INTRODUCTION

Royal jelly is produced by honeybees and is widely used as a dietary supplement. Nurse bees secrete royal jelly from the hypopharyngeal and mandibular glands [1] and feed it to all larvae for 3 days, but to queen bee larvae throughout their lives [2]. Royal jelly has been reported to have many pharmacological activities, including anti-inflammatory, anti-tumour and anti-bacterial activities [3–5]. It contains 10-hydroxy-trans-2-decenoic acid (10H2DA), which is a major and unique lipid component often referred to as ‘royal jelly acid’ [6]. 10H2DA is responsible for some of the pharmacological activities of royal jelly, including its anti-tumour and anti-bacterial activities [7–9].

We previously showed that 10H2DA exerted an inhibitory effect on lipopolysaccharide (LPS) and interferon (IFN)-γ signaling [10, 11]. Among the cytokines and chemokines upregulated by LPS stimulation, only interleukin (IL)-6 was inhibited by 10H2DA, by a mechanism involving specific inhibition of inhibitor of nuclear factor (NF)-κB (IκB)-ζ gene expression. In the case of IFN-γ-stimulation, 10H2DA inhibited nitric oxide (NO) production through inhibition of IFN regulatory factor (IRF)-8 induction, followed by reduction of tumour necrosis factor (TNF)-α production, which is an essential step in IFN-γ-induced NO production [12]. LPS is also known to induce NO production in murine macrophages. The promoter region of inducible NO synthase (iNOS), which is responsible for much of the NO production in macrophages, contains κB and IFN-stimulated response element (ISRE) sequences, which bind the transcription factors NF-κB and IFN-stimulated gene factor 3, respectively [13]. IRF-1 can also bind and activate ISRE in the iNOS promoter [14]. Activation of both κB and ISRE sequences is necessary for inducing iNOS transcription [15, 16]. In the case of LPS stimulation, IFN-β production and its autocrine stimulation is required for ISRE activation followed by iNOS induction [17].

The results of our previous study showed that LPS-induced TNF-α and IFN-β expression were not inhibited by 10H2DA [10]. Autocrine stimulation by TNF-α may activate NF-κB. It therefore remains unclear if 10H2DA inhibits LPS-induced NO production. In this study, we examined the effect of 10H2DA on LPS-induced NO production in murine macrophages and further investigated the mechanisms whereby 10H2DA inhibits LPS-stimulated iNOS induction at a transcriptional level. The selective inhibitory effect of 10H2DA on LPS- or cytokine-stimulated NF-κB activation is also discussed.

MATERIALS AND METHODS

Reagents

10H2DA (purity >98 % analysed by high-performance liquid chromatography) was a kind gift from Api Co. Ltd., Gifu, Japan. LPS from Escherichia coli O55 was from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Recombinant murine (r)IFN-β and rTNF-α were purchased from PBL Interferon Source (Piscataway, NJ, USA) and e-Bioscience (San Diego, CA, USA), respectively. Antibodies against signal transducer and activator of transcription (STAT)1, phospho-Ser727 STAT1 and phospho-Tyr701 STAT1 (Sigma-Aldrich, Inc.), IRF-1 (Santa Cruz Biotechnology, Inc., CA, USA) and IRF-3 (Cell Signaling Technology, Inc., Danvers, MA, USA) were used.

Plasmids

The reporter gene plasmids for iNOS and IFN-β promoter, piNOS-luc1974 and pGV-IFNb, respectively, have been described previously [11, 18]. NF-κB and ISRE reporter gene plasmids, pNF-κB-TA-luc and pISRE-TA-luc, respectively, were purchased from Clontech (Palo Alto, CA, USA). A transfection control reporter plasmid, pRL-TK, was from Promega (Madison, WI, USA). Plasmids were extracted using a GenElute HP endotoxin-free plasmid midiprep kit (Sigma-Aldrich, Inc.) for transfection.

Cell Culture, 10H2DA Treatment and Stimulation

The RAW264 murine macrophage cell line was obtained from Riken BioResource Center (Tokyo, Japan) and maintained in RPMI 1640 medium containing 5 % heat-inactivated fetal bovine serum. Cells were seeded in 96-well plates at 2 × 105 cells/well for NO and cytokine assays, or 5 × 104 cells/well for reporter gene assay. Cells were pre-incubated with or without the indicated concentrations of 10H2DA for 30 min, and then stimulated by the addition of LPS (100 ng/ml) or rIFN-β (10,000 U/ml) for the indicated periods.

Determination of Nitrite Concentration

Nitrite is the end product of NO metabolism, and was determined as described previously [19]. Briefly, culture supernatants were mixed with 100 μl of Griess reagent [20], and the nitrite concentration was measured at an absorbance of 540 nm. Data are expressed as the mean ± SD of triplicate sets representing at least two independent experiments.

Reverse Transcription–Polymerase Chain Reaction Analysis of iNOS mRNA Expression

RAW264 cells were cultured on six-well plates and stimulated with LPS (100 ng/ml). The cells were harvested, and total RNA was extracted using an RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) as recommended by the manufacturer. The RNA was reverse-transcribed with ReverTra-Plus- (Toyobo, Tokyo, Japan). Complementary iNOS DNA was amplified with specific primers for each gene using Quick Taq HS DyeMix (Toyobo). Primer sequences were as follows: iNOS, 5′-CTG CAG GTC TTT GAC GCT CG-3′ and 5′-GTG GAA CAC AGG GGT GAT GC-3′, hypoxanthine phosphoribosyltransferase (HPRT), 5′-GTA ATG ATC GTC AAC GGG GGA C-3′ and 5′-CCA GCA AGC TTG CAA CCT TAA C-3′. Amplified PCR products were analysed by agarose gel electrophoresis followed by ethidium bromide staining. Real-time PCR analysis was performed using ThunderBird SYBR Green Master Mix (Toyobo).

Reporter Gene Assay

Cells seeded in wells of a 96-well plate were transfected with one of the reporter gene constructs and pRL-TK plasmid using FuGENE-HD (Roche, Indianapolis, IN, USA) as recommended by the manufacturer, and incubated for 24 h. The cells were stimulated with LPS (100 ng/ml), rIFN-β (10,000 U/ml) or rTNF-α (50 ng/ml) for 24 h, and then lysed with Passive Lysis Buffer (Promega). The luciferase activity in the cell lysate was determined with the Dual-Luciferase Reporter Assay System (Promega).

Immunoblotting

RAW264 cells were pre-treated with 10H2DA (4 mM) for 30 min, and then cultured with LPS (100 ng/ml). The cells were harvested and lysed in lysis buffer (50 mM Tris–HCl buffer pH 7.5 containing 0.5 % Nonidet P-40, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride and phosphatase inhibitor cocktail II, Sigma-Aldrich, Inc.). After boiling at 100 °C for 5 min, the cell lysates containing an equal amount of protein (20 μg/lane) were loaded onto an 8 % gel, run under reducing conditions and transferred onto Immobilon Transfer Membranes (Millipore Co., Bedford, MA, USA). The membranes were set on a SNAP.i.d system (Millipore), blocked with 1 % bovine serum albumin (BSA) for 20 s and incubated with a first antibody for 10 min. The blots were further treated with a 1:2,000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology) for 10 min. The immune complexes on the blots were detected with ImmunoStar (Wako, Tokyo, Japan) and LAS-3000UVmini (Fujifilm, Tokyo, Japan).

Statistical Analysis

Experimental results are expressed as the mean value ± SD. Statistical analyses were performed using GraphPad PRISM software (GraphPad, San Diego, CA, USA). Significant differences were evaluated using Dunnett’s multiple comparison test after one-way analysis of variance. A value of p < 0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION

Inhibition of LPS-Induced NO Production by 10H2DA

The effect of 10H2DA on LPS-induced NO production was examined in RAW264 murine macrophages. LPS-induced NO production was significantly and dose-dependently inhibited by 0.5–4 mM 10H2DA (Fig. 1a). To reveal if NO production was inhibited at the transcriptional level of the iNOS gene, the levels of iNOS mRNA and iNOS promoter activity were measured by reverse transcription–polymerase chain reaction (RT-PCR) and reporter gene assay, respectively. Both semi-quantitative (Fig. 1b) and quantitative (Fig. 1c) RT-PCR analyses clearly showed that the LPS-induced increase in iNOS mRNA was reduced by 4 mM 10H2DA. LPS-induced iNOS promoter activation was also significantly inhibited by 4 mM 10H2DA in a dose-dependent manner (Fig. 1d). These results suggest that 10H2DA inhibits LPS-induced NO production at the translational level of the iNOS gene.

Fig. 1
figure 1

Effect of 10H2DA on LPS-induced NO production and iNOS and IFN-β promoter activation. a RAW264 cells were incubated with various concentrations of 10H2DA for 30 min, followed by stimulation with 100 ng/ml LPS (white circle) for 24 h. Data are expressed as mean ± SD (n = 3). Black circles no stimulation. b, c RAW264 cells were incubated with or without 4 mM 10H2DA for 30 min, followed by stimulation with 100 ng/ml LPS for the indicated periods. Total RNA was extracted and subjected to semi-quantitative RT-PCR (b) or quantitative RT-PCR (c). Samples were normalized to HPRT mRNA levels. Black circles LPS alone, white circles LPS + 10H2DA (4 mM). d, e RAW264 cells co-transfected with piNOS-luc1974 (d) or pGV-IFNb (e) and pRL-TK were incubated with or without 10H2DA (1, 2 and 4 mM) for 30 min, followed by stimulation with 100 ng/ml LPS for 24 h. Luciferase activity was measured by dual luciferase assay. Data are expressed as mean ± SD (n = 3). *p < 0.05.

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10H2DA Does Not Affect LPS-Induced IFN-β Production or ISRE Activation

LPS-induced IFN-β production is necessary for NO induction in murine macrophages [17]. Following LPS stimulation, Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF)-dependent signaling is activated resulting in activation and nuclear translocation of IRF-3 [21]. No effect of 10H2DA on nuclear translocation of IRF-3 was detected by immunocytochemistry [Electronic Supplementary Material (ESM) Fig. S1]. Further, 10H2DA had no effect on promoter activation of the IFN-β gene or the increase in IFN-β mRNA (Fig. 1e and ESM Fig. S2).

Autocrine stimulation of IFN-β activates the Janus kinase (JAK)–STAT signaling pathway [14, 17]. Because the induction of IFN-β by LPS stimulation was unaffected by 10H2DA, we examined the effects of 10H2DA on IFN-β signaling, including STAT1 phosphorylation and IRF-1 induction. LPS stimulation resulted in phosphorylation of STAT1 at Tyr701 and Ser727, and IRF-1 induction, both of which were unaffected by 10H2DA (Fig. 2a, b). Further, 10H2DA failed to inhibit LPS-stimulated ISRE activation, as determined by luciferase reporter gene assay (Fig. 2c), suggesting that transcription factors activating ISRE, such as STATs and IRF-1, were activated and internalized into the nucleus to transcribe the ISRE-dependent genes in the presence of 10H2DA. These results indicate that 10H2DA does not affect the activation of the JAK–STAT pathway following LPS stimulation.

Fig. 2
figure 2

Effect of 10H2DA on activation of JAK–STAT pathway following LPS stimulation. a, b RAW264 cells were incubated with or without 4 mM 10H2DA for 30 min, followed by stimulation with 100 ng/ml LPS for the indicated time periods. Cytoplasmic fractions obtained from the cells were separated by SDS–PAGE. Phospho-Tyr701-STAT1, phospho-Ser727-STAT1 and STAT1 (a), IRF-1 and ATAT1 (b) in the cytoplasmic fraction were detected by immunoblotting. c RAW264 cells transfected with pISRE-TA-luc and pRL-TK were incubated with or without 10H2DA (1, 2 and 4 mM) for 30 min, followed by stimulation with 100 ng/ml LPS for 24 h. Luciferase activity was measured by dual luciferase assay. Data are expressed as mean ± SD (n = 3).

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Inhibition of IFN-β-Induced NO Production by 10H2DA

LPS-induced IFN-β production and JAK–STAT activation were unaffected by 10H2DA, and we therefore examined the effect of 10H2DA on NO production induced by rIFN-β (Fig. 3a). 10H2DA significantly and dose-dependently inhibited rIFN-β-induced NO production. We further determined that 10H2DA inhibited rIFN-β-induced iNOS promoter activation, using a reporter gene assay (Fig. 3b), suggesting that inhibition of NO production occurred at the transcriptional level of the iNOS gene.

Fig. 3
figure 3

Effect of 10H2DA on IFN-β-induced NO production and iNOS promoter activation. a RAW264 cells were incubated with or without 10H2DA (1, 2 and 4 mM) for 30 min, followed by stimulation with 10,000 U/ml IFN-β for 24 h. Concentrations of nitrite ions in the culture supernatants were detected by the Griess method. Data are expressed as mean ± SD (n = 3). *p < 0.01; **p < 0.001. b RAW264 cells transfected with piNOS-luc1974 and pRL-TK were incubated with or without 10H2DA (1, 2 and 4 mM) for 30 min, followed by stimulation with 10,000 U/ml IFN-β for 24 h. Luciferase activity was measured by dual luciferase assay. Data are expressed as mean ± SD (n = 3). **p < 0.001.

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Inhibition of IFN-β-Induced NF-κB Activation and TNF-α Production by 10H2DA

In addition to the JAK–STAT pathway, NF-κB is also activated by type-I IFNs [22] including IFN-β, and may be involved in iNOS induction. The results of this study showed that 10H2DA significantly inhibited rIFN-β-induced NF-κB activation (Fig. 4a). We also examined the effect of 10H2DA on the production of TNF-α following stimulation with rIFN-β (Fig. 4b). TNF-α production was induced by rIFN-β and was partially inhibited by 4 mM 10H2DA. Our previous work has shown that 10H2DA does not affect the LPS-induced TNF-α production [10], while 10H2DA inhibited INF-γ-induced TNF-α production via inhibition of IRF-8 induction [11]. IFN-β-induced TNF-α production is mediated by NF-κB activation through phosphatidylinositol 3-kinase and Akt activation [22]. It would be possible that 10H2DA inhibits phosphatidylinositol 3-kinase–Akt pathway, which may not dominantly contribute to LPS-stimulated NF-κB activation. This pathway may also influence the expression of IRF-8 induced by INF-γ. Furthermore, rTNF-α activated NF-κB, and this activation was significantly inhibited by 10H2DA (Fig. 4c). These results together with our previous findings [10] indicate that 10H2DA inhibits NF-κB activation stimulated by IFN-β or TNF-α, without affecting LPS-induced IFN-β and TNF-α production.

Fig. 4
figure 4

Effect of 10H2DA on IFN-β- or TNF-α-induced NF-κB activation and IFN-β-induced TNF-α production. a, c RAW264 cells transfected with pNF-κB-TA-luc and pRL-TK were incubated with or without 10H2DA (1, 2 and 4 mM) for 30 min, followed by stimulation with 10,000 U/ml IFN-β (a) or 50 ng/ml TNF-α (c) for 24 h. Luciferase activity was measured by dual luciferase assay. Data are expressed as mean ± SD (n = 3). *p < 0.05; **p < 0.001. b RAW264 cells were incubated with or without 4 mM 10H2DA for 30 min, followed by stimulation with 10,000 U/ml IFN-β for the indicated periods. Concentrations of TNF-α in culture supernatants were measured by conventional sandwich enzyme-linked immunosorbent assays using specific antibodies and rTNF-α. Data are expressed as the mean ± SD (n = 3). *p < 0.05.

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Selective Inhibition of NF-κB Activation by 10H2DA

NF-κB is responsible for the expression of many cytokine and chemokine genes, including TNF-α, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory proteins (MIPs) [23]. The κB site is also present in the promoter region of the IFN-β gene, and could function in transcription [24]. We previously showed that 10H2DA inhibited LPS-induced IκB-ζ and IκB-ζ-dependent gene expression, but had no effect on other cytokines and chemokines, including TNF-α, MCP-1 and MIPs [10]. We speculate that the selective inhibition of NF-κB-dependent genes may be the result of inhibition of posttranslational modification of NF-κB by 10H2DA. Because iNOS gene expression is not dependent on IκB-ζ [25], the κB site in the iNOS promoter, as well as that in the IκB-ζ promoter, could be activated by a subset of NF-κB, the activation of which is sensitive to 10H2DA inhibition.

CONCLUSIONS

The results of the present study demonstrated that 10H2DA inhibited LPS- and IFN-β-induced NO production through inhibiting NF-κB activation induced by LPS, IFN-β or TNF-α, thus preventing upregulation of iNOS gene expression. In contrast, 10H2DA had no effect on LPS-induced IFN-β gene expression following activation of the JAK–STAT pathway and transcriptional activation of ISRE, suggesting that 10H2DA specifically inhibits NF-κB activation. The expression of NF-κB-dependent genes, except IκB-ζ and iNOS, was not inhibited by 10H2DA, suggesting that 10H2DA inhibits a subset of NF-κB, which is activated by not only LPS but also by IFN-β and TNF-α, and is responsible for the expression of IκB-ζ and iNOS genes. 10H2DA therefore represents a critical component of royal jelly, with unique anti-inflammatory and immunomodulating activities.