by subdiaphragmatic vagotomy: further evidence for vagal mediation of immune-brain communication, Neurosci. Lett. 185 (1995) 163–166. [3] L.V. Borovikova, S. Ivanova, M. Zhang, H. Yang, G.I. Botchkina, L.R. Watkins, et al., Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin, Nature 405 (2000) 458–462. [4] M.A. van Maanen, S.P. Stoof, E.P. van der Zanden, W.J. de Jonge, R.A. Janssen, D.F. Fischer, et al., The alpha7 nicotinic acetylcholine receptor on fibroblast-like synoviocytes and in synovial tissue from rheumatoid arthritis patients: a possible role for a key neurotransmitter in synovial inflammation, Arthritis Rheum. 60 (2009) 1272–1281. [5] J.M. Waldburger, D.L. Boyle, V.A. Pavlov, K.J. Tracey, G.S. Firestein, Acetylcholine regulation of synoviocyte cytokine expression by the alpha7 nicotinic receptor, Arthritis Rheum. 58 (2008) 3439–3449. [6] K.Z. Sato, T. Fujii, Y. Watanabe, S. Yamada, T. Ando, F. Kazuko, et al., Diversity of mRNA expression for muscarinic acetylcholine receptor subtypes and neuronal nicotinic acetylcholine receptor subunits in human mononuclear leukocytes and leukemic cell lines, Neurosci. Lett. 266 (1999) 17–20. [7] H. Wang, M. Yu, M. Ochani, C.A. Amella, M. Tanovic, S. Susarla, et al., Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation, Nature 421 (2003) 384–388.
[8] I.A. Giebelen, D.J. van Westerloo, G.J. LaRosa, A.F. de Vos, T. van der Poll, Stimulation of alpha 7 cholinergic receptors inhibits lipopolysaccharide-induced neutrophil recruitment by a tumor necrosis factor alpha-independent mechanism, Shock 27 (2007) 443–447. [9] V.A. Pavlov, M. Ochani, L.H. Yang, M. Gallowitsch-Puerta, K. Ochani, X. Lin, et al., Selective alpha7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis, Crit. Care Med. 35 (2007) 1139–1144. [10] D.J. van Westerloo, I.A. Giebelen, S. Florquin, M.J. Bruno, G.J. Larosa, L. Ulloa, et al., The vagus nerve and nicotinic receptors modulate experimental pancreatitis severity in mice, Gastroenterology 130 (2006) 1822–1830. [11] M.M. Yeboah, X. Xue, B. Duan, M. Ochani, K.J. Tracey, M. Susin, et al., Cholinergic agonists attenuate renal ischemia–reperfusion injury in rats, Kidney Int. 74 (2008) 62–69. [12] I.A. Giebelen, D.J. van Westerloo, G.J. LaRosa, A.F. de Vos, T. van der Poll, Local stimulation of alpha7 cholinergic receptors inhibits LPS-induced TNF-alpha release in the mouse lung, Shock 28 (2007) 700–703. [13] P.K. Chatterjee, Y. Al-Abed, B. Sherry, C.N. Metz, Cholinergic agonists regulate JAK2/ STAT3 signaling to suppress endothelial cell activation, Am. J. Physiol. Cell Physiol. 297 (2009) C1294–C1306. [14] M. Rosas-Ballina, R.S. Goldstein, M. Gallowitsch-Puerta, L. Yang, S.I. Valdes-Ferrer, N.B. Patel, et al., The selective alpha7 agonist GTS-21 attenuates cytokine production in human whole blood and human monocytes activated by ligands for TLR2, TLR3, TLR4, TLR9, and RAGE, Mol. Med. 15 (2009) 195–202. [15] M. Kox, J.F. van Velzen, J.C. Pompe, C.W. Hoedemaekers, J.G. van der Hoeven, P. Pickkers, GTS-21 inhibits pro-inflammatory cytokine release independent of the Toll-like receptor stimulated via a transcriptional mechanism involving JAK2 activation, Biochem. Pharmacol. 78 (2009) 863–872. [16] M.A. van Maanen, M.C. Lebre, T. van der Poll, G.J. LaRosa, D. Elbaum, M.J. Vervoordeldonk, et al., Stimulation of nicotinic acetylcholine receptors attenuates collagen-induced arthritis in mice, Arthritis Rheum. 60 (2009) 114–122. [17] T. Li, X. Zuo, Y. Zhou, Y. Wang, H. Zhuang, L. Zhang, et al., The vagus nerve and nicotinic receptors involve inhibition of HMGB1 release and early pro-inflammatory cytokines function in collagen-induced arthritis, J. Clin. Immunol. 30 (2010) 213–220. [18] W.R. Kem, The brain alpha 7 nicotinic receptor may be an important therapeutic target for the treatment of Alzheimer's disease: studies with DMXBA (GTS-21), Behav. Brain Res. 113 (2000) 169–181. [19] R. Freedman, A. Olincy, R.W. Buchanan, J.G. Harris, J.M. Gold, L. Johnson, et al., Initial phase 2 trial of a nicotinic agonist in schizophrenia, Am. J. Psychiatry 165 (2008) 1040–1047. [20] B. Jonsson, Patient access to rheumatoid arthritis treatments, Eur. J. Health Econ. 8 (Suppl. 2) (2008) S35–S38. [21] A. Morse, Services For People With Rheumatoid Arthritis International Comparisons, The National Audit Office, London, 2009. [22] Y. Hu, R. Liu, J. Li, Y. Yue, W. Cheng, P. Zhang, Attenuation of collagen-induced arthritis in rat by nicotinic alpha7 receptor partial agonist GTS-21, Biomed. Res. Int. 2014 (2014) 325875. [23] M. Ojaniemi, V. Glumoff, K. Harju, M. Liljeroos, K. Vuori, M. Hallman, Phosphatidylinositol 3-kinase is involved in Toll-like receptor 4-mediated cytokine expression in mouse macrophages, Eur. J. Immunol. 33 (2003) 597–605. [24] O.N. Ozes, L.D. Mayo, J.A. Gustin, S.R. Pfeffer, L.M. Pfeffer, D.B. Donner, NF-kappaB activation by tumour necrosis factor requires the Akt serine–threonine kinase, Nature 401 (1999) 82–85. [25] Z. Wei, F. Wang, J. Song, Q. Lu, P. Zhao, Y. Xia, et al., Norisoboldine inhibits the production of interleukin-6 in fibroblast-like synoviocytes from adjuvant arthritis rats through PKC/MAPK/NF-kappaB-p65/CREB pathways, J. Cell. Biochem. 113 (2012) 2785–2795. [26] Z.S. Nagy, M.J. LeBaron, J.A. Ross, A. Mitra, H. Rui, R.A. Kirken, STAT5 regulation of BCL10 parallels constitutive NFkappaB activation in lymphoid tumor cells, Mol. Cancer 8 (2009) 67. [27] M. Kox, J.C. Pompe, E. Peters, M. Vaneker, J.W. van der Laak, J.G. van der Hoeven, et al., alpha7 nicotinic acetylcholine receptor agonist GTS-21 attenuates ventilatorinduced tumour necrosis factor-alpha production and lung injury, Br. J. Anaesth. 107 (2011) 559–566. [28] H. Kawai, W. Zago, D.K. Berg, Nicotinic alpha 7 receptor clusters on hippocampal GABAergic neurons: regulation by synaptic activity and neurotrophins, J. Neurosci. 22 (2002) 7903–7912. [29] R.R. Jonnala, J.J. Buccafusco, Relationship between the increased cell surface alpha7 nicotinic receptor expression and neuroprotection induced by several nicotinic receptor agonists, J. Neurosci. Res. 66 (2001) 565–572. [30] M. Pohanka, Alpha7 nicotinic acetylcholine receptor is a target in pharmacology and toxicology, Int. J. Mol. Sci. 13 (2012) 2219–2238. [31] R. Doley, R.M. Kini, Protein complexes in snake venom, Cell. Mol. Life Sci. 66 (2009) 2851–2871. [32] A. Orr-Urtreger, F.M. Goldner, M. Saeki, I. Lorenzo, L. Goldberg, M. De Biasi, et al., Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alphabungarotoxin binding sites and hippocampal fast nicotinic currents, J. Neurosci. 17 (1997) 9165–9171. [33] P.A. Baeuerle, D. Baltimore, NF-kappa B: ten years after, Cell 87 (1996) 13–20. [34] S. Akira, K. Takeda, Toll-like receptor signalling, Nat. Rev. Immunol. 4 (2004) 499–511. [35] H.C. Dan, M.J. Cooper, P.C. Cogswell, J.A. Duncan, J.P. Ting, A.S. Baldwin, Aktdependent regulation of NF-{kappa}B is controlled by mTOR and Raptor in association with IKK, Genes Dev. 22 (2008) 1490–1500. [36] W. Chen, Z.J. Liu, J. Ye, Relationship among the expression of GSK3beta, PI3K/Akt, and IL-6 in chronic rhinosinusitis, Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 48 (2013) 128–134.
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005
Y. Yue et al. / International Immunopharmacology xxx (2015) xxx–xxx [37] M. Camps, T. Ruckle, H. Ji, V. Ardissone, F. Rintelen, J. Shaw, et al., Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis, Nat. Med. 11 (2005) 936–943. [38] Yang Y, Cheng Y, Lian QQ, Yang L, Qi W, Wu DR, et al. Contribution of CFTR to alveolar fluid clearance by lipoxin A4 via PI3K/Akt pathway in LPS-induced acute lung injury. Mediat. Inflamm. 2013;2013:862628. [39] M.P. Schon, W.H. Boehncke, Psoriasis, N. Engl. J. Med. 352 (2005) 1899–1912. [40] D. Stokoe, L.R. Stephens, T. Copeland, P.R. Gaffney, C.B. Reese, G.F. Painter, et al., Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B, Science 277 (1997) 567–570.
9
[41] A. Bellacosa, T.O. Chan, N.N. Ahmed, K. Datta, S. Malstrom, D. Stokoe, et al., Akt activation by growth factors is a multiple-step process: the role of the PH domain, Oncogene 17 (1998) 313–325. [42] A. Toker, L.C. Cantley, Signalling through the lipid products of phosphoinositide-3OH kinase, Nature 387 (1997) 673–676. [43] De Simone R, Ajmone-Cat MA, Carnevale D, Minghetti L. Activation of alpha7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures. J. Neuroinflammation 2005;2:4. [44] Bartok B, Firestein GS. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol. Rev. 2010;233:233–55.
Please cite this article as: Y. Yue, et al., GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling p..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.10.005