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Oxymatrine prevents synovial inflammation and migration via blocking NF-κB activation in rheumatoid fibroblast-like synoviocytes
International Immunopharmacology 55 (2018) 105–111
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Oxymatrine prevents synovial inflammation and migration via blocking NFκB activation in rheumatoid fibroblast-like synoviocytes ⁎
T
⁎⁎
Jiang Lianga, ,1, Boyang Changb,1, Mingcheng Huangc,1, Weichen Huangd, , Wukai Maa, Yang Liua, Wan Taia, Yi Longa, Yun Lua a
Collaborative Innovation Center of Miao medicine, Guiyang College of Traditional Chinese Medicine, Guiyang, Guizhou, China State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China c Department of Hematology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China d Department of the first Orthopedics, The 2nd Affiliated Hospital, Guiyang College of Traditional Chinese Medicine, Guiyang, Guizhou, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Oxymatrine Fibroblast-like synoviocytes Inflammation Migration NF-κB
The fibroblast-like synoviocytes (FLSs) has the aggressive phenotype, which is very important for cartilage destruction in rheumatoid arthritis (RA). To the pathology of RA, the increased FLSs migration, activation and proliferation are essential factors. Oxymatrine is a traditional Chinese herb, which is the extraction from the root of Sophora flavescens and regarded as quinolizidine alkaloid compounds and has been shown to inhibit inflammation, proliferation and migration in vitro or vivo. However, whether oxymatrine effects in the treatment of RA FLSs is undefined. In our study, the inhibition of oxymatrine in RA FLSs inflammation, proliferation and migration in RA FLS are evaluated. We found that oxymatrine decreased the IL-6 and IL-8 expression and the proliferation, migration and invasion of RA FLSs. We also evaluated the molecular mechanisms and we found the effect of oxymatrine on NF-κB activation. The results showed that oxymatrine inhibited the activity of NF-κB. And the treatment activity of oxymatrine on collagen-induced arthritis (CIA) was further explored by us. Thus, we conclude that oxymatrine may protect joint destruction of RA by inhibiting synoviocyte activation, migration, invasion, and proliferation.
1. Introduction Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterized by the aggressive inflammation and destruction of cartilage and bone [1]. Fibroblast-like synoviocytes (FLSs) are fundamental in the maintenance of synovial inflammation and aggressive joint destruction in RA [2,3]. Activated FLSs in RA show the tumor-like characteristics, for example, the constant growth and resistance to apoptosis; and in the formation of pannus by migration and invasion towards cartilage and bone, they are also in important position [4,5,6]. Evidence increasingly suggests that inhibiting FLS inflammation and migration may have novel therapeutic potential for RA [7]. Therefore, many efforts have been made to modulate the inflammatory mediators, proliferation and migration behaviors of RA FLSs and the inhibition of expression of inflammatory mediators, proliferation and migration of
FLSs might be a treatment for RA. Oxymatrine is the quinolizidine alkaloid extraction, which is from the Chinese herb Sophora flavescens Ait [8,9]. Oxymatrine has several pharmacological effects including anticancer, antiviral activities and anti-inflammatory [10,11]. Many studies have indicated that oxymatrine suppresses cancer cell proliferation, migration and invasion [12,13]. Oxymatrine also suppresses lipopolysaccharide (LPS) treated inflammation via regulating TLR4/NF-κB in macrophages and inhibits the inflammation of LPS-induced mastitis [14]. Recent studies have indicated that by regulating the imbalance between Treg and Th17 cells [15], oxymatrine has beneficial effects in an autoimmune arthritis mouse model. However, the anti-inflammatory, anti-proliferative and anti-migratory effects of oxymatrine on RA FLS have not been explored. Therefore, in our work, we determined whether oxymatrine reduced proinflammatory cytokines production and the proliferation and
⁎ Correspondence to: J. Liang, Collaborative Innovation Center of Miao Medicine, Guiyang College of Traditional Chinese Medicine, Huaxi University Town, Guiyang, Guizhou 550025, China. ⁎⁎ Correspondence to: W. Huang, Department of the First Orthopedics, The 2nd Affiliated Hospital, Guiyang College of Traditional Chinese Medicine, Guiyang, Guizhou, Feishan Street, 510000, China. E-mail addresses: [email protected] (J. Liang), [email protected] (W. Huang). 1 JL, BC and MH contributed equally to this work.
https://doi.org/10.1016/j.intimp.2017.12.006 Received 8 October 2017; Received in revised form 22 November 2017; Accepted 6 December 2017 1567-5769/ © 2017 Elsevier B.V. All rights reserved.
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migration of RA FLS and the molecular mechanisms involved. 2. Materials and methods 2.1. Patients and isolation of FLS We performed the study according to the recommendations of the Declaration of Helsinki and our study had got the consent from the Medical Ethical Committee of the Guiyang College of Traditional Chinese Medicine and the First Affiliated Hospital, Sun Yat-sen University. We also got the consent from all patients involved for taking part in the study. Synovial tissues were gained from 8 RA patients (3 men and 5 women aged from 35 to 60 years old), who were undergoing joint replacement or synovectomy. The synovial tissue was sliced into pieces and digested with collagenase I (Sigma-Aldrich, St. Louis, MO, USA) which was put in DMEM/F12 medium to isolate synoviocytes for 4 h at the incubator of 37 °C. They were washed with PBS and then grown in 10% fetal bovine serum of DMEM/F12, which contained 100 U/mL penicillin and 100 μg/mL streptomycin in a 5% CO2, 21% O2, and 75% N2 humidified incubator at 37 °C. Passages 3 to 5 of RA FLSs were used for experiments.
Fig. 1. Effects of oxymatrine on RA FLS. RA-FLSs were treated with indicated concentrations of oxymatrine for 48 h, and cell viability was measured using the MTT assay. Result shown is the mean ± SD. *P < 0.05, oxymatrine-treated versus vehicle-treated cells.
2.2. Cell viability assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to test cell viability. Firstly, RA FLSs were played in 96-well plates and they were incubated with either DMSO or the indicated concentration of oxymatrine. After 48 h and 72 h of treatment, MTT (0.5 mg/mL; Sigma-Aldrich, USA) was added to incubate for 4 h. The media containing MTT was removed, and the DMSO was used to dissolve the formed formazan crystals. The viable cells were tested by microplate reader (BioRad, Hercules, CA) to read the absorbance at 540 nm in. 2.3. Migration and invasion assay of FLS We performed the cell migration assays by using Transwell inserts (Transwell; Corning Inc., NY, USA). DMEM/F12 medium containing 10% FBS as the chemoattractant was used in the bottom chambers; and RA FLSs with oxymatrine was added in the upper chambers with oxymatrine for 24 h in serum free DMEM/F12 medium. The top membrane surface cells were removed when they were incubated for 12 h. The migrated cells which were on the bottom side of the membrane were fixed in methanol and they were stained with 0.1% Crystal Violet and counted under a microscope. For the invasion assay, we used inserts coated with a Matrigel basement membrane matrix (BD Biosciences, Oxford, UK) to perform similar experiments. The chemoattractant was DMEM/F12 medium, which contains 15% FBS. The 200 μL DMEM/F12 medium and FLS were seeded in the top chambers with oxymatrine for 24 h. And then after 24 h, the non-invaded cells were removed from the upper side of the filter by using a cotton swab. The invaded cells were dyed with 0.1% crystal violet and calculated by microscope.
Fig. 2. Effects of oxymatrine on pro-inflammatory cytokines expression in RA FLSs. After treatment with or without oxymatrine for 24 h, RA FLS were stimulated with TNF-α (10 ng/mL) for 12 (for IL-6 and IL-8 mRNA) or 24 (for IL-6 and IL-8 secretion) hours. The levels of IL-6 and IL-8 mRNA and in cultured cell supernatants were measured by quantitative real-time PCR and ELISA. Data are representative of five independent experiments from different patients. A, mRNA expression of IL-6 and IL-8 was determined by quantitative real-time PCR. Data were normalized to GAPDH. B, the levels of IL-6 and IL-8 in cultured cell supernatants were measured by ELISA. All values represent mean ± SD. *P < 0.05 vs control, #P < 0.05 vs TNF-α.
2.4. Wounding migration
without TNF-α (10 ng/mL) or oxymatrine for 24 h and the 5-Ethynyl-2′deoxyuridine (EdU) Cell Proliferation Assay Kit was used to measure the cell proliferation following the manufacturer's protocol.
In brief, RA-FLSs were planted onto 12-well plates and grown to confluence. A linear wound was generated on the cell monolayer by using pipette tips and treated with oxymatrine for 24 h. After 24 h of oxymatrine treatment, migration was evaluated by counting the cells which had shifted beyond a reference line. The cells were photographed by a microscope.
2.6. Confocal laser scanning fluorescence microscopy We grew cells on glass coverslips to have a detection of the p65 expression and cytoskeletal filamentous actin (F-actin) in RA FLSs. The RA FLSs were treated with oxymatrine for 24 h and then were treated with TNF-α (10 ng/mL) for 12 h. Then they were immobilized with paraformaldehyde; meanwhile, they were permeated with 0.1%
2.5. Proliferation assays RA FLSs were grown to 90% confluence and were stimulated with or 106
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Fig. 3. Effect of oxymatrine on proliferation in RA FLSs. EdU incorporation assays were conducted to assess cell proliferation in RA-FLSs after 24 h treatment with oxymatrine and then stimulated with TNF-α (10 ng/mL) for 24 h. Representative images from 3 independent experiments. All values represent mean ± SD. *P < 0.05 vs. control, #P < 0.05 vs. TNF-α.
Fig. 4. Effect of oxymatrine on migration and invasion of RA FLS. A, Migration was performed in a Boyden chamber and chemotaxis was quantified by counting the migrated cells. Photo images of migration of RA FLS treated with oxymatrine. B, In vitro wound-healing assays. Wound closure was quantified after 24 h culturing in the presence or absence of oxymatrine. C, Transwell invasion assays were done to determine the invasion of RA FLSs after treatment with oxymatrine for 24 h. Photo images of invasion of RA FLS treated with oxymatrine. All values represent mean ± SD. *P < 0.05 vs control.
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Fig. 5. Effect of oxymatrine on cytoskeleton alterations of RA FLS. Effect of oxymatrine on alterations of actin cytoskeleton in TNF-α-treated RA FLS. F-actin (red) and nucleus (blue) were stained with phalloidin and DAPI, respectively. Representative images from three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
times in TBST and then incubated with blocking buffer for 1 h at room temperature. The primary antibodies were purchased from Abcam (Cambridge, MA, USA) and were diluted 1:1000 for phospho-IKKβ, phospho-IκBα, phospho-JNK, phospho-p38, phospho-ERK, IκBα, p38, ERK, JNK and C/EBPbeta and they were used to incubate the membranes overnight at 4 °C. Then the secondary antibodies were used to incubate the membranes for 60 min at room temperature. They were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, USA) reaction.
TritonX-100 in PBS. For detecting F-actin, we incubated the cells with phalloidin for 12 h. We incubated the cells with DAPI and mounted the coverslips on glass slides by using antifade mounting media; and we examined the cells with a confocal fluorescence microscopy (Zeiss LSM710). ELISA. By using TNF-α (10 ng/mL), we stimulated the RA FLS for 24 h with oxymatrine. According to the usages of the manufacturer (R&D systems, Minneapolis, MO, USA), the cell culture supernatants were gathered for detection of IL-6 and IL-8 by ELISA kits.
2.10. Statistical analysis 2.7. RNA isolation and quantitative real-time PCR analysis All experiments and statistical analysis were carried under the software of SPSS 13.0. Data were shown as means ± SD. The MannWhitney U test or Student's t-test was used to evaluate the differences between the experimental groups. P < 0.05 were considered statistically significant.
After 24 hours' treatments with oxymatrine, we stimulated the cells with TNF-α for 12 h. TRIzol (Sigma) was used to extract total RNAs and by using the miScript Reverse Transcription Kit, we reverse transcribed the RNAs to cDNA. Then by using QuantiTect SYBR Green RT-PCR Kit on StepOnePlusTM Real Time PCR System (Applied Biosystems), we performed the quantitative real time polymerase chain reaction (PCR) tests for the expression of IL-6, IL-8, MMP1, MMP3, MMP9 and MMP13 on cDNA.
3. Results 3.1. Oxymatrine effect on RA FLS viability MTT assays showed that treatment with oxymatrine for 48 h had no effects on the viability of RA FLSs (Fig. 1). And the results showed that the inhibitory effect observed in the inflammation and migration was not due to cytotoxic effects.
2.8. CIA induction and oxymatrine treatment In brief, the DBA/1 mice of 8–9 weeks were immunized intradermally at the tail with 200 μg diluted in acetic acid of bovine type II collagen which was emulsified in same volume of Freund's complete adjuvant (2 mg/mL, chondrex). Mice were boosted by administering 200 μg type II collagen intraperitoneally on day 21. Treatment with 5% DMSO (as control) and 100 mg/kg oxymatrine daily was started on the day when arthritis occurred for 14 days. The mice were monitored daily for signs of arthritis, and arthritis severity was scored on a scale of 0–3, as described previously. The total score was recorded as the sum of the scores in the four limbs. And the paw thick was tested by the vernier caliper. After the mice killed, the whole hind and fore limbs were fixed in formalin for 24–48 h and they were paraffin embedded. Haematoxylin and eosin (H&E) or safranin O-fast green was used to stain the tissue sections. And we tested the serum levels of IL-6 and TNF-α by ELIAS kits according to the usages of the manufacturer.
3.2. Oxymatrine suppresses pro-inflammatory mediators in RA FLSs In pathogenesis of RA, IL-6 and IL-8 are critical proinflammatory cytokines. To explore the possible inhibitory effects of oxymatrine in pro-inflammatory cytokines production, we detected the effect of oxymatrine on TNF-α-induced IL-6 and IL-8 gene expression; meanwhile, we also examined the secretion of IL-6 and IL-8 in RA FLS. As expected in Fig. 2, TNF-α treatment increased the IL-6 and IL-8 gene expression and promoted the secretion of RA FLSs. Oxymatrine treatment inhibited TNF-α-treated IL6 and IL-8 mRNA expression and the secretion in RA FLS.(Fig. 2A and B). MMPs is an essential characteristic of the invasive phenotype of RA FLSs. We also examined the effect of oxymatrine on TNF-α-induced MMP gene expression. As shown in Fig. S4, oxymatrine markedly suppressed the TNF-α-induced mRNA expression of MMP-1, MMP-3, MMP-9, and MMP-13 compared with DMSO.
2.9. Western blot analysis We lysed the cells in a lysis buffer which contained protease inhibitor mixture which was purchased from Thermo Scientific. After the cells were incubated for 15 min at the temperature of 4 °C, the supernatant was gathered by centrifugation (12,000 rpm, 15 min, at 4 °C). The BCA protein assay reagent kit (Pierce, Rockford, IL, USA) was used to determine the protein concentrations and 12% SDS-PAGE gels were used to separate the equal amounts of protein and they were transferred onto Nitrocellulose membranes. Membranes were washed by three
3.3. Oxymatrine inhibits proliferation in RA FLSs Previous studies show that oxymatrine inhibits tumor cell proliferation and cell proliferation is critical for RA. Thus, EdU incorporation was used to test the effect of oxymatrine on TNF-α-treated RA FLSs proliferation. As shown in Fig. 3, oxymatrine treatment 108
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Fig. 6. Oxymatrine inhibits TNF-α induced relocation of p65 and degradation of IκBα. A, Immunofluorescence staining analysis of p65 localization. RA FLS were preincubated with oxymatrine for 24 h and TNF-α (10 ng/mL) for 15 min, fixed, and then underwent immunofluorescence analysis. B, After treatment with or without oxymatrine for 24 h, RA FLS were stimulated with TNF-α (10 ng/mL) for 15 min. Phosphorylation of IKK and degradation and phosphorylation of IκBα induced by TNF-α was analysed using western blot analysis. Densitometry was performed and fold change of protein expression is shown below the corresponding band. All values represent mean ± SD. *P < 0.05 vs control, #P < 0.05 vs TNF-α.
3.5. Oxymatrine suppresses cytoskeleton alterations of RA FLS
potently suppressed TNF-α-treated proliferation of FLSs in a concentration-dependent manner.
To further verify the effect of oxymatrine on FLS migration and invasion, we assessed whether oxymatrine had affection on F-actin reorganization in TNF-α-treated RA FLS. As shown in Fig. 5, TNF-α treatment increased actin stress fiber formation and treatment with oxymatrine reduced intensity of F-actin stress fibers.
3.4. Oxymatrine inhibits the migration and invasion of RA FLS The migration of RA FLSs is a critical process in cartilage and bone destruction and differs from proliferation functionally. In vitro woundhealing assays demonstrated that the oxymatrine significantly impaired the migration capacity of RA FLSs (Fig. 4B). In Fig. 4A and C, oxymatrine treatment also suppressed FBS-treated migration and invasion of the cells and the effect was dose-dependently inhibited by oxymatrine.
3.6. Oxymatrine regulates NF-κB activation and MAPK activation Since the NF-κB is a key regulator of proinflammatory cytokine and proliferation in TNF-α-induced FLS, we invested the effect of oxymatrine on the NF-κB pathway. We found a dramatically decrease in 109
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Fig. 7. Effect of oxymatrine on severity of arthritis and serum levels of cytokines in mice with collagen-induced arthritis (CIA). (A and B) Effect of oxymatrine on articular index and paw swelling (change in paw thick). C, Histologic appearance in the joints of CIA mice treated with DMSO or oxymatrine. H&E staining was used to observe synovial exudation, synovial infiltration, cartilage erosion and bone loss. Lower panel shows safranin O/Fast green staining to proteoglycan depletion. Original magnification × 100. Values in A and B are the mean ± SD. * = P < 0.05 versus DMSO. D, Effect of oxymatrine on serum levels of cytokines of CIA mice. The levels of IL-6 and TNF-α were measured by ELSIA. Values are the mean ± SD. * = P < 0.05 versus normal control, # = P < 0.05 versus DMSO. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
investigated whether oxymatrine had any effect on MAPK (p38, JNK and ERK) activation in response to TNF-α. As shown in Fig. S2, the phosphorylation of p38, JNK and ERK was clearly up-regulated after treatment with TNF-α. However, oxymatrine treatment robustly suppressed the TNF-α-induced phosphorylation of p38 and JNK. In contrast, oxymatrine had no role in TNF-α-induced ERK activation. These results indicated that oxymatrine inhibited NF-κB and MAPK
phosphorylated IKKβ after the treatment with oxymatrine in TNF-αinduced RA FLSs (Fig. 6A). Oxymatrine treatment also suppressed TNFα-stimulated phosphorylation and degradation of IκBα. The cells treated with TNF-α increased p65 nuclear accumulation and oxymatrine reduced p65 nuclear accumulation (Fig. 6B). Since MAPK signaling pathways are crucial for FLS migration, proliferation, and inflammatory cytokine expression, we next 110
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together, oxymatrine may be effective in inhibiting synovial activation of RA. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2017.12.006.
activation in response to TNF-α in RA FLSs. 3.7. Oxymatrine reduces synovial inflammation and suppresses serum cytokines in CIA mice
Acknowledgments
The result that oxymatrine inhibits in vitro RA FLSs makes us further evaluate its anti-inflammatory role in vivo. Collagen-induced arthritis (CIA) mice were used in our experiment. Intraperitoneal injection of oxymatrine suppressed the increase in the clinical score and the paw thick compared with that of animals treated with DMSO (Fig. 7A and B). Histologic analysis of CIA joints showed severe synovial hyperplasia, increasing inflammatory cells infiltration and bone erosion. However, these pathologic changes were inhibited in oxymatrinetreated animals (Fig. 7C). Meanwhile, the serum TNF-α and IL-6 levels were elevated in mice with CIA as compared with normal healthy mice. Treatment with oxymatrine resulted in markedly reduced in serum TNF-α and IL-6 levels (Fig. 7D).
This work was supported by National Natural Science Foundation of China (Less Developed regions) (Grant No 81560817); the Guizhou Provincial Science and Technology Funds, China (Grant No [2013] 2074); the Research Foundation for Young Scholars of Education Bureau of Guizhou Province (Grant No [2015]438); Project supported by the State Key Program for Basic Research of Guizhou Province (Grant No. [2015]2002). Competing interests None declared.
4. Discussion References Oxymatrine has been showed to have many effects on tumor disease activities and inflammatory associated diseases [8,11,14]. The present work of our study demonstrates that oxymatrine can effect prevent inflammation, proliferation migration and invasion of RA FLS. We also found that oxymatrine suppressed TNF-α-induced RA FLS NF-κB activation. Oxymatrine also attenuates the severity of synovial inflammation and joint destruction in mice with CIA. These studies show that oxymatrine effectively inhibits inflammation and migration and it might be a novel drug for RA. RA synovium produces many pro-inflammatory cytokines and chemokines, such as IL-6 and IL-8. And the high concentration inflammatory mediators in the synovial and serum play important roles in the pathogenesis of RA [16,17]. Under the condition of proinflammatory cytokines, FLSs can produce the chemokines and MMPs to promote inflammation and bone destruction [18]. In our study, we provided first evidence for the inflammatory mediators-suppressive activity of oxymatrine in RA-FLSs. Synovial hyperplasia is a key pathophysiological characteristic and causes the damage of articular cartilage and bone [19]. An overabundance of FLSs forms the synovial hyperplasia. The proliferation FLSs in RA is mainly caused by an imbalance of cell proliferation and death [20]. Apart from suppression of cell proinflammatory cytokines, oxymatrine showed the ability to inhibit proliferation of RA-FLSs. FLS motivation is seem as an critical mechanism to enhance the damage cartilage and bone erosion [21]. It is an important step in the aggravation of RA of the FLS migration to cartilage and bone [22]. In our study, we found that oxymatrine suppressed inflammation, proliferation and migration in RA FLS. The outcome from in vivo that oxymatrine decreased synovial inflammation and bone destruction in CIA mice further sustained that oxymatrine might be a therapeutic agent for RA. In TNF-α-induced inflammation diseases, NF-κB is an important signal transcription factor for controlling immune and inflammatory response [23]. NF-κB regulates the expression of pro-inflammatory genes and many studies have showed that it is regarded as a critical signaling factor in regulating synovial hyperplasia, inflammation and migration [24]. A former study showed that the NF-κB activity in intestinal inflammation was significantly suppressed by oxymatrine [10]. To confirm the mechanisms of oxymatrine in controlling synovial inflammatory responses, the effect of oxymatrine on NF-κB activation was observed. Our study demonstrated that oxymatrine suppressed NF-κB activation in TNF-α-induced FLS. It is suggested that the suppression of NF-κB activation might be a main mechanism of the suppression of proinflammatory medium production and proliferation by oxymatrine. In conclusion, we have found that oxymatrine could reduce TNF-αinduced production of IL-6 and IL-8 and proliferation and migration via inhibition of NF-κB signaling pathway in RA FLS. Taking these data
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Oxymatrine prevents synovial inflammation and migration via blocking NFκB activation in rheumatoid fibroblast-like synoviocytes ⁎
T
⁎⁎
Jiang Lianga, ,1, Boyang Changb,1, Mingcheng Huangc,1, Weichen Huangd, , Wukai Maa, Yang Liua, Wan Taia, Yi Longa, Yun Lua a
Collaborative Innovation Center of Miao medicine, Guiyang College of Traditional Chinese Medicine, Guiyang, Guizhou, China State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China c Department of Hematology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China d Department of the first Orthopedics, The 2nd Affiliated Hospital, Guiyang College of Traditional Chinese Medicine, Guiyang, Guizhou, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Oxymatrine Fibroblast-like synoviocytes Inflammation Migration NF-κB
The fibroblast-like synoviocytes (FLSs) has the aggressive phenotype, which is very important for cartilage destruction in rheumatoid arthritis (RA). To the pathology of RA, the increased FLSs migration, activation and proliferation are essential factors. Oxymatrine is a traditional Chinese herb, which is the extraction from the root of Sophora flavescens and regarded as quinolizidine alkaloid compounds and has been shown to inhibit inflammation, proliferation and migration in vitro or vivo. However, whether oxymatrine effects in the treatment of RA FLSs is undefined. In our study, the inhibition of oxymatrine in RA FLSs inflammation, proliferation and migration in RA FLS are evaluated. We found that oxymatrine decreased the IL-6 and IL-8 expression and the proliferation, migration and invasion of RA FLSs. We also evaluated the molecular mechanisms and we found the effect of oxymatrine on NF-κB activation. The results showed that oxymatrine inhibited the activity of NF-κB. And the treatment activity of oxymatrine on collagen-induced arthritis (CIA) was further explored by us. Thus, we conclude that oxymatrine may protect joint destruction of RA by inhibiting synoviocyte activation, migration, invasion, and proliferation.
1. Introduction Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterized by the aggressive inflammation and destruction of cartilage and bone [1]. Fibroblast-like synoviocytes (FLSs) are fundamental in the maintenance of synovial inflammation and aggressive joint destruction in RA [2,3]. Activated FLSs in RA show the tumor-like characteristics, for example, the constant growth and resistance to apoptosis; and in the formation of pannus by migration and invasion towards cartilage and bone, they are also in important position [4,5,6]. Evidence increasingly suggests that inhibiting FLS inflammation and migration may have novel therapeutic potential for RA [7]. Therefore, many efforts have been made to modulate the inflammatory mediators, proliferation and migration behaviors of RA FLSs and the inhibition of expression of inflammatory mediators, proliferation and migration of
FLSs might be a treatment for RA. Oxymatrine is the quinolizidine alkaloid extraction, which is from the Chinese herb Sophora flavescens Ait [8,9]. Oxymatrine has several pharmacological effects including anticancer, antiviral activities and anti-inflammatory [10,11]. Many studies have indicated that oxymatrine suppresses cancer cell proliferation, migration and invasion [12,13]. Oxymatrine also suppresses lipopolysaccharide (LPS) treated inflammation via regulating TLR4/NF-κB in macrophages and inhibits the inflammation of LPS-induced mastitis [14]. Recent studies have indicated that by regulating the imbalance between Treg and Th17 cells [15], oxymatrine has beneficial effects in an autoimmune arthritis mouse model. However, the anti-inflammatory, anti-proliferative and anti-migratory effects of oxymatrine on RA FLS have not been explored. Therefore, in our work, we determined whether oxymatrine reduced proinflammatory cytokines production and the proliferation and
⁎ Correspondence to: J. Liang, Collaborative Innovation Center of Miao Medicine, Guiyang College of Traditional Chinese Medicine, Huaxi University Town, Guiyang, Guizhou 550025, China. ⁎⁎ Correspondence to: W. Huang, Department of the First Orthopedics, The 2nd Affiliated Hospital, Guiyang College of Traditional Chinese Medicine, Guiyang, Guizhou, Feishan Street, 510000, China. E-mail addresses: [email protected] (J. Liang), [email protected] (W. Huang). 1 JL, BC and MH contributed equally to this work.
https://doi.org/10.1016/j.intimp.2017.12.006 Received 8 October 2017; Received in revised form 22 November 2017; Accepted 6 December 2017 1567-5769/ © 2017 Elsevier B.V. All rights reserved.
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migration of RA FLS and the molecular mechanisms involved. 2. Materials and methods 2.1. Patients and isolation of FLS We performed the study according to the recommendations of the Declaration of Helsinki and our study had got the consent from the Medical Ethical Committee of the Guiyang College of Traditional Chinese Medicine and the First Affiliated Hospital, Sun Yat-sen University. We also got the consent from all patients involved for taking part in the study. Synovial tissues were gained from 8 RA patients (3 men and 5 women aged from 35 to 60 years old), who were undergoing joint replacement or synovectomy. The synovial tissue was sliced into pieces and digested with collagenase I (Sigma-Aldrich, St. Louis, MO, USA) which was put in DMEM/F12 medium to isolate synoviocytes for 4 h at the incubator of 37 °C. They were washed with PBS and then grown in 10% fetal bovine serum of DMEM/F12, which contained 100 U/mL penicillin and 100 μg/mL streptomycin in a 5% CO2, 21% O2, and 75% N2 humidified incubator at 37 °C. Passages 3 to 5 of RA FLSs were used for experiments.
Fig. 1. Effects of oxymatrine on RA FLS. RA-FLSs were treated with indicated concentrations of oxymatrine for 48 h, and cell viability was measured using the MTT assay. Result shown is the mean ± SD. *P < 0.05, oxymatrine-treated versus vehicle-treated cells.
2.2. Cell viability assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to test cell viability. Firstly, RA FLSs were played in 96-well plates and they were incubated with either DMSO or the indicated concentration of oxymatrine. After 48 h and 72 h of treatment, MTT (0.5 mg/mL; Sigma-Aldrich, USA) was added to incubate for 4 h. The media containing MTT was removed, and the DMSO was used to dissolve the formed formazan crystals. The viable cells were tested by microplate reader (BioRad, Hercules, CA) to read the absorbance at 540 nm in. 2.3. Migration and invasion assay of FLS We performed the cell migration assays by using Transwell inserts (Transwell; Corning Inc., NY, USA). DMEM/F12 medium containing 10% FBS as the chemoattractant was used in the bottom chambers; and RA FLSs with oxymatrine was added in the upper chambers with oxymatrine for 24 h in serum free DMEM/F12 medium. The top membrane surface cells were removed when they were incubated for 12 h. The migrated cells which were on the bottom side of the membrane were fixed in methanol and they were stained with 0.1% Crystal Violet and counted under a microscope. For the invasion assay, we used inserts coated with a Matrigel basement membrane matrix (BD Biosciences, Oxford, UK) to perform similar experiments. The chemoattractant was DMEM/F12 medium, which contains 15% FBS. The 200 μL DMEM/F12 medium and FLS were seeded in the top chambers with oxymatrine for 24 h. And then after 24 h, the non-invaded cells were removed from the upper side of the filter by using a cotton swab. The invaded cells were dyed with 0.1% crystal violet and calculated by microscope.
Fig. 2. Effects of oxymatrine on pro-inflammatory cytokines expression in RA FLSs. After treatment with or without oxymatrine for 24 h, RA FLS were stimulated with TNF-α (10 ng/mL) for 12 (for IL-6 and IL-8 mRNA) or 24 (for IL-6 and IL-8 secretion) hours. The levels of IL-6 and IL-8 mRNA and in cultured cell supernatants were measured by quantitative real-time PCR and ELISA. Data are representative of five independent experiments from different patients. A, mRNA expression of IL-6 and IL-8 was determined by quantitative real-time PCR. Data were normalized to GAPDH. B, the levels of IL-6 and IL-8 in cultured cell supernatants were measured by ELISA. All values represent mean ± SD. *P < 0.05 vs control, #P < 0.05 vs TNF-α.
2.4. Wounding migration
without TNF-α (10 ng/mL) or oxymatrine for 24 h and the 5-Ethynyl-2′deoxyuridine (EdU) Cell Proliferation Assay Kit was used to measure the cell proliferation following the manufacturer's protocol.
In brief, RA-FLSs were planted onto 12-well plates and grown to confluence. A linear wound was generated on the cell monolayer by using pipette tips and treated with oxymatrine for 24 h. After 24 h of oxymatrine treatment, migration was evaluated by counting the cells which had shifted beyond a reference line. The cells were photographed by a microscope.
2.6. Confocal laser scanning fluorescence microscopy We grew cells on glass coverslips to have a detection of the p65 expression and cytoskeletal filamentous actin (F-actin) in RA FLSs. The RA FLSs were treated with oxymatrine for 24 h and then were treated with TNF-α (10 ng/mL) for 12 h. Then they were immobilized with paraformaldehyde; meanwhile, they were permeated with 0.1%
2.5. Proliferation assays RA FLSs were grown to 90% confluence and were stimulated with or 106
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Fig. 3. Effect of oxymatrine on proliferation in RA FLSs. EdU incorporation assays were conducted to assess cell proliferation in RA-FLSs after 24 h treatment with oxymatrine and then stimulated with TNF-α (10 ng/mL) for 24 h. Representative images from 3 independent experiments. All values represent mean ± SD. *P < 0.05 vs. control, #P < 0.05 vs. TNF-α.
Fig. 4. Effect of oxymatrine on migration and invasion of RA FLS. A, Migration was performed in a Boyden chamber and chemotaxis was quantified by counting the migrated cells. Photo images of migration of RA FLS treated with oxymatrine. B, In vitro wound-healing assays. Wound closure was quantified after 24 h culturing in the presence or absence of oxymatrine. C, Transwell invasion assays were done to determine the invasion of RA FLSs after treatment with oxymatrine for 24 h. Photo images of invasion of RA FLS treated with oxymatrine. All values represent mean ± SD. *P < 0.05 vs control.
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Fig. 5. Effect of oxymatrine on cytoskeleton alterations of RA FLS. Effect of oxymatrine on alterations of actin cytoskeleton in TNF-α-treated RA FLS. F-actin (red) and nucleus (blue) were stained with phalloidin and DAPI, respectively. Representative images from three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
times in TBST and then incubated with blocking buffer for 1 h at room temperature. The primary antibodies were purchased from Abcam (Cambridge, MA, USA) and were diluted 1:1000 for phospho-IKKβ, phospho-IκBα, phospho-JNK, phospho-p38, phospho-ERK, IκBα, p38, ERK, JNK and C/EBPbeta and they were used to incubate the membranes overnight at 4 °C. Then the secondary antibodies were used to incubate the membranes for 60 min at room temperature. They were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, USA) reaction.
TritonX-100 in PBS. For detecting F-actin, we incubated the cells with phalloidin for 12 h. We incubated the cells with DAPI and mounted the coverslips on glass slides by using antifade mounting media; and we examined the cells with a confocal fluorescence microscopy (Zeiss LSM710). ELISA. By using TNF-α (10 ng/mL), we stimulated the RA FLS for 24 h with oxymatrine. According to the usages of the manufacturer (R&D systems, Minneapolis, MO, USA), the cell culture supernatants were gathered for detection of IL-6 and IL-8 by ELISA kits.
2.10. Statistical analysis 2.7. RNA isolation and quantitative real-time PCR analysis All experiments and statistical analysis were carried under the software of SPSS 13.0. Data were shown as means ± SD. The MannWhitney U test or Student's t-test was used to evaluate the differences between the experimental groups. P < 0.05 were considered statistically significant.
After 24 hours' treatments with oxymatrine, we stimulated the cells with TNF-α for 12 h. TRIzol (Sigma) was used to extract total RNAs and by using the miScript Reverse Transcription Kit, we reverse transcribed the RNAs to cDNA. Then by using QuantiTect SYBR Green RT-PCR Kit on StepOnePlusTM Real Time PCR System (Applied Biosystems), we performed the quantitative real time polymerase chain reaction (PCR) tests for the expression of IL-6, IL-8, MMP1, MMP3, MMP9 and MMP13 on cDNA.
3. Results 3.1. Oxymatrine effect on RA FLS viability MTT assays showed that treatment with oxymatrine for 48 h had no effects on the viability of RA FLSs (Fig. 1). And the results showed that the inhibitory effect observed in the inflammation and migration was not due to cytotoxic effects.
2.8. CIA induction and oxymatrine treatment In brief, the DBA/1 mice of 8–9 weeks were immunized intradermally at the tail with 200 μg diluted in acetic acid of bovine type II collagen which was emulsified in same volume of Freund's complete adjuvant (2 mg/mL, chondrex). Mice were boosted by administering 200 μg type II collagen intraperitoneally on day 21. Treatment with 5% DMSO (as control) and 100 mg/kg oxymatrine daily was started on the day when arthritis occurred for 14 days. The mice were monitored daily for signs of arthritis, and arthritis severity was scored on a scale of 0–3, as described previously. The total score was recorded as the sum of the scores in the four limbs. And the paw thick was tested by the vernier caliper. After the mice killed, the whole hind and fore limbs were fixed in formalin for 24–48 h and they were paraffin embedded. Haematoxylin and eosin (H&E) or safranin O-fast green was used to stain the tissue sections. And we tested the serum levels of IL-6 and TNF-α by ELIAS kits according to the usages of the manufacturer.
3.2. Oxymatrine suppresses pro-inflammatory mediators in RA FLSs In pathogenesis of RA, IL-6 and IL-8 are critical proinflammatory cytokines. To explore the possible inhibitory effects of oxymatrine in pro-inflammatory cytokines production, we detected the effect of oxymatrine on TNF-α-induced IL-6 and IL-8 gene expression; meanwhile, we also examined the secretion of IL-6 and IL-8 in RA FLS. As expected in Fig. 2, TNF-α treatment increased the IL-6 and IL-8 gene expression and promoted the secretion of RA FLSs. Oxymatrine treatment inhibited TNF-α-treated IL6 and IL-8 mRNA expression and the secretion in RA FLS.(Fig. 2A and B). MMPs is an essential characteristic of the invasive phenotype of RA FLSs. We also examined the effect of oxymatrine on TNF-α-induced MMP gene expression. As shown in Fig. S4, oxymatrine markedly suppressed the TNF-α-induced mRNA expression of MMP-1, MMP-3, MMP-9, and MMP-13 compared with DMSO.
2.9. Western blot analysis We lysed the cells in a lysis buffer which contained protease inhibitor mixture which was purchased from Thermo Scientific. After the cells were incubated for 15 min at the temperature of 4 °C, the supernatant was gathered by centrifugation (12,000 rpm, 15 min, at 4 °C). The BCA protein assay reagent kit (Pierce, Rockford, IL, USA) was used to determine the protein concentrations and 12% SDS-PAGE gels were used to separate the equal amounts of protein and they were transferred onto Nitrocellulose membranes. Membranes were washed by three
3.3. Oxymatrine inhibits proliferation in RA FLSs Previous studies show that oxymatrine inhibits tumor cell proliferation and cell proliferation is critical for RA. Thus, EdU incorporation was used to test the effect of oxymatrine on TNF-α-treated RA FLSs proliferation. As shown in Fig. 3, oxymatrine treatment 108
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Fig. 6. Oxymatrine inhibits TNF-α induced relocation of p65 and degradation of IκBα. A, Immunofluorescence staining analysis of p65 localization. RA FLS were preincubated with oxymatrine for 24 h and TNF-α (10 ng/mL) for 15 min, fixed, and then underwent immunofluorescence analysis. B, After treatment with or without oxymatrine for 24 h, RA FLS were stimulated with TNF-α (10 ng/mL) for 15 min. Phosphorylation of IKK and degradation and phosphorylation of IκBα induced by TNF-α was analysed using western blot analysis. Densitometry was performed and fold change of protein expression is shown below the corresponding band. All values represent mean ± SD. *P < 0.05 vs control, #P < 0.05 vs TNF-α.
3.5. Oxymatrine suppresses cytoskeleton alterations of RA FLS
potently suppressed TNF-α-treated proliferation of FLSs in a concentration-dependent manner.
To further verify the effect of oxymatrine on FLS migration and invasion, we assessed whether oxymatrine had affection on F-actin reorganization in TNF-α-treated RA FLS. As shown in Fig. 5, TNF-α treatment increased actin stress fiber formation and treatment with oxymatrine reduced intensity of F-actin stress fibers.
3.4. Oxymatrine inhibits the migration and invasion of RA FLS The migration of RA FLSs is a critical process in cartilage and bone destruction and differs from proliferation functionally. In vitro woundhealing assays demonstrated that the oxymatrine significantly impaired the migration capacity of RA FLSs (Fig. 4B). In Fig. 4A and C, oxymatrine treatment also suppressed FBS-treated migration and invasion of the cells and the effect was dose-dependently inhibited by oxymatrine.
3.6. Oxymatrine regulates NF-κB activation and MAPK activation Since the NF-κB is a key regulator of proinflammatory cytokine and proliferation in TNF-α-induced FLS, we invested the effect of oxymatrine on the NF-κB pathway. We found a dramatically decrease in 109
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Fig. 7. Effect of oxymatrine on severity of arthritis and serum levels of cytokines in mice with collagen-induced arthritis (CIA). (A and B) Effect of oxymatrine on articular index and paw swelling (change in paw thick). C, Histologic appearance in the joints of CIA mice treated with DMSO or oxymatrine. H&E staining was used to observe synovial exudation, synovial infiltration, cartilage erosion and bone loss. Lower panel shows safranin O/Fast green staining to proteoglycan depletion. Original magnification × 100. Values in A and B are the mean ± SD. * = P < 0.05 versus DMSO. D, Effect of oxymatrine on serum levels of cytokines of CIA mice. The levels of IL-6 and TNF-α were measured by ELSIA. Values are the mean ± SD. * = P < 0.05 versus normal control, # = P < 0.05 versus DMSO. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
investigated whether oxymatrine had any effect on MAPK (p38, JNK and ERK) activation in response to TNF-α. As shown in Fig. S2, the phosphorylation of p38, JNK and ERK was clearly up-regulated after treatment with TNF-α. However, oxymatrine treatment robustly suppressed the TNF-α-induced phosphorylation of p38 and JNK. In contrast, oxymatrine had no role in TNF-α-induced ERK activation. These results indicated that oxymatrine inhibited NF-κB and MAPK
phosphorylated IKKβ after the treatment with oxymatrine in TNF-αinduced RA FLSs (Fig. 6A). Oxymatrine treatment also suppressed TNFα-stimulated phosphorylation and degradation of IκBα. The cells treated with TNF-α increased p65 nuclear accumulation and oxymatrine reduced p65 nuclear accumulation (Fig. 6B). Since MAPK signaling pathways are crucial for FLS migration, proliferation, and inflammatory cytokine expression, we next 110
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together, oxymatrine may be effective in inhibiting synovial activation of RA. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2017.12.006.
activation in response to TNF-α in RA FLSs. 3.7. Oxymatrine reduces synovial inflammation and suppresses serum cytokines in CIA mice
Acknowledgments
The result that oxymatrine inhibits in vitro RA FLSs makes us further evaluate its anti-inflammatory role in vivo. Collagen-induced arthritis (CIA) mice were used in our experiment. Intraperitoneal injection of oxymatrine suppressed the increase in the clinical score and the paw thick compared with that of animals treated with DMSO (Fig. 7A and B). Histologic analysis of CIA joints showed severe synovial hyperplasia, increasing inflammatory cells infiltration and bone erosion. However, these pathologic changes were inhibited in oxymatrinetreated animals (Fig. 7C). Meanwhile, the serum TNF-α and IL-6 levels were elevated in mice with CIA as compared with normal healthy mice. Treatment with oxymatrine resulted in markedly reduced in serum TNF-α and IL-6 levels (Fig. 7D).
This work was supported by National Natural Science Foundation of China (Less Developed regions) (Grant No 81560817); the Guizhou Provincial Science and Technology Funds, China (Grant No [2013] 2074); the Research Foundation for Young Scholars of Education Bureau of Guizhou Province (Grant No [2015]438); Project supported by the State Key Program for Basic Research of Guizhou Province (Grant No. [2015]2002). Competing interests None declared.
4. Discussion References Oxymatrine has been showed to have many effects on tumor disease activities and inflammatory associated diseases [8,11,14]. The present work of our study demonstrates that oxymatrine can effect prevent inflammation, proliferation migration and invasion of RA FLS. We also found that oxymatrine suppressed TNF-α-induced RA FLS NF-κB activation. Oxymatrine also attenuates the severity of synovial inflammation and joint destruction in mice with CIA. These studies show that oxymatrine effectively inhibits inflammation and migration and it might be a novel drug for RA. RA synovium produces many pro-inflammatory cytokines and chemokines, such as IL-6 and IL-8. And the high concentration inflammatory mediators in the synovial and serum play important roles in the pathogenesis of RA [16,17]. Under the condition of proinflammatory cytokines, FLSs can produce the chemokines and MMPs to promote inflammation and bone destruction [18]. In our study, we provided first evidence for the inflammatory mediators-suppressive activity of oxymatrine in RA-FLSs. Synovial hyperplasia is a key pathophysiological characteristic and causes the damage of articular cartilage and bone [19]. An overabundance of FLSs forms the synovial hyperplasia. The proliferation FLSs in RA is mainly caused by an imbalance of cell proliferation and death [20]. Apart from suppression of cell proinflammatory cytokines, oxymatrine showed the ability to inhibit proliferation of RA-FLSs. FLS motivation is seem as an critical mechanism to enhance the damage cartilage and bone erosion [21]. It is an important step in the aggravation of RA of the FLS migration to cartilage and bone [22]. In our study, we found that oxymatrine suppressed inflammation, proliferation and migration in RA FLS. The outcome from in vivo that oxymatrine decreased synovial inflammation and bone destruction in CIA mice further sustained that oxymatrine might be a therapeutic agent for RA. In TNF-α-induced inflammation diseases, NF-κB is an important signal transcription factor for controlling immune and inflammatory response [23]. NF-κB regulates the expression of pro-inflammatory genes and many studies have showed that it is regarded as a critical signaling factor in regulating synovial hyperplasia, inflammation and migration [24]. A former study showed that the NF-κB activity in intestinal inflammation was significantly suppressed by oxymatrine [10]. To confirm the mechanisms of oxymatrine in controlling synovial inflammatory responses, the effect of oxymatrine on NF-κB activation was observed. Our study demonstrated that oxymatrine suppressed NF-κB activation in TNF-α-induced FLS. It is suggested that the suppression of NF-κB activation might be a main mechanism of the suppression of proinflammatory medium production and proliferation by oxymatrine. In conclusion, we have found that oxymatrine could reduce TNF-αinduced production of IL-6 and IL-8 and proliferation and migration via inhibition of NF-κB signaling pathway in RA FLS. Taking these data
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