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Anti-inflammatory effects of adenosine N1-oxide.

Kohno K, Ohashi E, Sano O, Kusano H, Kunikata T, Arai N, Hanaya T, Kawata T, Nishimoto T, Fukuda S - J Inflamm (Lond) (2015)

Bottom Line: Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly.We found that it is refractory to adenosine deaminase-mediated conversion to inosine.Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock.

View Article: PubMed Central - PubMed

Affiliation: Core Technology Division, Research and Development Center, Hayashibara Co., Ltd, Okayama, Japan.

ABSTRACT

Background: Adenosine is a potent endogenous anti-inflammatory and immunoregulatory molecule. Despite its promise, adenosine's extremely short half-life in blood limits its clinical application. Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly. ANO is an oxidized product of adenosine at the N1 position of the adenine base moiety. We found that it is refractory to adenosine deaminase-mediated conversion to inosine. We further examined the anti-inflammatory activities of ANO in vitro and in vivo.

Methods: The effect of ANO on pro-inflammatory cytokine secretion was examined in mouse peritoneal macrophages and the human monocytic cell line THP-1, and compared with that of adenosine, synthetic adenosine receptor (AR)-selective agonists and dipotassium glycyrrhizate (GK2). The anti-inflammatory activity of ANO in vivo was examined in an LPS-induced endotoxin shock model in mice.

Results: ANO inhibited secretion of inflammatory mediators at much lower concentrations than adenosine and GK2 when used with peritoneal macrophages and THP-1 cells that were stimulated by LPS plus IFN-γ. The potent anti-inflammatory activity of ANO could not be solely accounted for by its refractoriness to adenosine deaminase. ANO was superior to the synthetic A1 AR-selective agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), A2A AR-selective agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamideadenosine hydrochloride (CGS21680), and A3 AR-selective agonist, N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), in suppressing the secretion of a broad spectrum of pro-inflammatory cytokines by peritoneal macrophages. The capacities of ANO to inhibit pro-inflammatory cytokine production by THP-1 cells were comparable with those of CCPA and IB-MECA. Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock. A significant increase in survival rate was also observed by oral administration of ANO. Mechanistic analysis suggested that the up-regulation of the anti-inflammatory transcription factor c-Fos was, at least in part, involved in the ANO-induced suppression of pro-inflammatory cytokine secretion.

Conclusions: Our data suggest that ANO, a naturally occurring molecule that is structurally close to adenosine but is functionally more potent, presents potential strategies for the treatment of inflammatory disorders.

No MeSH data available.


Related in: MedlinePlus

Inhibitory effects of ANO, adenosine, adenine and adenine N1-oxide on TNF-α and IL-6 secretion by LPS/muIFN-γ-stimulated peritoneal macrophages. Peritoneal macrophages (5 × 104/well) were stimulated with LPS (1 μg/mL) and muIFN-γ (10 IU/mL) in the presence or absence of various concentrations of ANO, adenosine, adenine and adenine N1-oxide at 37°C for 24 h. Levels of TNF-α (A) and IL-6 (B) in the culture supernatants and growth of macrophages (C) were determined as described in Figure 1. Values represent the means ± S.D. of quadruplicate cultures. Results are representative of two separate experiments with similar results. *p < 0.05; **p < 0.01, significantly different when compared with control culture.
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Fig2: Inhibitory effects of ANO, adenosine, adenine and adenine N1-oxide on TNF-α and IL-6 secretion by LPS/muIFN-γ-stimulated peritoneal macrophages. Peritoneal macrophages (5 × 104/well) were stimulated with LPS (1 μg/mL) and muIFN-γ (10 IU/mL) in the presence or absence of various concentrations of ANO, adenosine, adenine and adenine N1-oxide at 37°C for 24 h. Levels of TNF-α (A) and IL-6 (B) in the culture supernatants and growth of macrophages (C) were determined as described in Figure 1. Values represent the means ± S.D. of quadruplicate cultures. Results are representative of two separate experiments with similar results. *p < 0.05; **p < 0.01, significantly different when compared with control culture.

Mentions: ANO is an oxidized product of adenosine at the N1 position of the adenine moiety. To explore the relationship between structure and activity, we compared the inhibitory effects of ANO on pro-inflammatory cytokine production with those of adenosine, adenine and adenine N1-oxide. For this purpose, murine peritoneal macrophages were stimulated with 1 μg/mL LPS plus 10 IU/mL murine IFN-γ (LPS/muIFN-γ) to induce multiple pro-inflammatory mediators. IFN-γ primes macrophages, enhancing their response to LPS [21]. Moreover, IFN-γ is a critical mediator of endotoxin hypersensitivity induced by bacteria [22]. Figure 2A and B show the dose–response curves of the inhibitory effects of ANO and related compounds on the release of TNF-α and IL-6. ANO efficiently inhibited both TNF-α and IL-6 production in a dose-dependent fashion. Although minimal inhibition of macrophage growth was observed at 1 μM ANO, significant reductions in the production of both TNF-α and IL-6 were observed at concentrations that had no effect on macrophage growth. Therefore, it seems likely that inhibition of pro-inflammatory cytokines by ANO was not due to a reduction of cell proliferation (Figure 2C). Adenosine also inhibited the secretion of both TNF-α and IL-6 in a dose-dependent manner, but much higher concentrations were required to achieve reductions observed with ANO (Figure 2A and B). Unexpectedly, adenine significantly inhibited the release of TNF-α at 10 times higher concentrations than adenosine. IL-6 production was also significantly inhibited by adenine, while no clear dose–response effect was observed. Addition of adenine N1-oxide also resulted in reduction of both TNF-α and IL-6. However, the inhibitory action of adenine N1-oxide was comparable or inferior to that of adenine (Figure 2A and B).Figure 2


Anti-inflammatory effects of adenosine N1-oxide.

Kohno K, Ohashi E, Sano O, Kusano H, Kunikata T, Arai N, Hanaya T, Kawata T, Nishimoto T, Fukuda S - J Inflamm (Lond) (2015)

Inhibitory effects of ANO, adenosine, adenine and adenine N1-oxide on TNF-α and IL-6 secretion by LPS/muIFN-γ-stimulated peritoneal macrophages. Peritoneal macrophages (5 × 104/well) were stimulated with LPS (1 μg/mL) and muIFN-γ (10 IU/mL) in the presence or absence of various concentrations of ANO, adenosine, adenine and adenine N1-oxide at 37°C for 24 h. Levels of TNF-α (A) and IL-6 (B) in the culture supernatants and growth of macrophages (C) were determined as described in Figure 1. Values represent the means ± S.D. of quadruplicate cultures. Results are representative of two separate experiments with similar results. *p < 0.05; **p < 0.01, significantly different when compared with control culture.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4308844&req=5

Fig2: Inhibitory effects of ANO, adenosine, adenine and adenine N1-oxide on TNF-α and IL-6 secretion by LPS/muIFN-γ-stimulated peritoneal macrophages. Peritoneal macrophages (5 × 104/well) were stimulated with LPS (1 μg/mL) and muIFN-γ (10 IU/mL) in the presence or absence of various concentrations of ANO, adenosine, adenine and adenine N1-oxide at 37°C for 24 h. Levels of TNF-α (A) and IL-6 (B) in the culture supernatants and growth of macrophages (C) were determined as described in Figure 1. Values represent the means ± S.D. of quadruplicate cultures. Results are representative of two separate experiments with similar results. *p < 0.05; **p < 0.01, significantly different when compared with control culture.
Mentions: ANO is an oxidized product of adenosine at the N1 position of the adenine moiety. To explore the relationship between structure and activity, we compared the inhibitory effects of ANO on pro-inflammatory cytokine production with those of adenosine, adenine and adenine N1-oxide. For this purpose, murine peritoneal macrophages were stimulated with 1 μg/mL LPS plus 10 IU/mL murine IFN-γ (LPS/muIFN-γ) to induce multiple pro-inflammatory mediators. IFN-γ primes macrophages, enhancing their response to LPS [21]. Moreover, IFN-γ is a critical mediator of endotoxin hypersensitivity induced by bacteria [22]. Figure 2A and B show the dose–response curves of the inhibitory effects of ANO and related compounds on the release of TNF-α and IL-6. ANO efficiently inhibited both TNF-α and IL-6 production in a dose-dependent fashion. Although minimal inhibition of macrophage growth was observed at 1 μM ANO, significant reductions in the production of both TNF-α and IL-6 were observed at concentrations that had no effect on macrophage growth. Therefore, it seems likely that inhibition of pro-inflammatory cytokines by ANO was not due to a reduction of cell proliferation (Figure 2C). Adenosine also inhibited the secretion of both TNF-α and IL-6 in a dose-dependent manner, but much higher concentrations were required to achieve reductions observed with ANO (Figure 2A and B). Unexpectedly, adenine significantly inhibited the release of TNF-α at 10 times higher concentrations than adenosine. IL-6 production was also significantly inhibited by adenine, while no clear dose–response effect was observed. Addition of adenine N1-oxide also resulted in reduction of both TNF-α and IL-6. However, the inhibitory action of adenine N1-oxide was comparable or inferior to that of adenine (Figure 2A and B).Figure 2

Bottom Line: Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly.We found that it is refractory to adenosine deaminase-mediated conversion to inosine.Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock.

View Article: PubMed Central - PubMed

Affiliation: Core Technology Division, Research and Development Center, Hayashibara Co., Ltd, Okayama, Japan.

ABSTRACT

Background: Adenosine is a potent endogenous anti-inflammatory and immunoregulatory molecule. Despite its promise, adenosine's extremely short half-life in blood limits its clinical application. Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly. ANO is an oxidized product of adenosine at the N1 position of the adenine base moiety. We found that it is refractory to adenosine deaminase-mediated conversion to inosine. We further examined the anti-inflammatory activities of ANO in vitro and in vivo.

Methods: The effect of ANO on pro-inflammatory cytokine secretion was examined in mouse peritoneal macrophages and the human monocytic cell line THP-1, and compared with that of adenosine, synthetic adenosine receptor (AR)-selective agonists and dipotassium glycyrrhizate (GK2). The anti-inflammatory activity of ANO in vivo was examined in an LPS-induced endotoxin shock model in mice.

Results: ANO inhibited secretion of inflammatory mediators at much lower concentrations than adenosine and GK2 when used with peritoneal macrophages and THP-1 cells that were stimulated by LPS plus IFN-γ. The potent anti-inflammatory activity of ANO could not be solely accounted for by its refractoriness to adenosine deaminase. ANO was superior to the synthetic A1 AR-selective agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), A2A AR-selective agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamideadenosine hydrochloride (CGS21680), and A3 AR-selective agonist, N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), in suppressing the secretion of a broad spectrum of pro-inflammatory cytokines by peritoneal macrophages. The capacities of ANO to inhibit pro-inflammatory cytokine production by THP-1 cells were comparable with those of CCPA and IB-MECA. Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock. A significant increase in survival rate was also observed by oral administration of ANO. Mechanistic analysis suggested that the up-regulation of the anti-inflammatory transcription factor c-Fos was, at least in part, involved in the ANO-induced suppression of pro-inflammatory cytokine secretion.

Conclusions: Our data suggest that ANO, a naturally occurring molecule that is structurally close to adenosine but is functionally more potent, presents potential strategies for the treatment of inflammatory disorders.

No MeSH data available.


Related in: MedlinePlus