NCX 4040, a nitric oxide-donating aspirin derivative, inhibits Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages

Author’s Accepted Manuscript NCX 4040, a nitric oxide-donating aspirin derivative, inhibits Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages Eun-Young Choi, So-Hui Choe, Jin-Yi Hyeon, Hae Ryoun Park, Jeom-Il Choi, In Soon Choi, Sung-Jo Kim

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S0014-2999(15)30313-7 http://dx.doi.org/10.1016/j.ejphar.2015.10.032 EJP70289

To appear in: European Journal of Pharmacology Received date: 25 July 2015 Revised date: 15 September 2015 Accepted date: 19 October 2015 Cite this article as: Eun-Young Choi, So-Hui Choe, Jin-Yi Hyeon, Hae Ryoun Park, Jeom-Il Choi, In Soon Choi and Sung-Jo Kim, NCX 4040, a nitric oxidedonating aspirin derivative, inhibits Prevotella intermedia lipopolysaccharideinduced production of proinflammatory mediators in murine macrophages, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.10.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NCX 4040, a nitric oxide-donating aspirin derivative, inhibits Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages

Eun-Young Choi1, So-Hui Choe1, Jin-Yi Hyeon1, Hae Ryoun Park2,5, Jeom-Il Choi3,4, In Soon Choi1 and Sung-Jo Kim,3,4,5

1

Department of Biological Science, College of Medical and Life Sciences, Silla University,

Busan, Korea; 2Department of Oral Pathology, School of Dentistry, Pusan National University, Yangsan, Gyeongsangnam-do, Korea;3Department of Periodontology, School of Dentistry, Pusan National University, Yangsan, Gyeongsangnam-do, Korea; 4Dental Research Institute, Pusan National University Dental Hospital, Yangsan, Gyeongsangnam-do, Korea; 5Institute of Translational Dental Sciences, Pusan National University, Yangsan, Gyeongsangnam-do, Korea

Correspondence:

Sung-Jo Kim Department of Periodontology, School of Dentistry Pusan National University 49 Busandaehak-ro, Mulgeum-eup, Yangsan Gyeongsangnam-do 626-870, Korea Tel: +82 55 360 5201, Fax: +82 55 360 5194 e-mail: [email protected]

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Abstract In this study, the effects and underlying mechanisms of NCX 4040, a nitric oxide (NO)donating aspirin derivative, on the production of proinflammatory mediators were examined using murine macrophages exposed to lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in the etiology of periodontal disease. NCX 4040 significantly reduced P. intermedia LPS-induced production of inducible NO synthase (iNOS)-derived NO, IL-1β and IL-6 as well as their mRNA expression in RAW264.7 cells. Notably, NCX 4040 was much more effective than the parental compound aspirin in reducing LPS-induced production of inflammatory mediators. NCX 4040 induced the expression of heme oxygenase-1 (HO-1) in cells treated with P. intermedia LPS, and the suppressive effect of NCX 4040 on LPS-induced NO production was significantly reversed by SnPP, a competitive HO-1 inhibitor. NCX 4040 did not influence LPS-induced phosphorylation of JNK and p38. IκB-α degradation as well as nuclear translocation and DNA-binding activity of NF-κB p65 and p50 subunits induced by P. intermedia LPS were significantly reduced by NCX 4040. Besides, LPS-induced phosphorylation of STAT1 and STAT3 was significantly down-regulated by NCX 4040. Further, NCX 4040 elevated the SOCS1 mRNA in cells stimulated with LPS. This study indicates that NCX 4040 inhibits P. intermedia LPS-induced production of NO, IL-1β and IL6 in murine macrophages through anti-inflammatory HO-1 induction and suppression of NFκB, STAT1 and STAT3 activation, which is associated with the activation of SOCS1 signaling. NCX 4040 could potentially be a promising tool in the treatment of periodontal disease, although further studies are required to verify this.

Key words: NCX 4040, lipopolysaccharide, proinflammatory mediators, HO-1, NF-κB, STAT, SOCS1 2

1. Introduction

Periodontal disease is defined as chronic inflammatory disease induced and perpetuated by several specific pathogenic bacteria present in the plaque biofilm. Periodontal disease leads to loss of connective tissue attachment, progressive destruction of alveolar bone and, ultimately, tooth loss (Williams, 1990). Periodontal disease is associated with an increased risk of several systemic disorders such as cardiovascular disease, diabetes mellitus and stroke (Seymour et al., 2007). Prevotella intermedia, a gram-negative anaerobe, has been implicated in the etiology of chronic periodontitis (Braga et al., 2010), necrotizing ulcerative gingivitis (Chung et al., 1983) and pregnancy gingivitis (Kornman and Loesche, 1980). This bacterium has a variety of virulence factors important in the pathogenesis of periodontal disease (Shenker et al., 1991; Beem et al., 1998; Guan et al., 2009). Lipopolysaccharide (LPS) is a dominant constituent of outer membrane of gramnegative bacteria, including P. intermedia. It leads to the production of various proinflammatory mediators, such as TNF-α, IL-1β and IL-6, and nitric oxide (NO), in a variety of immune cells (Morrison and Ryan, 1987). Host response against periodontal microbiota and their products is manifested by increased production of various proinflammatory mediators by the cells of periodontal tissues (Rossomando et al., 1990; Matejka et al., 1998; Mogi et al., 1999), and these mediators are important for bone destruction in periodontal disease (Tatakis, 1993; Kobayashi et al., 2000; Pelt et al., 2002; Liu et al., 2005). Therefore, host modulatory strategies targeting these destructive mediators can lead to better clinical outcome in the management of periodontal 3

disease (Reddy et al., 2003; Preshaw, 2008). Non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, are a class of drugs that commonly used to treat acute or chronic conditions where pain and inflammation are present. Unfortunately, several important side-effects associated with the long-term use have been reported for NSAIDs, such as gastrointestinal toxicity, which considerably limit their clinical use (Segasothy et al., 1994; Soll, 1998). In an effort to overcome these adverse side-effects, NO-donating NSAIDs (NO-NSAIDs) were developed as hybrid drugs. NONSAIDs are synthesized by coupling an NO-donating moiety to a conventional NSAID molecule (Burgaud et al., 2002). NO-NSAIDs have been shown to exhibit a significantly higher activity and a much lower toxicity compared to the parental drug (Williams et al., 2001). NCX 4040, a NO-donating aspirin derivative, consists of an aspirin backbone to which a NO-donating group is covalently linked via a spacer molecule (Tesei et al., 2008). NCX 4040 has been reported to have antitumor activities against colon, pancreatic and ovarian cancer (Tesei et al., 2005; Rosetti et al., 2006; Bratasz et al., 2008). In addition, there is evidence that NCX 4040 is potentially anti-inflammatory in LPS-stimulated cells, which suggests that this agent has potential in the treatment of chronic inflammatory diseases, including periodontal disease (Ricciotti et al., 2010). In this study, the effects and underlying mechanisms of NCX 4040 on the production of proinflammatory mediators were examined using the mouse macrophage cell line RAW264.7 exposed to LPS isolated from P. intermedia, a pathogen implicated in the etiology of inflammatory periodontal disease.

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2. Materials and methods

2.1. Reagents and antibodies We obtained NCX 4040 from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against HO-1, NF-κB p65, NF-κB p50, β-actin and PCNA were the products of Santa Cruz Biotechnology (Santa Cruz, CA, USA), and those to JNK, phospho-JNK, p38, phospho-p38, IκB-α, STAT1, phospho-STAT1, STAT3 and phospho-STAT3 were obtained from Cell Signaling Technology (Beverly, MA, USA). Tin protoporphyrin IX (SnPP) was acquired from Frontier Scientific Inc. (Logan, UT, USA). Unless stated otherwise, all other reagents used in this study were purchased from Sigma-Aldrich.

2.2. Bacterial culture and isolation of LPS P. intermedia ATCC 25611 was cultured using GAM broth (Nissui, Tokyo, Japan) supplemented with hemin (5 μg/ml) and vitamin K (1 μg/ml) in an anaerobic atmosphere at 37 °C. LPS was obtained by standard hot phenol-water extraction from freeze-dried bacterial cells, and any contaminating proteins and nucleic acids were removed from LPS preparation by treatment with proteinase K, DNase and RNase as previously described in our work (Choi et al., 2011).

2.3. Cell culture and cytotoxicity assay The murine macrophage cell line RAW264.7 was purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), which was supplemented with 10% heat-inactivated FBS, 100 5

U/ml of penicillin and 100 μg/ml of streptomycin, at 37°C and 5% CO2 in humidified air as described previously (Choi et al., 2011). The cytotoxicity induced by NCX 4040 was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (Choi et al., 2011).

2.4. Measurement of NO generation The amount of NO in the culture media was determined by measuring the amount of accumulated nitrite (NO2-), a stable end product of NO, as described previously (Choi et al., 2011). Briefly, cells were treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml) for 24 h. After incubation, aliquots of 100 μl of cultured medium were added to the same volume of Griess reagent and incubated at room temperature for 10 min. The optical density value at a wavelength of 540 nm was read using a Spectra Max 250 ELISA Reader (Molecular Devices, Sunnyvale, CA, USA), and nitrite concentration was calculated using a NaNO2 standard calibration curve.

2.5. Measurement of IL-1β and IL-6 generation Cells were treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml) for 24 h (for IL-6) or 48 h (for IL-1β), after which the IL-1β and IL-6 levels in the culture supernatant were quantified using the corresponding enzyme-linked immunosorbent assay (ELISA) kits (OptEIA; BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions.

2.6. Real-time polymerase chain reaction analysis Cells were seeded into 60-mm tissue culture dishes at a density of 4×106 cells in a 6

volume of 4 ml and treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml) for the indicated periods of time. Real-time PCR analysis was carried out following the procedures as described earlier (Choi et al., 2011). Briefly, total RNA was isolated from the incubated cells by using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. An iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) was used for reverse transcription of total RNA into cDNA, and cDNA was amplified using a SsoFast EvaGreen Supermix (Bio-Rad) and CFX96 real-time PCR detection system (Bio-Rad). Real-time PCR conditions were as follows: After denaturing at 98°C for 30 s, PCR was performed for 45 cycles, each of which consisted of denaturing at 95°C for 1 s, annealing/extending at 60°C for 5 s. The following specific oligonucleotide primers were used: iNOS (130 bp) forward, 5´GCACCACCCTCCTCGTTCAG-3´ and reverse, 5´-TCCACAACTCGCTCCAAGATTCC3´; IL-1β (131 bp) forward, 5´-TTCAGGCAGGCAGTATCA-3´ and reverse, 5´AGGATGGGCTCTTCTTCAA-3´; IL-6 (162 bp) forward, 5´GCCAGAGTCCTTCAGAGAGATACAG-3´ and reverse, 5´GAATTGGATGGTCTTGGTCCTTAGC-3´; HO-1 (149 bp) forward, 5´CAATGTGGCCTTCTCTCTGT-3´ and reverse, 5´-TTTTGGTGAGGGAACTGTGT-3´; SOCS1 (133 bp) forward, 5´-CACTTCTGGCTGGAGACC-3´ and reverse, 5´TGGAGAGGTAGGAGTGGAA-3´; β-actin (149 bp) forward, 5´TGAGAGGGAAATCGTGCGTGAC-3´ and reverse, 5´GCTCGTTGCCAATAGTGATGACC-3´. The gene expression levels were normalized to the expression of β-actin.

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2.7. Immunoblot analysis Cells were seeded into 60-mm tissue culture dishes at a density of 4×106 cells in a volume of 4 ml and treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml) for the indicated periods of time. After incubation, immunoblot analysis was performed according to standard procedures as previously reported in our work (Choi et al., 2011). Briefly, nuclear and whole-cell protein extracts were prepared using the nuclear extract kit (Active Motif, Carlsbad, CA, USA) and ice-cold lysis buffer (50 mM Tris·Cl, 150 mM NaCl, 0.002% sodium azide, 0.1% SDS and 1% Nonidet P-40), respectively. Equal amounts of protein (30 μg for each lane) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and then blotted onto a nitrocellulose membrane. The membrane was probed with the relevant primary antibodies and then incubated with secondary antibodies that are horseradish peroxidase-conjugated conjugated. Immunoreactive bands were detected using the enhanced chemiluminescence detection reagent (Cell Signaling Technology).

2.8. Determination of DNA-binding activities of NF-κB subunits Cells were seeded into 60-mm tissue culture dishes at a density of 4×106 cells in a volume of 4 ml and treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml) for the indicated periods of time. The nuclear proteins were extracted as described above and analyzed for DNA-binding activities using the ELISA-based NF-κB transcription factor assay kit (Active Motif) as described previously (Choi et al., 2011).

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2.9. Statistical analysis Data are expressed as means  S.E.M. One-way analysis of variance (ANOVA) was used to analyze the significant differences within the groups. The comparison between two groups was determined by Tukey’s post-hoc comparisons. P values of <0.05 were considered statistically significant.

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3. Results

3.1. Effects of NCX 4040 on P. intermedia LPS-induced production of proinflammatory mediators The levels of NO, IL-1β and IL-6 were markedly increased when the cells were activated with 10 μg/ml of P. intermedia LPS compared to the control, and it was found that treatment of cells with NCX 4040 significantly reduced these proinflammatory mediators induced by LPS in a dose-dependent manner (Fig. 1). The inhibition of NO and IL-6 release at 30 μM of NCX 4040 was 89% and 97%, respectively. In addition, a 95% reduction in LPSinduced IL-1β production was noted in cells treated with 20 μM of NCX 4040. NCX 4040 did not exhibit any toxicity at the concentrations used in this study as assessed by MTT assay, suggesting that the inhibitory effect of NCX 4040 was not related to its nonspecific cytotoxicity (Fig. S1). We also determined the effects of the parental compound aspirin in reducing P. intermedia LPS-induced generation of proinflammatory mediators. It was found that aspirin reduced the production of IL-1β approximately by 24% at 20 μM (Fig. 1). However , at the highest concentration of 30 μM, aspirin failed to suppress NO and IL-6 production (Fig. 1). Immunoblot analysis results revealed that NCX 4040 dose-dependently reduced P. intermedia LPS-induced iNOS protein expression (Fig. 2). NCX 4040 also caused a marked decrease in the mRNA levels of iNOS, IL-1β and IL-6, which were significantly increased by P. intermedia LPS treatment. (Fig. 3).

3.2. Effect of NCX 4040 on HO-1 induction in cells treated with P. intermedia LPS To determine the potential mediation of HO-1 in the suppression by NCX 4040 of P. 10

intermedia LPS-induced generation of NO, IL-1β and IL-6, HO-1 induction was measured using immunoblot and real-time PCR analyses. NCX 4040 induced the expression of HO-1 protein as well as its mRNA in a dose-dependent manner in RAW264.7 cells treated with LPS from P. intermedia (Fig. 4A,B).

3.3. Effect of NCX 4040-mediated HO-1 induction on inhibition of P. intermedia LPSinduced production of proinflammatory mediators Because NCX 4040 provoked the expression of HO-1 in cells activated with P. intermedia LPS, we evaluated the likelihood that the NCX 4040-mediated suppression of NO, IL-1β and IL-6 is ascribed to HO-1 induction. Cells were stimulated with P. intermedia LPS (10 μg/ml) and NCX 4040 (30 or 20 μM) in the presence of different concentrations of SnPP, a synthetic heme analog that competitively inhibits HO-1, and levels of inflammatory mediators in the culture supernatant were quantified. As shown in Fig. 4C, the suppressive effect of NCX 4040 on P. intermedia LPS-induced production of NO was significantly reversed by SnPP. Nevertheless, attenuation of IL-1β and IL-6 production mediated by NCX 4040 was not influenced by SnPP treatment (data not shown).

3.4. Effects of NCX 4040 on P. intermedia LPS-induced phosphorylation of JNK and p38 The JNK, p38, NF-κB and JAK2/STAT1 pathways play a role in the generation of NO and IL-6 in RAW264.7 cells activated with P. intermedia LPS (Choi et al., 2011). These pathways, except JNK, are also involved in IL-1β production induced by P. intermedia LPS (data not shown). Besides, the STAT3 pathway mediates P. intermedia LPS-induced production of NO, IL-1β and IL-6 (data not shown). We first evaluated the likelihood that the 11

suppressive outcomes of NCX 4040 on P. intermedia LPS-induced elaboration of NO, IL-1β and IL-6 are associated with inactivation of JNK and p38 signaling. As shown in Fig. 5, LPSinduced phosphorylation of JNK and p38 was not affected by NCX 4040 treatment.

3.5. Effects of NCX 4040 on P. intermedia LPS-induced NF-κB transcriptional activity We analyzed the effect of NCX 4040 on NF-κB signaling pathway that is known to mediate P. intermedia LPS-induced production of NO, IL-1β and IL-6 in RAW264.7 cells. The cytoplasmic level of IκB-α protein was markedly diminished when the cells were activated with P. intermedia LPS compared to the control, and co-treatment of cells with NCX 4040 significantly and dose-dependently inhibited IκB-α degradation induced by LPS (Fig. 6A). We also assessed the influence of NCX 4040 on the translocation of NF-κB subunits into the nucleus in P. intermedia LPS-stimulated cells. LPS treatment induced nuclear translocation of NF-κB p65 and p50 subunits, and it was found that NCX 4040 dosedependently decreased translocation of p65 and p50 subunits into the nucleus (Fig. 6B). Further, we investigated whether the DNA-binding activitities of NF-κB subunits are influenced by NCX 4040. As shown in Fig. 6C, NCX 4040 significantly diminished NF-κB p65 and p50 binding to DNA induced by P. intermedia LPS.

3.6. Effects of NCX 4040 on P. intermedia LPS-induced phosphorylation of STAT1 and STAT3 We also evaluated the likelihood that NCX 4040 suppresses P. intermedia LPSinduced production of NO, IL-1β and IL-6 through inhibition of STAT1 and STAT3. The 12

data presented in Fig. 7 show that NCX 4040 down-regulated STAT1 and STAT3 phosphorylation in a dose-dependent manner in RAW264.7 cells treated with LPS isolated from P. intermedia.

3.7. Effect of NCX 4040 on SOCS1 expression in cells activated by P. intermedia LPS Finally, we tested the hypothesis that NCX 4040 would modulate the SOCS signaling that is known to play a role in cytokine signaling as negative regulators. Real-time PCR analysis showed that treatment with NCX 4040 dose-dependently elevated the SOCS1 mRNA in cells stimulated with P. intermedia LPS (Fig. 8). However, NCX 4040 did not influence the mRNA levels of SOCS2 and SOCS3 in LPS-activated cells (data not shown).

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4. Discussion

In this study, we examined the effects of NCX 4040, a NO-donating aspirin derivative, on the production of proinflammatory mediators in macrophages activated with P. intermedia LPS. NO, a gaseous inorganic free radical, is a signaling molecule that is known to play a crucial role in diverse physiological and pathological processes (Moncada et al., 1991) and is synthesized from the substrate L-arginine by a family of NO synthases (NOS) (Stuehr, 1999). Three isoforms of NOS are known to exist in mammalian tissues: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Both nNOS and eNOS are constitutive calcium-dependent forms of the enzyme, whereas iNOS, which is independent of calcium, is highly induced in response to a range of inflammatory stimuli such as LPS and cytokines in a variety of cells (Stuehr, 1997). Low concentrations of NO induced by constitutive NOS are believed to be beneficial (Ignarro et al., 1987; Garthwaite, 1995), whereas massive amounts of NO generated by iNOS play an important role in the initiation and progression of inflammation, septic shock, atherosclerosis and other pathologies (Wong and Billiar, 1995; Takahashi et al., 1997). It was reported that the structure and function of P. intermedia LPS is dissimilar to those of LPS preparations isolated from enteric bacteria, such as Escherichia coli and Salmonella species (Hamada et al., 1990). In addition, unlike Salmonella LPS, P. intermedia LPS induces the activation of macrophages of classical LPS-refractory C3H/HeJ mice (Kirikae et al., 1999). Moreover, while the biological activities of LPS isolated from Enterobacteriaceae were repressed by polymyxin B treatment, P. intermedia LPS was not prone to polymyxin B (Kirikae et al., 1999). 14

Our previous studies clearly demonstrate that P. intermedia LPS is able to induce the production of the major inflammatory mediators, such as NO, IL-1β, IL-6 and TNF-α, in macrophages (Kim et al., 2004, 2007; Choi et al., 2011, 2014). In addition, P. intermedia LPS was found to provoke the generation of NO and IL-6 by fetal mouse osteoblasts in organoid culture (Pelt et al., 2002). P. intermedia LPS has also been shown to suppress bone formation by diminishing alkaline phosphatase activity and calcium incorporation (Pelt et al., 2002). In this study, NCX 4040 significantly reduced P. intermedia LPS-induced production of iNOS-derived NO, IL-1β and IL-6 as well as their mRNA expression in RAW264.7 cells. These findings indicate that transcriptional mechanisms are involved in the attenuation of these proinflammatory mediators by NCX 4040. NCX 4040 exerted a strong inhibitory effects on P. intermedia LPS-induced production of NO, IL-1β and IL-6, in striking contrast to the activities shown by the parental compound aspirin. The enhanced effect of NCX 4040 seems to be due to NO-releasing moiety of this drug (Del Soldato et al., 1999; Fiorucci et al., 2000). One important point to be discussed is how NO, released from NCX 4040, counteracts P. intermedia LPS-induced production of proinflammatory mediators. While NO donors, including NCX 4040, are capable of providing NO when applied to biological systems, their actions are more complicated than just merely liberating NO (Sarkar et al., 2001). Hence, caution should be applied in equating the effect of exogenous NO donors with that of endogenous NO, such as iNOS-derived NO. Studies conducted so far indicate that NO donors may down-regulate the proinflammatory responses to LPS presumably by its activities on key LPS-dependent signaling pathways such as NF-κB (Peng et al., 1995; Matthews et al., 1996; Anuar et al., 2006; Ricciotti et al., 2010). HO-1 is the inducible isoform of heme oxygenase that catabolizes conversion of free 15

heme into carbon monoxide (CO), biliverdin and ferrous iron. HO-1 possesses numerous potentially protective actions including anti-apoptotic, antioxidant and anti-inflammatory properties (Morse and Choi, 2002; Otterbein et al., 2003; Ryter et al., 2006). HO-1-deficient mice exhibited severe inflammation, while overexpression of this enzyme was shown to reduce inflammation (Poss and Tonegawa, 1997; Otterbein et al., 2003). In this study, we found that NCX 4040 induced the expression of HO-1 in cells treated with P. intermedia LPS, and the suppressive effect of NCX 4040 on LPS-induced production of NO was significantly reversed by SnPP, supporting the idea that HO-1 expression induced by NCX 4040 plays a role in the inhibition of P. intermedia LPS-induced production of NO. However, HO-1 was not involved in the attenuation of IL-1β and IL-6 production mediated by NCX 4040. Studies have demonstrated that the biological activities of HO-1 are mediated by the heme degradation byproducts CO and bilirubin (Otterbein et al., 2000; Morse et al., 2003; SaradyAndrews et al., 2005). We therefore propose that the suppressive effect of NCX 4040 on NO production is partly due to CO and bilirubin generated as a consequence of HO-1 induction. Next, we examined whether the suppressive outcomes of NCX 4040 on P. intermedia LPS-induced production of NO, IL-1β and IL-6 are associated with inhibition of JNK and p38 signaling. NCX 4040 did not influence JNK and p38 phosphorylation induced by P. intermedia LPS, indicating that these pathways do not play a role in the suppression of LPS-induced generation of NO, IL-1β and IL-6 by NCX 4040. NF-κB is a major transcription factor that plays a central role in the regulation of genes involved in the inflammatory responses and has been implicated in the pathogenesis of various inflammatory diseases (Baeuerle and Henkel, 1994; Rothwarf and Karin, 1999; Karin and Ben-Neriah, 2000). In the present study, we showed that the degradation of IκB-α as well 16

as nuclear translocation and DNA-binding activity of NF-κB p65 and p50 subunits induced by P. intermedia LPS were significantly reduced by NCX 4040. There are several reports in the literature that NO donors, including NCX 4040, suppress NF-κB activation either by inhibition of IκB-α degradation (Peng et al., 1995; Ricciotti et al., 2010) or by interfering with the DNA binding activity of NF-κB subunits (Matthews et al., 1996). Signal transducer and activator of transcription (STAT) is another key transcription factor that has been implicated in LPS-induced inflammatory responses (Pfitzner et al., 2004). In particular, STAT1 and STAT3 play pivotal roles in modulating LPS-induced expression of genes encoding various proinflammatory mediators, including iNOS and inflammatory cytokines, in macrophages (Gao et al., 1998; Samavati et al., 2009). Present study showed that phosphorylation of STAT1 and STAT3 was significantly down-regulated by NCX 4040 in cells treated with P. intermedia LPS, suggesting that NCX 4040 exerts its antiinflammatory action by attenuating the activation of the STAT1 and STAT3 signaling. The suppressors of cytokine signaling (SOCS) family, which inhibits cytokine signal transduction in a variety of cell types, has eight members, including SOCS1 to 7 and cytokine-inducible Src homology 2 (SH2) domain-containing protein (CIS) (Alexander, 2002; Alexander and Hilton, 2004). SOCS proteins can be induced by various cytokines and also by toll-like receptor (TLR) ligands, such as LPS (Stoiber et al., 1999; Cassatella et al., 1999). These proteins are primarily up-regulated by the activation of JAK/STAT signaling and provide negative feedback to cytokine signaling by suppressing the interactions of activated JAK or STAT with receptors (Tamiya et al., 2011). In particular, SOCS-1 has been shown to directly bind to JAK2 and plays a key role in the inhibition of inflammation (Waiboci et al., 2007). Additionally, SOCS-1 also restricts TLR activation and down-regulates NF-κB17

dependent transcription by decreasing p65 subunit stability (Strebovsky et al., 2011). Garlet et al. (2006) showed increased expression of SOCS1 mRNA in diseased periodontal tissues than in healthy gingival biopsies of control subjects, implying a possibly active role for SOCS1 in the pathogenesis of human periodontal disease. In addition, the kinetic profile of SOCS1 expression in the ligature-induced periodontitis in rats was directly correlated with inflammation severity and alveolar bone loss, as well as with the activation status of STAT1 and STAT3 (de Souza et al., 2011). Hence, the up-regulation SOCS1 could be useful in suppressing the inflammation associated with periodontal diseases. In this study, we found that NCX 4040 significantly raised the expression of SOCS1 mRNA in cells activated with P. intermedia LPS. This is, to our knowledge, the first observation about the involvement of NCX 4040 in SOCS modulation. Overall, the results from the current study indicate that NCX 4040 inhibits P. intermedia LPS-induced production of iNOS-derived NO, IL-1β and IL-6 in murine macrophages through anti-inflammatory HO-1 induction and suppression of NF-κB, STAT1 and STAT3 activation, which is associated with the activation of SOCS1 signaling. Accordingly, we conclude that NCX 4040 could potentially be a promising tool in the prevention and/or treatment of periodontal disease, although further study will be required to verify this.

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Acknowledgements

“This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013R1A1A2007625)”

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References

Alexander, W.S., 2002. Suppressors of cytokine signalling (SOCS) in the immune system. Nat. Rev. Immunol. 2, 410-416. Alexander, W.S., Hilton, D.J., 2004. The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response. Annu. Rev. Immunol. 22, 503-529. Anuar, F., Whiteman, M., Siau, J.L., Kwong, S.E., Bhatia, M., Moore, P.K., 2006. Nitric oxide-releasing flurbiprofen reduces formation of proinflammatory hydrogen sulfide in lipopolysaccharide-treated rat. Br. J. Pharmacol. 147, 966-974. Baeuerle, P.A., Henkel, T., 1994. Function and activation of NF-κB in the immune system. Annu. Rev. Immunol. 12, 141-179. Beem, J.E., Nesbitt, W.E., Leung, K.P., 1998. Identification of hemolytic activity in Prevotella intermedia. Oral Microbiol. Immunol. 13, 97-105. Braga, R.R., Carvalho, M.A., Bruña-Romero, O., Teixeira, R.E., Costa, J.E., Mendes, E.N., Farias, L.M., Magalhães, P.P., 2010. Quantification of five putative periodontal pathogens in female patients with and without chronic periodontitis by real-time polymerase chain reaction. Anaerobe 16, 234-239. Bratasz, A., Selvendiran, K., Wasowicz, T., Bobko, A., Khramtsov, V.V., Ignarro, L.J., Kuppusamy, P. , 2008. NCX-4040, a nitric oxide-releasing aspirin, sensitizes drugresistant human ovarian xenograft tumors to cisplatin by depletion of cellular thiols. J. Transl. Med.6, 9. Burgaud, J.L., Riffaud, J.P., del Soldato, P., 2002. Nitric-oxide releasing molecules: a new class of drugs with several major indications. Curr. Pharm. Des. 8, 201-213. 20

Cassatella, M.A., Gasperini, S., Bovolenta, C., Calzetti, F., Vollebregt, M., Scapini, P., Marchi, M., Suzuki, R., Suzuki, A., Yoshimura, A., 1999. Interleukin-10 (IL-10) selectively enhances CIS3/SOCS3 mRNA expression in human neutrophils: evidence for an IL-10-induced pathway that is independent of STAT protein activation. Blood 94, 2880-2889. Choi, E.Y., Jin, J.Y., Lee, J.Y., Choi, J.I., Choi, I.S., Kim, S.J., 2011. Melatonin inhibits Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-6 in murine macrophages by suppressing NF-κB and STAT1 activity. J. Pineal. Res. 50, 197-206. Choi, E.Y., Jin, J.Y., Choi, J.I., Choi, I.S., Kim, S.J., 2014. DHA suppresses Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages. Br. J. Nutr. 111, 1221-1230. Chung, C.P., Nisengard, R.J., Slots, J., Genco, R.J., 1983. Bacterial IgG and IgM antibody titers in acute necrotizing ulcerative gingivitis. J. Periodontol. 54, 557-562. de Souza, J.A., Nogueira, A.V., de Souza, P.P., Cirelli, J.A., Garlet, G.P., Rossa, C. Jr., 2011. Expression of suppressor of cytokine signaling 1 and 3 in ligature-induced periodontitis in rats. Arch. Oral Biol. 56, 1120-1128. Del Soldato, P., Sorrentino, R., Pinto, A., 1999. NO-aspirins: a class of new antiinflammatory and anti-thrombotic agents. Trends Pharmacol. Sci. 20, 319-323. Fiorucci, S., Santucci, L., Cirino, G., Mencarelli, A., Familiari, L., Soldato, P.D., Morelli, A., 2000. IL-1β converting enzyme is a target for nitric oxide-releasing aspirin: new insights in the anti-inflammatory mechanism of nitric oxide-releasing nonsteroidal antiinflammatory drugs. J. Immunol. 165, 5245-5254. 21

Gao, J.J., Filla, M.B., Fultz, M.J., Vogel, S.N., Russell, S.W., Murphy, W.J., 1998. Autocrine/paracrine IFN-αβ mediates the lipopolysaccharide-induced activation of transcription factor Stat1α in mouse macrophages: pivotal role of Stat1α in induction of the inducible nitric oxide synthase gene. J. Immunol. 161, 4803-4810. Garlet, G.P., Cardoso, C.R., Campanelli, A.P., Martins, W. Jr., Silva, J.S., 2006. Expression of suppressors of cytokine signaling in diseased periodontal tissues: a stop signal for disease progression? J. Periodontal Res. 41, 580-584. Garthwaite, J., 1995. Neural nitric oxide signaling. Trends Neurosci. 18, 51-52. Guan, S.M., Shu, L., Fu, S.M., Liu, B., Xu, X.L., Wu, J.Z., 2009. Prevotella intermedia upregulates MMP-1 and MMP-8 expression in human periodontal ligament cells. FEMS Microbiol. Lett. 299, 214-222. Hamada, S., Takada, H., Ogawa, T., Fujiwara, T., Mihara, J., 1990. Lipopolysaccharides of oral anaerobes associated with chronic inflammation: chemical and immunomodulating properties. Int. Rev. Immunol. 6, 247-261. Ignarro, L.J., Buga, G.M., Wood, K.S., Byrns, R.E., Chaudhuri, G., 1987. Endotheliumderived relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci. U.S.A. 84, 9265-9269. Karin, M., Ben-Neriah, Y., 2000. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu. Rev. Immunol. 18, 621-663. Kim, S.J., Ha, M.S., Choi, E.Y., Choi, J.I., Choi, I.S., 2004. Prevotella intermedia lipopolysaccharide stimulates release of nitric oxide by inducing expression of inducible nitric oxide synthase. J. Periodontal Res. 39, 424-431. Kim, S.J., Choi, E.Y., Kim, E.G., Shin, S.H., Lee, J.Y., Choi, J.I., Choi, I.S., 2007. Prevotella 22

intermedia lipopolysaccharide stimulates release of tumor necrosis factor-alpha through mitogen-activated protein kinase signaling pathways in monocyte-derived macrophages. FEMS Immunol. Med. Microbiol. 51, 407-413. Kirikae, T., Nitta, T., Kirikae, F., Suda, Y., Kusumoto, S., Qureshi, N., Nakano, M., 1999. Lipopolysaccharides (LPS) of oral black-pigmented bacteria induce tumor necrosis factor production by LPS-refractory C3H/HeJ macrophages in a way different from that of Salmonella LPS. Infect. Immun. 67, 1736-1742. Kobayashi, K., Takahashi, N., Jimi, E., Udagawa, N., Takami, M., Kotake, S., Nakagawa, N., Kinosaki, M., Yamaguchi, K., Shima, N., Yasuda, H., Morinaga, T., Higashio, K., Martin, T.J., Suda, T., 2000. Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J. Exp. Med. 191, 275286. Kornman, K.S., Loesche, W.J., 1980. The subgingival microbial flora during pregnancy. J. Periodontal Res. 15, 111-122. Liu, X.H., Kirschenbaum, A., Yao, S., Levine, A.C., 2005. Cross-talk between the interleukin-6 and prostaglandin E2 signaling systems results in enhancement of osteoclastogenesis through effects on the osteoprotegerin/receptor activator of NF-κB (RANK) ligand/RANK system. Endocrinology 146, 1991-1998. Matejka, M., Partyka, L., Ulm, C., Solar, P., Sinzinger, H., 1998. Nitric oxide synthesis is increased in periodontal disease. J. Periodontal Res. 33, 517-518. Matthews, J.R., Botting, C.H., Panico, M., Morris, H.R., Hay, R.T., 1996. Inhibition of NFκB binding by nitric oxide. Nucleic Acids Res. 24, 2236-2242. Mogi, M., Otogoto, J., Ota, N., Inagaki, H., Minami, M., Kojima, K., 1999. Interleukin 1β, 23

interleukin 6, β2-microglobulin, and transforming growth factor-α in gingival crevicular fluid from human periodontal disease. Arch. Oral Biol. 44, 535-539. Moncada, S., Palmer, R.M., Higgs, E.A., 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109-142. Morrison, D.C., Ryan, J.L., 1987. Endotoxins and disease mechanisms. Annu. Rev. Med. 38, 417-432. Morse, D., Choi, A.M., 2002. Heme oxygenase-1: the “emerging molecule” has arrived. Am. J. Respir. Cell. Mol. Biol. 27, 8-16. Morse, D., Pischke, S.E., Zhou, Z., Davis, R.J., Flavell, R.A., Loop, T., Otterbein, S.L., Otterbein, L.E., Choi, A.M., 2003. Suppression of inflammatory cytokine production by carbon monoxide involves the JNK pathway and AP-1. J. Biol. Chem. 278, 36993-36998. Otterbein, L.E., Bach, F.H., Alam, J., Soares, M., Tao Lu, H., Wysk, M., Davis, R.J., Flavell, R.A., Choi, A.M., 2000. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat. Med. 6, 422-428. Otterbein, L.E., Soares, M.P., Yamashita, K., Bach, F.H., 2003. Heme oxygenase-1: unleashing the protective properties of heme. Trends Immunol. 24, 449-455. Pelt, P., Zimmermann, B., Ulbrich, N., Bernimoulin, J.P., 2002. Effects of lipopolysaccharide extracted from Prevotella intermedia on bone formation and on the release of osteolytic mediators by fetal mouse osteoblasts in vitro. Arch. Oral Biol. 47, 859-866. Peng, H.B., Libby, P., Liao, J.K., 1995. Induction and stabilization of IκB-α by nitric oxide mediates inhibition of NF-κB. J. Biol. Chem. 270, 14214-14219. Pfitzner, E., Kliem, S., Baus, D., Litterst, C.M., 2004. The role of STATs in inflammation and inflammatory diseases. Curr. Pharm. Des. 10, 2839-2850. 24

Poss, K.D., Tonegawa, S., 1997. Reduced stress defense in heme oxygenase 1-deficient cells. Proc. Natl. Acad. Sci. U.S.A. 94, 10925-10930. Preshaw, P.M., 2008. Host response modulation in periodontics. Periodontol. 2000 48, 92-110. Reddy, M.S., Geurs, N.C., Gunsolley, J.C., 2003. Periodontal host modulation with antiproteinase, anti-inflammatory, and bone-sparing agents. A systematic review. Ann. Periodontol. 8, 12-37. Ricciotti, E., Dovizio, M., Di Francesco, L., Anzellotti, P., Salvatore, T., Di Francesco, A., Sciulli, M.G., Pistritto, G., Monopoli, A., Patrignani, P., 2010. NCX 4040, a nitric oxidedonating aspirin, exerts anti-inflammatory effects through inhibition of IκB-α degradation in human monocytes. J. Immunol. 184, 2140-2147. Rosetti, M., Tesei, A., Ulivi, P., Fabbri, F., Vannini, I., Brigliadori, G., Amadori, D., Bolla, M., Zoli, W., 2006. Molecular characterization of cytotoxic and resistance mechanisms induced by NCX 4040, a novel NO-NSAID, in pancreatic cancer cell lines. Apoptosis 11, 1321-1330. Rossomando, E.F., Kennedy, J.E., Hadjimichael, J., 1990. Tumour necrosis factor alpha in gingival crevicular fluid as a possible indicator of periodontal disease in humans. Arch. Oral. Biol. 35, 431-434. Rothwarf, D.M., Karin, M., 1999. The NF-κB activation pathway: a paradigm in information transfer from membrane to nucleus. Sci. STKE 1999, RE1. Ryter, S.W., Alam, J., Choi, A.M., 2006. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol. Rev. 86, 583-650. Samavati, L., Rastogi, R., Du, W., Hüttemann, M., Fite, A., Franchi, L., 2009. STAT3 tyrosine phosphorylation is critical for interleukin 1 beta and interleukin-6 production in 25

response to lipopolysaccharide and live bacteria. Mol. Immunol. 46, 1867-1877. Sarady-Andrews, J.K., Liu, F., Gallo, D., Nakao, A., Overhaus, M., Ollinger, R., Choi, A.M., Otterbein, L.E., 2005. Biliverdin administration protects against endotoxin-induced acute lung injury in rats. Am. J. Physiol. Lung Cell. Mol. Physiol. 289, L1131- L1137. Sarkar, D., Vallance, P., Harding, S.E., 2001. Nitric oxide: not just a negative inotrope. Eur. J. Heart. Fail. 3, 527-534. Segasothy, M., Samad, S.A., Zulfigar, A., Bennett, W.M., 1994. Chronic renal disease and papillary necrosis associated with the longterm use of nonsteroidal anti-inflammatory drugs as the sole or predominant analgesic. Am. J. Kidney Dis. 24, 17-24. Seymour, G.J., Ford, P.J., Cullinan, M.P., Leishman, S., Yamazaki, K., 2007. Relationship between periodontal infections and systemic disease. Clin. Microbiol. Infect. 13, 3-10. Shenker, B.J., Vitale, L., Slots, J., 1991. Immunosuppressive effects of Prevotella intermedia on in vitro human lymphocyte activation. Infect. Immun. 59, 4583-4589. Soll, A., 1998. Pathogenesis of nonsteroidal anti-inflammatory drug-related upper gastrointestinal toxicity. Am. J. Med. 105, 10S-16S. Stoiber, D., Kovarik, P., Cohney, S., Johnston, J.A., Steinlein, P., Decker, T., 1999. Lipopolysaccharide induces in macrophages the synthesis of the suppressor of cytokine signaling 3 and suppresses signal transduction in response to the activating factor IFN-γ. J. Immunol. 163, 2640-2647. Strebovsky, J., Walker, P., Lang, R., Dalpke, A.H., 2011. Suppressor of cytokine signaling 1 (SOCS1) limits NF-κB signaling by decreasing p65 stability within the cell nucleus. FASEB J. 25, 863-874. Stuehr, D.J. 1997. Structure-function aspects in the nitric oxide synthases. Annual Rev. 26

Pharmacol. Toxicol. 37, 339-359. Stuehr, D.J., 1999. Mammalian nitric oxide synthases. Biochim. Biophys. Acta 1411, 217230. Takahashi, M., Fukuda, K., Ohata, T., Sugimura, T., Wakabayashi, K., 1997. Increased expression of inducible and endothelial constitutive nitric oxide synthases in rat colon tumors induced by azoxymethane. Cancer Res. 57, 1233-1237. Tamiya, T., Kashiwagi, I., Takahashi, R., Yasukawa, H., Yoshimura, A., 2011. Suppressors of cytokine signaling (SOCS) proteins and JAK/STAT pathways: regulation of T-cell inflammation by SOCS1 and SOCS3. Arterioscler. Thromb. Vasc. Biol. 31, 980-985. Tatakis, D.N., 1993. Interleukin-1 and bone metabolism: a review. J. Periodontol. 64, 416431. Tesei, A., Ulivi, P., Fabbri, F., Rosetti, M., Leonetti, C., Scarsella, M., Zupi, G., Amadori, D., Bolla, M., Zoli, W., 2005. In vitro and in vivo evaluation of NCX 4040 cytotoxic activity in human colon cancer cell lines. J. Transl. Med. 3, 7. Tesei, A., Zoli, W., Fabbri, F., Leonetti, C., Rosetti, M., Bolla, M., Amadori, D., Silvestrini, R., 2008. NCX 4040, an NO-donating acetylsalicylic acid derivative: efficacy and mechanisms of action in cancer cells. Nitric Oxide 19, 225-236. Waiboci, L.W., Ahmed, C.M., Mujtaba, M.G., Flowers, L.O., Martin, J.P., Haider, M.I., Johnson, H.M., 2007. Both the suppressor of cytokine signaling 1 (SOCS-1) kinase inhibitory region and SOCS-1 mimetic bind to JAK2 autophosphorylation site: implications for the development of a SOCS-1 antagonist. J. Immunol. 178, 5058-5068. Williams, J.L., Borgo, S., Hasan, I., Castillo, E., Traganos, F., Rigas, B., 2001. Nitric oxidereleasing nonsteroidal anti-inflammatory drugs (NSAIDs) alter the kinetics of human 27

colon cancer cell lines more effectively than traditional NSAIDs: implications for colon cancer chemoprevention. Cancer Res. 61, 3285-3289. Williams, R.C., 1990. Periodontal disease. N. Engl. J. Med. 322, 373-381. Wong, J.M., Billiar, T.R., 1995. Regulation and function of inducible nitric oxide synthase during sepsis and acute inflammation. Adv. Pharmacol. 34, 155-170.

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Figure legends

Fig. 1. Effects of NCX 4040 on Prevotella intermedia LPS-induced production of NO (A), IL-1β (B) and IL-6 (C) in RAW264.7 cells. Cells were treated with different concentrations of NCX 4040 or aspirin in the absence or presence of P. intermedia LPS (10 μg/ml) for 24 h ((for NO and IL-6) or 48 h (for IL-1β), after which the NO, IL-1β and IL-6 levels in the culture supernatant were quantified. Data are presented as percentage of P. intermedia LPS alone. The results are means ± S.E.M. of three independent experiments. * P < 0.05 versus P. intermedia LPS alone; ** P < 0.01 versus P. intermedia LPS alone.

Fig.2. Effect of NCX 4040 on Prevotella intermedia LPS-induced iNOS protein expression in RAW264.7 cells. Cells were treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml) for 24 h, after which iNOS protein synthesis was measured by immunoblot analysis of cell lysates using iNOS-specific antibody. The expression of iNOS protein was quantified by densitometric scanning. A representative immunoblot from three separate experiments with similar results is shown. ** p < 0.01 versus P. intermedia LPS alone.

Fig. 3. Effects of NCX 4040 on Prevotella intermedia LPS-induced iNOS (A), IL-1β (B) and IL-6 (C) mRNA expression in RAW264.7 cells. Cells were treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml) for 6 h (for IL-1β) or 24 h (for iNOS and IL-6), after which real-time PCR was carried out with EvaGreen Supermix, β-actin being used as an endogenous control. The results are means ± 29

S.E.M. of three independent experiments. * P < 0.05 versus P. intermedia LPS alone; ** P < 0.01 versus P. intermedia LPS alone.

Fig. 4. Involvement of HO-1 in the inhibitory effects of NCX 4040 on Prevotella intermedia LPS-induced production of NO in RAW264.7 cells. (A, B) Cells were treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml) for 6 h (for HO-1 protein) or 3 h (for HO-1 mRNA). (A) HO-1 protein synthesis was measured by immunoblot analysis of cell lysates using HO-1-specific antibody. A representative immunoblot from three separate experiments with similar results is shown. (B) Real-time PCR was performed with EvaGreen Supermix, β-actin being used as an endogenous control. The results are means ± S.E.M. of three independent experiments. ** P < 0.01 versus P. intermedia LPS alone. (C) Cells were treated with NCX 4040 (30 μM) and P. intermedia LPS (10 μg/ml) for 24 h in the presence of different doses of SnPP, after which the NO level in the culture supernatant were quantified. The results are means ± S.E.M. of three independent experiments. ** P < 0.01 versus P. intermedia LPS alone; † P < 0.05 versus P. intermedia LPS plus NCX 4040; †† P < 0.01 versus P. intermedia LPS plus NCX 4040.

Fig. 5. Effects of NCX 4040 on Prevotella intermedia LPS-induced phosphorylation of JNK (A) and p38 (B) in RAW264.7 cells. Cells were treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml) for 15 min (for p38) or 30 min (for JNK), after which cell lysates were subjected to immunoblot analysis using specific antibodies. A representative immunoblot from three separate experiments with similar results is shown. 30

Fig. 6. Effects of NCX 4040 on Prevotella intermedia LPS-induced activation of NF-κB in RAW264.7 cells. (A, B) Cells were treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml). (A) After 30 min of incubation, IκB-α degradation was determined by immunoblot analysis of cell lysates using antibody against IκB-α. A representative immunoblot from three separate experiments with similar results is shown. (B) After 30 min (for NF-κB p65) or 8 h (for NF-κB p50) of incubation, the nuclear fraction was isolated from cells. Nuclear translocation of NF-κB subunits was assessed by immunoblot analysis using antibodies against NF-κB p65 and p50. A representative immunoblot from three separate experiments with similar results is shown. (C) Cells were treated with different concentrations of NCX 4040 (20 and 30 μM) in the absence or presence of P. intermedia LPS (10 μg/ml). After 30 min (for NF-κB p65) or 8 h (for NF-κB p50) of incubation, the nuclear fraction was isolated from cells. DNA-binding activity of NF-κB in nuclear extracts was assessed by using the ELISA-based NF-κB p65/NF-κB p50 transcription factor assay kits. The results are means ± S.E.M. of two independent experiments. ** P < 0.01 versus P. intermedia LPS alone.

Fig. 7. Effects of NCX 4040 on Prevotella intermedia LPS-induced phosphorylation of STAT1 (A) and STAT3 (B) in RAW264.7 cells. Cells were treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml) for 4 h, after which cell lysates were subjected to immunoblot analysis using specific antibodies. A representative immunoblot from three separate experiments with similar results is shown.

Fig. 8. Effect of NCX 4040 on SOCS1 expression in RAW264.7 cells activated by Prevotella 31

intermedia LPS. Cells were treated with different concentrations of NCX 4040 in the absence or presence of P. intermedia LPS (10 μg/ml) for 1 h, after which real-time PCR was carried out with EvaGreen Supermix, β-actin being used as an endogenous control. The results are means ± S.E.M. of three independent experiments. * P < 0.05 versus P. intermedia LPS alone; **

P < 0.01 versus P. intermedia LPS alone.

Supporting information Fig. S1. Effect of NCX 4040 on cell viability in RAW264.7 cells activated by Prevotella intermedia LPS. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay. The results are means ± S.E.M. of three independent experiments.

, 24 h culture;

,48 h culture.

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