背景:腺苷是一种有效的内源性抗炎和免疫调节分子。尽管承诺,腺苷在血液中极短的半衰期限制了其临床应用。这里,我们检查了腺苷N1-氧化物(ANO),在蜂王浆中发现。ANO是腺苷在腺嘌呤碱基部分的N1位置的氧化产物。我们发现腺苷脱氨酶介导的向肌苷的转化是难治的。我们进一步检查了ANO在体外和体内的抗炎活性。
方法:在小鼠腹膜巨噬细胞和人单核细胞系THP-1中检查了ANO对促炎细胞因子分泌的影响,并与腺苷进行了比较。合成腺苷受体(AR)-选择性激动剂和甘草酸二钾(GK2)。在LPS诱导的小鼠内毒素休克模型中检查了ANO的体内抗炎活性。
结果:当与LPS加IFN-γ刺激的腹膜巨噬细胞和THP-1细胞一起使用时,ANO抑制炎症介质的分泌的浓度远低于腺苷和GK2。ANO的有效抗炎活性不能仅归因于其对腺苷脱氨酶的难治性。ANO优于合成的A1AR选择性激动剂,2-氯-N(6)-环戊基腺苷(CCPA),A2AAR选择性激动剂,2-[对(2-羧乙基)苯乙基氨基]-5'-N-乙基甲酰胺腺苷盐酸盐(CGS21680),和A3AR选择性激动剂,N(6)-(3-碘苄基)腺苷-5'-N-甲基糖醛酸酰胺(IB-MECA),抑制腹膜巨噬细胞分泌广谱促炎细胞因子。ANO抑制THP-1细胞产生促炎细胞因子的能力与CCPA和IB-MECA相当。反映其在体外的有效抗炎作用,静脉注射ANO可显著降低LPS诱导的内毒素休克的致死率。通过口服ANO也观察到存活率的显著增加。机制分析提示抗炎转录因子c-Fos的上调是,至少在某种程度上,参与ANO诱导的促炎细胞因子分泌抑制。
结论:我们的数据表明ANO,一种天然存在的分子,在结构上接近腺苷,但在功能上更有效,提出了治疗炎症性疾病的潜在策略。
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.